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Applications of natural silk protein sericin in biomaterials
Silk sericin is a natural macromolecular protein derived from silkworm Bombyx mori. During the various stages of producing raw silk and textile, sericin can be recovered for other uses. Also, sericin recovery reduces the environmental impact of silk manufacture. Sericin protein is useful because of its properties. The protein resists oxidation, is antibacterial, UV resistant, and absorbs and releases moisture
Yu-Qing Zhang
Consumption of silk protein, sericin elevates intestinal absorption of zinc, iron, magnesium and calcium in rats
Rats were examined for the effect of consumption of silk protein, sericin on the intestinal absorption of Zn, Fe, Mg and Ca. Male Wistar rats were fed on the diet containing either 23% egg albumin or 20% egg albumin plus 3% sericin for 12 d. Consumption of sericin elevated the apparent absorption of Zn, Fe, Mg and Ca (41%, 41%,
Masahiro Sasaki; Hideyuki Yamada; Norihisa Kato
Immobilization of l-asparaginase on the microparticles of the natural silk sericin protein and its characters
The natural silk sericin recovered from Bombyx mori silk waste by the degumming processing in the high-temperature and high-pressure is a macromolecular protein. Amino acid composition and molecular weight range of the sericin protein as a vector for enzyme immobilization were investigated. The silk sericin protein with different molecular mass from 50 to 200kDa was poorly soluble microparticles with an
Yu-Qing Zhang; Mei-Lin Tao; Wei-De Shen; Yu-Zhen Zhou; Yue Ding; Yan Ma; Wen-Lin Zhou
Optimization of the silk scaffold sericin removal process for retention of silk fibroin protein structure and mechanical properties.
In the process of removing sericin (degumming) from a raw silk scaffold, the fibroin structural integrity is often challenged, leading to mechanical depreciation. This study aims to identify the factors and conditions contributing to fibroin degradation during alkaline degumming and to perform an optimization study of the parameters involved to achieve preservation of fibroin structure and properties. The methodology involves degumming knitted silk scaffolds for various durations (5-90 min) and temperatures (60-100 degrees C). Mechanical agitation and use of the refreshed solution during degumming are included to investigate how these factors contribute to degumming efficiency and fibroin preservation. Characterizations of silk fibroin morphology, mechanical properties and protein components are determined by scanning electron microscopy (SEM), single fiber tensile tests and gel electrophoresis (SDS-PAGE), respectively. Sericin removal is ascertained via SEM imaging and a protein fractionation method involving SDS-PAGE. The results show that fibroin fibrillation, leading to reduced mechanical integrity, is mainly caused by prolonged degumming duration. Through a series of optimization, knitted scaffolds are observed to be optimally degummed and experience negligible mechanical and structural degradation when subjected to alkaline degumming with mechanical agitation for 30 min at 100 degrees C. PMID:20460689
Teh, Thomas K H; Toh, Siew-Lok; Goh, James C H
The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture
Sericin is the silk protein that covers fibroin fibers and functions as a `glue' in the cocoons of silkworms, and its most abundant component, Ser1, contains repeats of Ser- and Thr-rich 38 amino acid residues. The viability of Sf9 insect cells was 20, 57 and 49% on the fifth day and 41, 91 and 70% on the ninth day after
Masakazu Takahashi; Kazuhisa Tsujimoto; Hideyuki Yamada; Hiroshi Takagi; Shigeru Nakamori
Isolation, purification and characterization of silk protein sericin from cocoon peduncles of tropical tasar silkworm, Antheraea mylitta
A high molecular weight water-soluble glue protein, sericin was identified in the cocoon peduncle (a strong thread connecting the cocoons to the branches of the tree with a ring) of the tropical tasar silkworm, Antheraea mylitta. The sericin was isolated by 8M urea containing 1% sodium dodecyl sulfate and ?-mercaptoethenol (2%) or by 1% sodium chloride. The protein was purified
Rupesh Dash; Soumen Mukherjee; S. C. Kundu
Preparation of gel film from Bombyx mori silk sericin and its characterization as a wound dressing.
Sericin is a highly hydrophilic protein family acting as the glue in Bombyx mori silk. In order to apply sericin as a wound dressing, a novel sericin film named gel film was prepared by a simple process without using any chemical modifications: sericin solution was gelled with ethanol into a sheet shape and then dried. Infrared analysis revealed that the sericin gel film contained water-stable beta-sheet networks formed in the gelation step. This structural feature rendered the gel film morphologically stable against swelling and gave it good handling properties in the wet state. The sericin gel film rapidly absorbed water, equilibrating at a water content of about 80%, and exhibited elastic deformation up to a strain of about 25% in the wet state. A culture of mouse fibroblasts on the gel film indicated that it had low cell adhesion properties and no cytotoxicity. These characteristics of sericin gel film suggest its potential as a wound dressing. PMID:19060395
Teramoto, Hidetoshi; Kameda, Tsunenori; Tamada, Yasushi
Cryopreservation for bovine embryos in serum-free freezing medium containing silk protein sericin.
Because the use of serum in the embryo cryopreservation increases the probability of animal health problems such as bovine spongiform encephalopathy (BSE) and viral infections, this study was conducted to examine the effects of sericin supplementation for serum-free freezing medium on the survival and development of bovine embryos after freezing-thawing and direct transfer to recipients. When in vitro-produced bovine embryos were frozen conventionally in the freezing medium supplemented with various concentrations (0.1%, 0.5%, and 1.0%) of sericin, the percentages of damaged zona pellucida, survival, and development of frozen-thawed embryos were similar to those of embryos frozen in freezing medium supplemented with 0.4% bovine serum albumin (BSA) and 20% fetal bovine serum (FBS) (0.4BSA/20F; control). When in vivo-derived embryos were frozen with 0.4BSA/20F (control), 0.5% sericin +20% FBS (0.5S/20F) or 0.5% sericin (0.5S) and were subsequently transferred directly to recipients, the percentages of recipients with pregnancy and normal calving in the 0.5S/20F group were higher than those in the control group (47.3% vs. 40.1% and 94.6% vs. 87.3%, respectively). Moreover, the percentages of recipients with pregnancy and normal calving (42.2% and 92.4%, respectively) in the 0.5S group were similar with those of other groups. In conclusion, these results indicate that serum-free freezing medium supplemented with sericin is available for the cryopreservation of bovine embryos and that it is beneficial for the elimination of a potential source of biological contamination by serum or BSA. PMID:23850826
Isobe, Tomohiro; Ikebata, Yoshihisa; Onitsuka, Takeshi; Do, Lanh Thi Kim; Sato, Yoko; Taniguchi, Masayasu; Otoi, Takeshige
Dietary silk protein, sericin, improves epidermal hydration with increased levels of filaggrins and free amino acids in NC/Nga mice.
Epidermal hydration is maintained primarily by natural moisturising factors (NMF), of which free amino acids (AA) are major constituents that are generated by filaggrin degradation. To identify dietary sources that may improve skin dryness of atopic dermatitis (AD), we investigated dietary effects of silk proteins, sericin and fibroin, on epidermal levels of hydration, filaggrins and free AA, as well as PPAR?, peptidylarginine deiminase-3 (PAD3) and caspase-14 proteins involved in filaggrin expression and degradation processes. NC/Nga mice, an animal model of AD, were fed a control diet (group CA: atopic control) or diets with 1 % sericin (group S) or fibroin (group F) for 10 weeks. In group S, epidermal levels of hydration, total filaggrins and total free AA, as well as PPAR?, PAD3 and caspase-14, which were reduced in group CA, were increased to higher or similar levels of a normal control group of BALB/c mice (group C). Furthermore, profilaggrin, a precursor with multiple filaggrin repeats, and three repeat intermediates were increased, while two repeat intermediates and filaggrin were decreased in parallel with increased levels of glutamate and serine, major AA of NMF in group S. Despite increased levels of total filaggrins, total free AA, PPAR? and PAD3, epidermal levels of hydration, glutamate, serine and caspase-14 were not increased, but other minor AA of NMF were highly detected in group F. Dietary sericin improves epidermal hydration in parallel with enhancing profilaggrin expression and degradation into free AA that is coupled with elevated levels of PPAR?, PAD3 and caspase-14 proteins. PMID:22244094
Kim, Hyunae; Lim, Yu-ji; Park, Ji-Ho; Cho, Yunhi
Production of silk sericin/silk fibroin blend nanofibers
NASA Astrophysics Data System (ADS)
Silk sericin (SS)/silk fibroin (SF) blend nanofibers have been produced by electrospinning in a binary SS/SF trifluoroacetic acid (TFA) solution system, which was prepared by mixing 20 wt.% SS TFA solution and 10 wt.% SF TFA solution to give different compositions. The diameters of the SS/SF nanofibers ranged from 33 to 837 nm, and they showed a round cross section. The surface of the SS/SF nanofibers was smooth, and the fibers possessed a bead-free structure. The average diameters of the SS/SF (75/25, 50/50, and 25/75) blend nanofibers were much thicker than that of SS and SF nanofibers. The SS/SF (100/0, 75/25, and 50/50) blend nanofibers were easily dissolved in water, while the SS/SF (25/75 and 0/100) blend nanofibers could not be completely dissolved in water. The SS/SF blend nanofibers could not be completely dissolved in methanol. The SS/SF blend nanofibers were characterized by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry, and differential thermal analysis. FTIR showed that the SS/SF blend nanofibers possessed a random coil conformation and �-sheet structure.
Zhang, Xianhua; Tsukada, Masuhiro; Morikawa, Hideaki; Aojima, Kazuki; Zhang, Guangyu; Miura, Mikihiko
Effect of Sericin on Mechanical Behavior of Composite Material Reinforced by Silk Woven Fabric
NASA Astrophysics Data System (ADS)
Recent, attention has been given to shift from glass fibers and carbon fibers to natural fibers for FRP composites for the goal of protecting the environment. This paper concerned with the application of silk fabric for composite materials. Polypropylene (PP) was used for the matrix material and the silk fabric composites were molded using a compression molding method. Especially, the effect of sericin on mechanical behaviors of composite materials was discussed. Good adhesion between silk and PP was obtained by removing the sericin existing around the fibroin. The tensile modulus of composite decreased with decreasing the sericin because of the flexibility of silk fibers without sericin. In particular, the higher Izod impact value was obtained for the composites containing the silk fibers without sericin.
Kimura, Teruo; Ino, Haruhiro; Hanada, Koji; Katori, Sigetaka
Biomimetic Nucleation of Hydroxyapatite Crystals Mediated by Antheraea pernyi Silk Sericin Promotes Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stem Cells.
Biomacromolecules have been used as templates to grow hydroxyapatite crystals (HAps) by biomineralization to fabricate mineralized materials for potential application in bone tissue engineering. Silk sericin is a protein with features desirable as a biomaterial, such as increased hydrophilicity and biodegradation. Mineralization of the silk sericin from Antheraea pernyi (A. pernyi) silkworm has rarely been reported. Here, for the first time, nucleation of HAps on A. pernyi silk sericin (AS) was attempted through a wet precipitation method and consequently the cell viability and osteogenic differentiation of BMSCs on mineralized AS were investigated. It was found that AS mediated the nucleation of HAps in the form of nanoneedles while self-assembling into ?-sheet conformation, leading to the formation of a biomineralized protein based biomaterial. The cell viability assay of BMSCs showed that the mineralization of AS stimulated cell adhesion and proliferation, showing that the resultant AS biomaterial is biocompatible. The differentiation assay confirmed that the mineralized AS significantly promoted the osteogenic differentiation of BMSCs when compared to nonmineralized AS as well as other types of sericin (B. mori sericin), suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. This result represented the first work proving the osteogenic differentiation of BMSCs directed by silk sericin. Therefore, the biomineralization of A. pernyi silk sericin coupled with seeding BMSCs on the resultant mineralized biomaterials is a useful strategy to develop the potential application of this unexplored silk sericin in the field of bone tissue engineering. This study lays the foundation for the use of A. pernyi silk sericin as a potential scaffold for tissue engineering. PMID:24666022
Yang, Mingying; Shuai, Yajun; Zhang, Can; Chen, Yuyin; Zhu, Liangjun; Mao, Chuanbin; Ouyang, Hongwei
Formulation and characterization of silk sericin-PVA scaffold crosslinked with genipin.
A porous-three-dimensional scaffold shows several advantages in terms of tissue engineering since it can provide a framework for cells to attach, proliferate and form an extracellular matrix. Sericin, a by-product from the silk industry, can form a three-dimensional scaffold with PVA after freeze-drying but has a fragile structure. Glycerin (as a plasticizer) and genipin (a crosslinking agent) are necessary to make a strong and stable matrix. Our objective was to investigate the properties of a three-dimensional silk sericin and PVA scaffold with and without glycerin and genipin at various concentrations. SEM showed that adding glycerin into scaffold gave better uniformity and porosity. Smaller pore sizes and better uniformity were found as the concentration of genipin in the scaffold increased. The results of FTIR indicated that glycerin retained a high moisture content and had a major effect at 3286 cm(-1), indicating the presence of water molecule in the matrix structure. Adding genipin into the scaffold resulted in a higher degree of crosslinking or fewer free ?-amino groups, as shown by the decrease in the stretching (=C-H) peak and absorption peaks around 1370-1650 cm(-1), respectively. The sericin/PVA scaffold had a low water sorption capacity, but adding glycerin significantly increased this property. Genipin further enhanced the moisture absorption capacity of the scaffold and extended the time taken to reach equilibrium. After immersing the sericin/PVA scaffold into purified water, the scaffold completely dissolved within an hour, whereas the scaffolds containing glycerin or glycerin with 0.1% genipin swelled 8 and 11 times, respectively, compared with the initial stage after 6h of immersion. In terms of mechanical properties, the sericin/PVA/glycerin scaffold exhibited a similar compressive strength to the scaffold with a high genipin concentration, whereas a low concentration of genipin softened and reduced the compressive strength of the scaffold. A small amount of sericin was released from the scaffold and a higher concentration of genipin, resulting in less protein leaching compared to non-crosslinked sericin/PVA. The fraction of protein released from the sericin/PVA/glycerin scaffold was about 4%, with values of about 1 and 0.04% in the case of scaffolds with 0.01 and 0.1% genipin, respectively. All results indicated that the composition of the scaffolds had a significant effect on their physical properties, and that can easily be tuned to obtain scaffolds suitable for biological applications. PMID:20804781
Aramwit, Pornanong; Siritientong, Tippawan; Kanokpanont, Sorada; Srichana, Teerapol
Fibroin and Sericin from Bombyx mori Silk Stimulate Cell Migration through Upregulation and Phosphorylation of c-Jun
Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. An important phase of wound healing is the generation of a basal epithelium able to wholly replace the epidermis of the wound. A broad range of products derived from fibroin and sericin from Bombyx mori silk are used to stimulate wound healing. However, so far the molecular mechanism underlying this phenomenon has not been elucidated. The aim of this work was to determine the molecular basis underlying wound healing properties of silk proteins using a cell model. For this purpose, we assayed fibroin and sericin in a wound healing scratch assay using MDA-MB-231 and Mv1Lu cells. Both proteins stimulated cell migration. Furthermore, treatment with sericin and fibroin involved key factors of the wound healing process such as upregulation of c-Jun and c-Jun protein phosphorylation. Moreover, fibroin and sericin stimulated the phosphorylation of ERK 1/2 and JNK 1/2 kinases. All these experiments were done in the presence of specific inhibitors for some of the cell signalling pathways referred above. The obtained results revealed that MEK, JNK and PI3K pathways are involved in fibroin and sericin stimulated cells migration. Inhibition of these three kinases prevented c-Jun upregulation and phosphorylation by fibroin or sericin. Fibroin and sericin were tested in the human keratinocyte cell line, HaCaT, with similar results. Altogether, our results showed that fibroin and sericin initiate cell migration by activating the MEK, JNK and PI3K signalling pathways ending in c-Jun activation.
Garcia-Vizcaino, Eva Maria; Alcaraz, Antonia; Cenis, Jose Luis; Nicolas, Francisco Jose
Accelerated healing of full-thickness wounds by genipin-crosslinked silk sericin/PVA scaffolds.
Silk sericin has recently been studied for its advantageous biological properties, including its ability to promote wound healing. This study developed a delivery system to accelerate the healing of full-thickness wounds. Three-dimensional scaffolds were fabricated from poly(vinyl alcohol) (PVA), glycerin (as a plasticizer) and genipin (as a crosslinking agent), with or without sericin. The physical and biological properties of the genipin-crosslinked sericin/PVA scaffolds were investigated and compared with those of scaffolds without sericin. The genipin-crosslinked sericin/PVA scaffolds exhibited a higher compressive modulus and greater swelling in water than the scaffolds without sericin. Sericin also exhibited controlled release from the scaffolds. The genipin-crosslinked sericin/PVA scaffolds promoted the attachment and proliferation of L929 mouse fibroblasts. After application to full-thickness rat wounds, the wounds treated with genipin-crosslinked sericin/PVA scaffolds showed a significantly greater reduction in wound size, collagen formation and epithelialization compared with the control scaffolds without sericin but lower numbers of macrophages and multinucleated giant cells. These results indicate that the delivery of sericin from the novel genipin-crosslinked scaffolds efficiently healed the wound. Therefore, these genipin-crosslinked sericin/PVA scaffolds represent a promising candidate for the accelerated healing of full-thickness wounds. PMID:23307034
Aramwit, Pornanong; Siritienthong, Tippawan; Srichana, Teerapol; Ratanavaraporn, Juthamas
Effect of silk sericin on morphology and structure of calcium carbonate crystal
NASA Astrophysics Data System (ADS)
In this paper, silk sericin was employed to regulate the mineralization of calcium carbonate (CaCO3). CaCO3 composite particles were prepared by the precipitation reaction of sodium carbonate with calcium chloride solution in the presence of silk sericin. The as-prepared samples were collected at different reaction time to study the crystallization process of CaCO3 by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). The results showed that silk sericin significantly affected the morphology and crystallographic polymorph of CaCO3. With increasing the reaction time, the crystal phase of CaCO3 transferred from calcite dominated to vaterite dominated mixtures, while the morphology of CaCO3 changed from disk-like calcite crystal to spherical vaterite crystal. These studies showed the potential of silk sericin used as a template molecule to control the growth of inorganic crystal.
Zhao, Rui-Bo; Han, Hua-Feng; Ding, Shao; Li, Ze-Hao; Kong, Xiang-Dong
Inhibitory effects of silk protein, sericin on UVB-induced acute damage and tumor promotion by reducing oxidative stress in the skin of hairless mouse
This study was conducted to assess protective effect of an antioxidant protein, sericin, on UVB-induced acute damage and tumor promotion in mouse skin. In experiment 1, HR-1 hairless mice were treated with 180 mJ\\/cm2 of ultraviolet B light (UVB) once daily for 1 and 7 days. The treatment for 7 days caused red sunburn lesions of the skin. The intensity
Siqin Zhaorigetu; Noriyuki Yanaka; Masahiro Sasaki; Hiromitsu Watanabe; Norihisa Kato
Sericin removal from raw Bombyx mori silk scaffolds of high hierarchical order.
Silk fibroin has previously been described as a promising candidate for ligament tissue engineering (TE) approaches. For biocompatibility reasons, silkworm silk requires removal of sericin, which can elicit adverse immune responses in the human body. One disadvantage of the required degumming process is the alteration of the silk fiber structural properties, which can hinder textile engineering of high order hierarchical structures. Therefore, the aim of this study was to find a way to remove sericin from a compact and highly ordered raw silk fiber matrix. The wire rope design of the test model scaffold comprises several levels of geometric hierarchy. Commonly used degumming solutions fail in removing sericin in this wire rope design. Weight loss measurements, picric acid and carmine staining as well as scanning electron microscopy demonstrated that the removal of sericin from the model scaffold of a wire rope design can be achieved through a borate buffer-based system. Furthermore, the borate buffer degummed silks were shown to be nontoxic and did not alter cell proliferation behavior. The possibility to remove sericin after the textile engineering process has taken place eases the production of highly ordered scaffold structures and may expand the use of silk as scaffold material in further TE and regenerative medicine applications. PMID:24066942
Teuschl, Andreas Herbert; van Griensven, Martijn; Redl, Heinz
Effect of Silk Protein Processing on Drug Delivery from Silk Films
Sericin removal from the core fibroin protein of silkworm silk is a critical first step in the use of silk for biomaterial-related applications, but degumming can affect silk biomaterial properties, including molecular weight, viscosity, diffusivity and degradation behavior. Increasing the degumming time (10, 30, 60 and 90 min) decreases the average molecular weight of silk protein in solution, silk solution viscosity, and silk film glass transition temperature, and increases the rate of degradation of silk film by protease. Model compounds spanning a range of physical-chemical properties generally showed an inverse relationship between degumming time and release rate through a varied degumming time silk coating. Degumming provides a useful control point to manipulate silk�s material properties.
Pritchard, Eleanor M.; Hu, Xiao; Finley, Violet; Kuo, Catherine K.; Kaplan, David L.
Treatment of waste water from silk degumming processes for protein recovery and water reuse
Large volumes of wastewaters containing proteins (mainly sericin), salts and tensioactives are produced in the degumming process (washing) of both yarn and tissues made from natural silk. These wastewaters bearing a COD above 6000 mg\\/l are generally treated in biological plants with increasing costs. Sericin represents, on the contrary, a valuable by-product that can be used in cosmetics and pharmaceutical
C. Fabiani; M. Pizzichini; M. Spadoni; G. Zeddita
Identification of a Novel Type of Silk Protein and Regulation of Its Expression
The silk of lepidopteran insects has been studied ex- tensively as proteins of two categories: the fibroins, which are produced in the posterior section of silk glands, and the sericins, which are secreted in the mid- dle section. We now describe a third category that is named seroins to accentuate the fact that both the ser- icin- and the fibroin-producing
M. Zurovec; Changsong Yang; Dalibor Kodrik
National Technical Information Service (NTIS)
Spider silk proteins from major and minor ampullate silk have been sequenced via their cDNAs. One of the proteins from major ampullate silk has been expressed in bacteria to levels of 10-20 mg/mL. Studies using fiber X-ray diffraction and solid state NMR ...
R. V. Lewis
Evaluation of sericin\\/collagen membranes as prospective wound dressing biomaterial
Sericin, a silk protein, has high potential for use in biomedical applications. In this study, wound dressing membranes of Sericin (S) and Collagen (C) were prepared by glutaraldehyde cross-linking at S\\/C; 2:1, 1:1, 1:2, and 0:1 weight ratios. They were stable in water for 4weeks. However, increasing the proportion of sericin had decreasing effect on the membrane stability. Water swelling
Omer Akturk; Aysen Tezcaner; Hasan Bilgili; M. Salih Deveci; M. Rusen Gecit; Dilek Keskin
Green synthesis of silk sericin-capped silver nanoparticles and their potent anti-bacterial activity
In this study, a �green chemistry� approach was introduced to synthesize silk sericin (SS)-capped silver nanoparticles (AgNPs) under an alkaline condition (pH�11) using SS as a reducing and stabilizing agent instead of toxic chemicals. The SS-capped AgNPs were successfully synthesized at various concentrations of SS and AgNO3, but the yields were different. A higher yield of SS-capped AgNPs was obtained when the concentrations of SS and AgNO3 were increased. The SS-capped AgNPs showed a round shape and uniform size with diameter at around 48 to 117�nm. The Fourier transform infrared (FT-IR) spectroscopy result proved that the carboxylate groups obtained from alkaline degradation of SS would be a reducing agent for the generation of AgNPs while COO? and NH2?+ groups stabilized the AgNPs and prevented their precipitation or aggregation. Furthermore, the SS-capped AgNPs showed potent anti-bacterial activity against various gram-positive bacteria (minimal inhibitory concentration (MIC) 0.008�mM) and gram-negative bacteria (MIC ranging from 0.001 to 0.004�mM). Therefore, the SS-capped AgNPs would be a safe candidate for anti-bacterial applications.
National Technical Information Service (NTIS)
Flagelliform silk proteins have been studied by cloning the cDNAs for the major protein in that silk as well as the gene. The protein consists of three sequence segments which compose a repeat These repeats appear numerous times in the protein The three s...
R. V. Lewis
Potential of 2D crosslinked sericin membranes with improved biostability for skin tissue engineering.
Silk sericin protein is a natural, hydrophilic, macromolecular glycoprotein mainly synthesized in the middle silk gland of the silkworm. It constitutes 25-30% of the silk cocoon. Sericin proteins have antioxidant, antimicrobial, UV-resistant properties, promote wound healing and support cell proliferation even in serum-free media. Most of the sericin is discarded as waste in silk processing industries. This study aims at improving the mechanical strength and stability of sericin extracted from the silk cocoons during processing and utilize it as a biocompatible natural biopolymer in biomedical applications. Crosslinked sericin membranes, from the cocoon of non-mulberry tropical silkworm, Antheraea mylitta, were prepared using gluteraldehyde as the crosslinking agent. Physical and structural characteristics of the membranes were analyzed using scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy and X-ray diffraction along with swelling and degradation studies. The secondary structure of the membrane indicates that crosslinking provides a more integrated structure that significantly improves the stability and mechanical strength of the membranes. In vitro cytocompatibility of the membranes was evaluated by MTT assay and cell cycle analysis of feline fibroblast cells. The adherence, growth and proliferation patterns of cells on membranes were assessed by confocal microscopy, which demonstrated that the latter is non-toxic and supports cell growth. Cell cycle analyses indicate cytocompatibility with normal cell cycle pattern. This study reveals that silk sericin protein can be used as a biocompatible natural biopolymer for various applications in the biomedical field. PMID:22327482
Nayak, Sunita; Talukdar, Sarmistha; Kundu, Subhas C
Antioxidant activities of two sericin proteins extracted from cocoon of silkworm (Bombyx mori) measured by DPPH, chemiluminescence, ORAC and ESR methods
Recent efforts have focused on the use of sericin proteins extracted from cocoons of silkworm as a healthy food source for human consumption. In this study, we focused on the antioxidative properties of sericin proteins. The antioxidative properties were measured in sericin proteins extracted from the shell of the cocoon, designated hereafter as white sericin protein and yellow-green sericin protein, as well as bread without sericin protein and bread to which white sericin powder had been added using four measurement methods: 1,1-Diphenyl-2-picrylhydrazyl (DPPH), chemiluminescence, oxygen radical absorbance capacity (ORAC) and electron spin resonance (ESR). High antioxidative properties of sericin proteins were indicated by all four methods. A comparison of the two types of sericin proteins revealed that yellow-green sericin protein exhibited high antioxidative properties as indicated by the DPPH, chemiluminescence and ORAC methods. By contrast, a higher antioxidative property was determined in white sericin protein by the ESR method. Consequently, our findings confirmed that sericin proteins have antioxidative properties against multiple radicals. In addition, the antioxidative property of bread was enhanced by the addition of sericin powder to the bread. Therefore, findings of this study suggest that sericin proteins may be efficiently used as beneficial food for human health.
Evaluation of sericin/collagen membranes as prospective wound dressing biomaterial.
Sericin, a silk protein, has high potential for use in biomedical applications. In this study, wound dressing membranes of Sericin (S) and Collagen (C) were prepared by glutaraldehyde cross-linking at S/C; 2:1, 1:1, 1:2, and 0:1 weight ratios. They were stable in water for 4 weeks. However, increasing the proportion of sericin had decreasing effect on the membrane stability. Water swelling property of membranes was enhanced with sericin. The highest water swelling was obtained in 1:1 group (9.06 g/g), but increasing collagen or sericin content in the membranes had a diminishing effect. Highest water vapor transmission rate was obtained with 1:2 group (1013.80 g/m(2)/day). Oxygen permeability results showed that 1:2 (7.67 mg/L) and 2:1 (7.85 mg/L) S/C groups were better than the other groups. While sericin decreased the tensile strength and elongation of membranes, it increased modulus. Sericin also increased brittleness of membranes, but their UTS range (24.93-44.92 MPa) was still suitable for a wound dressing. Membranes were not penetrable to microorganisms. Cytotoxicity studies showed that fibroblasts and keratinocytes attached and gained their characteristic morphologies. They also proliferated on membranes significantly. After 1 week of subcutaneous implantation, a fibrous capsule formed around all membranes with an acute inflammation. Sericin containing membranes showed signs of degradation (at 2nd week), while collagen only membranes remained largely intact. Eventually, sericin containing membranes degraded in 3 weeks with moderate inflammatory response. Overall results suggest that sericin/collagen membranes would be favorable as wound dressing material when sericin ratio is less than or equal to the collagen component. PMID:21697006
Akturk, Omer; Tezcaner, Aysen; Bilgili, Hasan; Deveci, M Salih; Gecit, M Rusen; Keskin, Dilek
Polymeric materials based on silk proteins
Silks are protein-based fibers made by arthropods for a variety of task-specific applications. In this article, we review the key features of silk proteins. This article initially focuses on the structure and function of silk proteins produced naturally by silkworms and spiders, followed by the biological and technical processing of silk proteins into a variety of morphologies (including capsules, fibers,
John G. Hardy; Lin M. R�mer; Thomas R. Scheibel
Recombinant DNA production of spider silk proteins
Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.
Tokareva, Olena; Michalczechen-Lacerda, Valquiria A; Rech, Elibio L; Kaplan, David L
Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders.
The known silk fibroins and fibrous glues are thought to be encoded by members of the same gene family. All silk fibroins sequenced to date contain regions of long-range order (crystalline regions) and/or short-range order (non-crystalline regions). All of the sequenced fibroin silks (Flag or silk from flagelliform gland in spiders; Fhc or heavy chain fibroin silks produced by Lepidoptera larvae) are made up of hierarchically organized, repetitive arrays of amino acids. Fhc fibroin genes are characterized by a similar molecular genetic architecture of two exons and one intron, but the organization and size of these units differs. The Flag, Ser (sericin gene) and BR (Balbiani ring genes; both fibrous proteins) genes are made up of multiple exons and introns. Sequences coding for crystalline and non-crystalline protein domains are integrated in the repetitive regions of Fhc and MA exons, but not in the protein glues Ser1 and BR-1. Genetic 'hot-spots' promote recombination errors in Fhc, MA, and Flag. Codon bias, structural constraint, point mutations, and shortened coding arrays may be alternative means of stabilizing precursor mRNA transcripts. Differential regulation of gene expression and selective splicing of the mRNA transcript may allow rapid adaptation of silk functional properties to different physical environments. PMID:12470814
Craig, Catherine L; Riekel, Christian
Microbial production of spider silk proteins
The remarkable properties of spider dragline silk and related protein polymers will find many applications if the materials can be produced economically. We have demonstrated the production of high molecular weight spider dragline silk analog proteins encoded by synthetic genes in several microbial systems, including Escherichia coli and Pichia pastoris. In E. coli, proteins of up to 1000 amino acids
Stephen R Fahnestock; Zhongjie Yao; Laura A Bedzyk
Silk cocoon of Bombyx mori: proteins and posttranslational modifications--heavy phosphorylation and evidence for lysine-mediated cross links.
Although silk is used to produce textiles and serves as a valuable biomaterial in medicine, information on silk proteins of the cocoon is limited. Scanning electron microscopy was applied to morphologically characterise the sample and the solubility of cocoon in lithium thiocyanate and 2-DE was carried out with multi-enzyme in-gel digestion followed by MS identification of silk-peptides. High-sequence coverage of the silk cocoon proteins fibroin light and heavy chain, sericins and fibrohexamerins was revealed and PTMs as heavy phosphorylation of silk fibroin heavy chain; lysine hydroxylation and Lys->allysine formation have been observed providing evidence for lysine-mediated cross linking of silk as found in collagens, which has not been reported so far. Tyrosine oxidation verified the presence of di-tyrosine cross links. A high degree of sequence conflicts probably representing single-nucleotide polymorphisms was observed. PTM and sequence conflicts may be modulating structure and physicochemical properties of silk. PMID:20029844
Chen, Wei-Qiang; Priewalder, Helga; John, Julius Paul Pradeep; Lubec, Gert
Control of expression of silk protein genes
At least three silk genes are specifically expressed in the posterior, and five other genes in middle, silk glands. The products of genes active in PSG include fibroin, L-chain fibroin and P25 protein. PSG genes as well as the Ser-1 gene, differing in structure, exhibit a striking degree of homology of their 5? flanking sequences. This suggests the presence of
Krystyna Grzelak
Assembly mechanism of recombinant spider silk proteins
Spider silk threads are formed by the irreversible aggregation of silk proteins in a spinning duct with dimensions of only a few micrometers. Here, we present a microfluidic device in which engineered and recombinantly produced spider dragline silk proteins eADF3 (engineered Araneus diadematus fibroin) and eADF4 are assembled into fibers. Our approach allows the direct observation and identification of the essential parameters of dragline silk assembly. Changes in ionic conditions and pH result in aggregation of the two proteins. Assembly of eADF3 fibers was induced only in the presence of an elongational flow component. Strikingly, eADF4 formed fibers only in combination with eADF3. On the basis of these results, we propose a model for dragline silk aggregation and early steps of fiber assembly in the microscopic regime.
Rammensee, S.; Slotta, U.; Scheibel, T.; Bausch, A. R.
Sequence Requirements of Spider Silk Elastic Proteins.
National Technical Information Service (NTIS)
Sequence for flagelliform silk proteins from Nephila, Argiope, and Araneus species have been obtained. These sequences show significant sequence differences although the changes are not predicted to affect the secondary structure of the protein and theref...
R. V. Lewis
Effect of Processing on Silk-Based Biomaterials: Reproducibility and Biocompatibility
Silk fibroin has been successfully used as a biomaterial for tissue regeneration. In order to prepare silk fibroin biomaterials for human implantation a series of processing steps are required to purify the protein. Degumming to remove inflammatory sericin is a crucial step related to biocompatibility and variability in the material. Detailed characterization of silk fibroin degumming is reported. The degumming conditions significantly affected cell viability on the silk fibroin material and the ability to form three-dimensional porous scaffolds from the silk fibroin, but did not affect macrophage activation or ?-sheet content in the materials formed. Methods are also provided to determine the content of residual sericin in silk fibroin solutions and to assess changes in silk fibroin molecular weight. Amino acid composition analysis was used to detect sericin residuals in silk solutions with a detection limit between 1.0% and 10% wt/wt, while fluorescence spectroscopy was used to reproducibly distinguish between silk samples with different molecular weights. Both methods are simple and require minimal sample volume, providing useful quality control tools for silk fibroin preparation processes.
Wray, Lindsay S.; Hu, Xiao; Gallego, Jabier; Georgakoudi, Irene; Omenetto, Fiorenzo G.; Schmidt, Daniel; Kaplan, David L.
Functional expression of a Bombyx mori cocoonase: potential application for silk degumming.
Cocoon, a shelter for larva development to silk moth, contains the fibrous protein fibroin, which is coated by the globular protein sericin. Emergence of the silk moth requires the action of cocoonase, a protease secreted by the pupa. The full-length prococoonase cDNA, with 780 bp open reading frame encoding 260 amino acids, was cloned by reverse transcription from total RNA of the head of 6-day-old Thai-silk Bombyx mori pupa. Only the gene fragment lacking the propeptide encoding sequence was successfully expressed in Pichia pastoris, yielding an extracellularly active cocoonase. The recombinant cocoonase was purified to homogeneity by 80% ammonium-sulfate fractionation and CM-Sepharose chromatography, and its internal peptide sequences were analyzed by nano liquid chromatography-mass spectrometry/mass spectrometry. This monomeric protein has native molecular weight of 26 kDa by gel exclusion analysis and 25 kDa subunit size by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme hydrolyses sericin but does not hydrolyse fibroin, as shown by radial diffusion on thin-layer enzyme assay (RD-TEA). Scanning electron microscopy showed that purified recombinant cocoonase could remove sericin from natural silk completely in 24 h, without damaging fibroin, using only 1 immobilized sericin unit (ISU) of enzyme as determined by RD-TEA. Natural cocoonase isolated from B. mori pupa could also digest sericin effectively, but required more enzymes (2 ISU) and longer time (48 h). In comparison, a commercial enzyme, alcalase, with the same activity not only showed less complete digestion of sericin but also caused damage of fibroin. These results suggest that recombinant B. mori cocoonase is potentially useful for silk degumming. PMID:23169343
Rodbumrer, Prangprapai; Arthan, Dumrongkiet; Uyen, Utai; Yuvaniyama, Jirundon; Svasti, Jisnuson; Wongsaengchantra, Pramvadee Y
Protein-protein nanoimprinting of silk fibroin films.
Protein-protein imprinting of silk fibroin is introduced as a rapid, high-throughput method for the fabrication of nanoscale structures in silk films, through the application of heat and pressure. Imprinting on conformal surfaces is demonstrated with minor adjustments to the system, at resolutions comparable to other currently available nonplanar nanoimprint lithography techniques. PMID:23483712
Brenckle, Mark A; Tao, Hu; Kim, Sunghwan; Paquette, Mark; Kaplan, David L; Omenetto, Fiorenzo G
Silks produced by insect labial glands
Insect silks are secreted from diverse gland types; this chapter deals with the silks produced by labial glands of Holometabola (insects with pupa in their life cycle). Labial silk glands are composed of a few tens or hundreds of large polyploid cells that secrete polymerizing proteins which are stored in the gland lumen as a semi-liquid gel. Polymerization is based on weak molecular interactions between repetitive amino acid motifs present in one or more silk proteins; cross-linking by disulfide bonds may be important in the silks spun under water. The mechanism of long-term storage of the silk dope inside the glands and its conversion into the silk fiber during spinning is not fully understood. The conversion occurs within seconds at ambient temperature and pressure, under minimal drawing force and in some cases under water. The silk filament is largely built of proteins called fibroins and in Lepidoptera and Trichoptera coated by glue-type proteins known as sericins. Silks often contain small amounts of additional proteins of poorly known function. The silk components controlling dope storage and filament formation seem to be conserved at the level of orders, while the nature of polymerizing motifs in the fibroins, which determine the physical properties of silk, differ at the level of family and even genus. Most silks are based on fibroin ?-sheets interrupted with other structures such as ?-helices but the silk proteins of certain sawflies have predominantly a collagen-like or polyglycine II arrangement and the silks of social Hymenoptera are formed from proteins in a coiled coil arrangement.
Sutherland, Tara
High molecular mass complexes of aquatic silk proteins
Little is known about specific protein�protein associations that take place during formation of Chironomus tentans silk. The aim of this study was to learn if C. tentans salivary glands contain biochemically discrete silk protein complexes. Examination of native extracts by non-denaturing agarose gel electrophoresis and immunoblotting revealed two SDS-resistant complexes: C1a, nominally containing silk proteins spIa, sp185 and sp140, and
Steven T Case; Jennifer R Thornton
Production And Characterization Of Synthetic Spider Silks Based On Nephila Clavipes Major Ampullate Silk Proteins
NASA Astrophysics Data System (ADS)
The extraordinary mechanical properties of orb-weaving spider silks have served spiders for over 400 million years. However, only in the late 20th century did we start to understand the molecular nature of spider silk that contributes to its incredible properties as biomaterials. Among all seven types of spider silks, major ampullate silk from typical orb-weaving spiders is the toughest of all, it consists of primarily two proteins: MaSp1 and MaSp2. Variable ratios and conserved motifs of these two proteins in all the native spider silks demonstrate the significant role of MaSp1 and MaSp2 in controlling the mechanical properties of the fiber. The amino acid sequences of the orb weaving spider silk proteins have remained almost unchanged for more than 100 million years. Interestingly, MaSp1 and MaSp2 are the only two components in all studied dragline silk fibers from these spiders. The mechanical properties of native dragline silk vary slightly between species, which are believed to relate to the ratio of MaSp1 to MaSp2 in the silk. Both of these facts clearly indicate the importance of these two proteins to the mechanical properties of the fiber. Various types of synthetic spider silk fibers have been produced and studied in an effort to mass-produce man-made fibers with qualities comparable to native spider silk. To investigate the roles of MaSp1 and MaSp2 in silk fiber, synthetic MaSp1 (major abundant protein in Nephila clavipes major ampullate silks) only fibers, MaSp1/MaSp2 protein mixture fibers and chimeric protein fibers with both MaSp1 and MaSp2 sequence features have been produced and tested for mechanical properties. Solid-State Nuclear Magnetic Resonance was used to characterize the structure of silk fibers and reveal the relation between fiber spatial structure and mechanical properties.
An, Bo
From the Cover: Assembly mechanism of recombinant spider silk proteins
Spider silk threads are formed by the irreversible aggregation of silk proteins in a spinning duct with dimensions of only a few micrometers. Here, we present a microfluidic device in which engineered and recombinantly produced spider dragline silk proteins eADF3 (engineered Araneus diadematus fibroin) and eADF4 are assembled into fibers. Our approach allows the direct observation and identification of the
S. Rammensee; U. Slotta; T. Scheibel; A. R. Bausch
Protein-protein nanoimprinting of silk fibroin films
Control of the interface between biological tissue and high technology materials is paramount for the development of future applications in biomedicine, especially in the case of implantable integrated devices for signal transduction.[1]-[3] Such work requires careful materials design to develop devices that can efficiently perform technological functions while retaining biocompatibility and biological integration. Silk fibroin protein from the Bombyx mori silkworm has come of considerable interest in this context, owing to its attractive mechanical,[4]-[7] biological, [8][9] and optical properties.[10][11] Recent work has shown adaptation of common micro- and nano-fabrication tools to silk films,[12]-[18] as well as silk protein secondary structure patterning techniques,[19] leading to biocompatible and degradable electronic and photonic devices which can simultaneously act as a carrier and stabilizer for protein pharmaceuticals and other bioactive reagents.[20]-[23] In particular, silk based nanoscale photonic devices face the challenge of sub-wavelength resolution fabrication on a soft polymeric substrate.[15][24] Previous work introduced the possibility of direct, rapid nanoimprinting in silk for the fabrication of photonic structures by leveraging the material properties of this protein.[25
Brenckle, Mark A; Tao, Hu; Kim, Sunghwan; Paquette, Mark; Kaplan, David L; Omenetto, Fiorenzo G
Silk--a new substrate for UDP-d-xylose:proteoglycan core protein beta-D-xylosyltransferase.
The formation of most connective tissue polysaccharides is initiated by transfer of D-xylose from UDP-D-xylose to specific serine residues in the core proteins of the putative proteoglycans. The substrate specificity of the xylosyltransferase catalyzing this reaction has not yet been examined in detail, but it appears that a -Ser-Gly- pair is an essential part of the substrate structure. Since the preparation of the known acceptors (e.g., Smith-degraded or HF-treated cartilage proteoglycan) involves a substantial effort, we have searched for readily available proteins with the -Ser-Gly-sequence, which might serve as alternative substrates. In the present work, it was found that silk fibroin from Bombyx mori, which consists, in large part, of the repeating hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly, is an excellent substrate for the xylosyltransferase from embryonic chick cartilage. Pieces of silk were used directly in the reaction mixtures, and [14C]xylose transferred from UDP-D-[14C]xylose was measured by liquid scintillation spectrometry after rinsing the silk in 1 M NaCl and water. Substantially greater incorporation was observed with preparations of silk or fibroin which had been dissolved in 60% LiSCN and subsequently dialyzed exhaustively or diluted appropriately. Under standard reaction conditions, the Vmax for fibroin was 531 pmol/h/mg enzyme protein, as compared to 223 pmol/h/mg for Smith-degraded proteoglycan. Km values were 182 mg/liter (fibroin) and 143 mg/liter (Smith-degraded proteoglycan). The product of [14C]xylose transfer to silk was alkali labile, and [14C]xylitol was formed when [14C]xylosylsilk was treated with borohydride in alkali. Proteolytic digestion with papain, Pronase, leucine aminopeptidase, and carboxypeptidase A yielded a radioactive product which was identified as [14C]xylosylserine by electrophoresis and chromatography. The identity of the isolated [14C]xylosylserine was further supported by its resistance to treatment with alkali (0.5 M KOH; 100 degrees C; 8 h) and by acid hydrolysis which yielded [14C]xylose. Tryptic and chymotryptic fragments from fibroin were also good xylose acceptors and had Vmax values 60-70% of that observed for the intact protein. Substantial acceptor activity was displayed also by the sericin fraction of silk and by the silk sequence hexapeptide. Ser-Gly-Ala-Gly-Ala-Gly; the latter had a Vmax value close to 20% of that of intact fibroin. PMID:6731831
Campbell, P; Jacobsson, I; Benzing-Purdie, L; Rod�n, L; Fessler, J H
Conformation transition kinetics ofBombyx mori silk protein
Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein con- centration also had an
Xin Chen; Zhengzhong Shao; David P. Knight; Fritz Vollrath
Production of synthetic spider dragline silk protein in Pichia pastoris
The methylotrophic yeast Pichia pastoris was tested as a host for the production of long, repetitive protein polymers. Synthetic genes for a designed analog of a\\u000a spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered
S. R. Fahnestock; L. A. Bedzyk
Local tolerance to spider silks and protein polymers in vivo.
Spider silks were implanted subcutaneously in pigs for a study of the tolerance against this material. Four types of spider silks of high purity and cleanliness were implanted: (i) major ampullate dragline silk reeled from the golden silk spider Nephila clavipes, (ii) native (unsterilised) silk reeled from a Brachypelma spider, (iii) native silk taken from this spider's web and (iv) its web silk thermally treated at 80 degrees C. For comparison we used fibrous silk analogue protein polymers and four already marketed wound dressings (polyurethane film, collagen dressings, gauze pads). All materials were applied epicutaneously to split skin wounds. The implants were examined macroscopically as well as by light microscopy. Superficially, all sites healed rapidly. There were marked inflammatory reactions in all sites with lympho-plasmacellular infiltrations, evidence of phagocytosis and granuloma formation as indicated by the appearance of giant cells. However there was a marked absence of epitheloid cells indicating that the observed reaction was a foreign body granuloma. Furthermore, the histopathological images recorded after 14 days revealed no marked differences between the dressings. Polyurethane films, however, seemed to be superior with respect to the duration of the wound healing process. PMID:12224131
Vollrath, F; Barth, P; Basedow, A; Engstr�m, W; List, H
Bioengineered Chimeric Spider Silk-Uranium Binding Proteins
Heavy metals constitute a source of environmental pollution. Here, novel functional hybrid biomaterials for specific interactions with heavy metals are designed by bioengineering consensus sequence repeats from spider silk of Nephila clavipes with repeats of a uranium peptide recognition motif from a mutated 33-residue of calmodulin protein from Paramecium tetraurelia. The self-assembly features of the silk to control nanoscale organic/inorganic material interfaces provides new biomaterials for uranium recovery. With subsequent enzymatic digestion of the silk to concentrate the sequestered metals, options can be envisaged to use these new chimeric protein systems in environmental engineering, including to remediate environments contaminated by uranium.
Krishnaji, Sreevidhya Tarakkad; Kaplan, David L.
New silk protein: modification of silk protein by gene engineering for production of biomaterials
The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme
Hajime Mori; Masuhiro Tsukada
Spider silk as archetypal protein elastomer
We present an overview of the physical properties of spider silks, and introduce a model designed to study the energy absorbed by the material as it stretches before breaking. Of particular interest are the inter- and intramolecular hydrogen bonds as well as the role of water in modifying the material properties of silk. A solid understanding of this interaction is
Fritz Vollrath; David Porter
Nanostructure and molecular mechanics of spider dragline silk protein assemblies.
Spider silk is a self-assembling biopolymer that outperforms most known materials in terms of its mechanical performance, despite its underlying weak chemical bonding based on H-bonds. While experimental studies have shown that the molecular structure of silk proteins has a direct influence on the stiffness, toughness and failure strength of silk, no molecular-level analysis of the nanostructure and associated mechanical properties of silk assemblies have been reported. Here, we report atomic-level structures of MaSp1 and MaSp2 proteins from the Nephila clavipes spider dragline silk sequence, obtained using replica exchange molecular dynamics, and subject these structures to mechanical loading for a detailed nanomechanical analysis. The structural analysis reveals that poly-alanine regions in silk predominantly form distinct and orderly beta-sheet crystal domains, while disorderly regions are formed by glycine-rich repeats that consist of 3?-helix type structures and beta-turns. Our structural predictions are validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots, alpha-carbon atomic distances, as well as secondary structure content. Mechanical shearing simulations on selected structures illustrate that the nanoscale behaviour of silk protein assemblies is controlled by the distinctly different secondary structure content and hydrogen bonding in the crystalline and semi-amorphous regions. Both structural and mechanical characterization results show excellent agreement with available experimental evidence. Our findings set the stage for extensive atomistic investigations of silk, which may contribute towards an improved understanding of the source of the strength and toughness of this biological superfibre. PMID:20519206
Keten, Sinan; Buehler, Markus J
Designing Spider Silk Proteins for Materials Applications.
National Technical Information Service (NTIS)
Spider silks have the potential to provide new bio-based materials for numerous military applications ranging from protective clothing to parachute cords to composite materials in aircraft. Specific amino acid motifs have been identified which have been c...
R. V. Lewis
A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning
The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and
Florence Teul�; Alyssa R Cooper; William A Furin; Daniela Bittencourt; Amanda Brooks; Elibio L Rech; Randolph V Lewis
Method for Purifying and Recovering Silk Proteins Using Magnetic Affinity Separation.
National Technical Information Service (NTIS)
A method for the purification of recombinant silk proteins from a sample using magnetic affinity separation is described. The recombinant silk protein is expressed with an affinity tag which has a high binding affinity for an affinity ligand immobilized o...
C. Hoffmann K. Keller
Differential usage of iso-accepting tRNASer species in silk glands of Bombyx mori.
The rapid development of the silk glands of Bombyx mori during the last larval instar shows two phases. During the first 4 days, in both the middle and posterior parts of the silk glands, the ribosomal machinery is assembled and the synthesis of housekeeping proteins starts. During the second phase (the last 4 days), the middle part of the gland synthesis approximately 45 mg of the silk protein sericin (31% serine) and the posterior part of the gland synthesizes approximately 130 mg of the silk protein fibroin (46% glycine, 29% alanine and 12% serine). Silk fibroin and sericin are detectable by the second day and represent 80 and 50% respectively of the total proteins produced at day 8 (refs 1--4). It is known that the tRNA population of the posterior part of the gland is quantitatively adapted to fibroin codon frequency during this period but little is known about the situation in the middle part except for the observation that it contains more tRNASer than does the posterior part. We show here that the two parts contain, and presumably use, different iso-accepting species of tRNASer, the middle part using tRNASer1, which recognizes AGU and AGC codons, and the posterior part using tRNASer2 which recognizes UCA. We also suggest that this differential adaptation of the tRNASer species is under transcriptional control as the two species are accumulated at different rates, but degraded at the same rate. PMID:6782490
Hentzen, D; Chevallier, A; Garel, J P
Conformation transition kinetics of Bombyx mori silk protein.
Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which beta-sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of beta-sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations > 40% in films or > 50% in silk fibroin solutions. Our results indicate that the ethanol-induced conformation transition of silk fibroin in films and solutions is a three-phase process. The first phase is the initiation of beta-sheet structure (nucleation), the second is a fast phase of beta-sheet growth while the third phase represents a slow perfection of previously formed beta-sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation-dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber. PMID:17436322
Chen, Xin; Shao, Zhengzhong; Knight, David P; Vollrath, Fritz
Production of spider silk proteins in tobacco and potato
Spider dragline silk is a proteinaceous fiber with remarkable mechanical properties that make it attractive for technical applications. Unfortunately, the material cannot be obtained in large quantities from spiders. We have therefore generated transgenic tobacco and potato plants that express remarkable amounts of recombinant Nephila clavipes dragline proteins. Using a gene synthesis approach, the recombinant proteins exhibit homologies of >90%
J�rgen Scheller; Karl-Heinz G�hrs; Frank Grosse; Udo Conrad
Novel Assembly Properties of Recombinant Spider Dragline Silk Proteins
Spider dragline silk, which exhibits extraordinary strength and toughness, is primarily composed of two related proteins that largely consist of repetitive sequences. In most spiders, the repetitive region of one of these proteins is rich in prolines, which are not present in the repetitive region of the other [1]. The absence of prolines in one component was previously speculated to
Daniel Huemmerich; Thomas Scheibel; Fritz Vollrath; Shulamit Cohen; Uri Gat; Shmulik Ittah
Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins
Several types of silks and silk protein coding genes have been characterized from orb-web weaving spiders. When the protein sequences of major ampullate, minor ampullate, and flagelliform silks from Nephila clavipes are compared, they can be summarized as sets of shared amino acid motifs. Four of these motifs and their likely secondary structures are described. Each structural element, termed a
Cheryl Y. Hayashi; Nichola H. Shipley; Randolph V. Lewis
Bilayered vascular grafts based on silk proteins.
A major block in the development of small diameter vascular grafts is achieving suitable blood vessel regeneration while minimizing the risk of thrombosis, intimal hyperplasia, suture retention, and mechanical failure. Silk-based tubular vessels for tissue engineering have been prepared by molding, dipping, electrospinning, or gel spinning, however, further studies are needed to improve the mechanical and blood compatibility properties. In the present study a bilayered vascular graft based on silk fibroin (SF) was developed. The graft was composed of an inner silk fiber-reinforced SF tube containing heparin and a highly porous SF external layer. Compared with previously fabricated SF tubes the fiber-reinforcement provided a comparable or higher mechanical strength, burst pressure, and suture retention strength, as well as mechanical compliance, to saphenous veins for vascular grafts. Heparin release was sustained for at least 1 month, affording blood compatibility to the grafts. The outer layer of the grafts prepared through lyophilization had a highly porous structure in which the macropore walls were composed of nanofibers similar to extracellular matrix, which offered an excellent environment for cell growth. In vitro studies showed good cytocompatibility and hemocompatibility. PMID:23851155
Liu, Shanshan; Dong, Chaofei; Lu, Guozhong; Lu, Qiang; Li, Zhanxiong; Kaplan, David L; Zhu, Hesun
Giant wood spider Nephila pilipes alters silk protein in response to prey variation
Recent studies have demonstrated that orb-weaving spiders may alter web structures, foraging localities or silk output in response to prey variations. In this study we conducted field surveys and food manipulations to examine whether orb-weaving spiders may also adjust the protein of silk to prey variations. A comparison of dragline silks collected from nine giant wood spider Nephila pilipes populations
I-Min Tso; Hsuan-Chen Wu; In-Ru Hwang
Bombyx mori silk protein films microprocessing with a nanosecond ultraviolet laser and a femtosecond laser workstation: theory and experiments
NASA Astrophysics Data System (ADS)
Laser microprocessing of several biopolymers from renewable resources is studied. Three proteinic materials were either extracted from the extracellular matrix like Silk Fibroin/Sericin and collagen, or coming from a commercial source like gelatin. All can find future applications in biomedical experimentation, in particular for cell scaffolding. Films of �hundred of microns thick were made by aqueous solution drying and laser irradiation. Attention is paid to the properties making them processable with two laser sources: the ultraviolet and nanosecond (ns) KrF (248 nm) excimer and the infrared and femtosecond (fs) Yb:KGW laser. The UV radiation is absorbed in a one-photon resonant process to yield ablation and the surface foaming characteristics of a laser-induced pressure wave. To the contrary, resonant absorption of the IR photons of the fs laser is not possible and does not take place. However, the high field of the intense I>�1012 W/cm2 femtosecond laser pulse ionizes the film by the multiphoton absorption followed by the electron impact mechanism, yielding a dense plasma capable to further absorb the incident radiation of the end of the pulse. The theoretical model of this absorption is described in detail, and used to discuss the presented experimental effects (cutting, ablation and foaming) of the fs laser. The ultraviolet laser was used to perform simultaneous multiple spots experiments in which energetic foaming yields melt ejection and filament spinning. Airborne nanosize filaments "horizontally suspended by both ends" (0.25 ?m diameter and 10 ?m length) of silk biopolymer were observed upon irradiation with large fluences.
Lazare, S.; Sionkowska, A.; Zaborowicz, M.; Planecka, A.; Lopez, J.; Dijoux, M.; Loum�na, C.; Hernandez, M.-C.
A Materiomics Approach to Spider Silk: Protein Molecules to Webs
NASA Astrophysics Data System (ADS)
The exceptional mechanical properties of hierarchical self-assembling silk biopolymers have been extensively studied experimentally and in computational investigations. A series of recent studies has been conducted to examine structure-function relationships across different length scales in silk, ranging from atomistic models of protein constituents to the spider web architecture. Silk is an exemplary natural material because its superior properties stem intrinsically from the synergistic cooperativity of hierarchically organized components, rather than from the superior properties of the building blocks themselves. It is composed of beta-sheet nanocrystals interspersed within less orderly amorphous domains, where the underlying molecular structure is dominated by weak hydrogen bonding. Protein chains are organized into fibrils, which pack together to form threads of a spider web. In this article we survey multiscale studies spanning length scales from angstroms to centimeters, from the amino acid sequence defining silk components to an atomistically derived spider web model, with the aim to bridge varying levels of hierarchy to elucidate the mechanisms by which structure at each composite level contributes to organization and material phenomena at subsequent levels. The work demonstrates that the web is a highly adapted system where both material and hierarchical structure across all length scales is critical for its functional properties.
Tarakanova, Anna; Buehler, Markus J.
Differential Scanning Fluorimetry provides high throughput data on silk protein transitions.
Here we present a set of measurements using Differential Scanning Fluorimetry (DSF) as an inexpensive, high throughput screening method to investigate the folding of silk protein molecules as they abandon their first native melt conformation, dehydrate and denature into their final solid filament conformation. Our first data and analyses comparing silks from spiders, mulberry and wild silkworms as well as reconstituted �silk' fibroin show that DSF can provide valuable insights into details of silk denaturation processes that might be active during spinning. We conclude that this technique and technology offers a powerful and novel tool to analyse silk protein transitions in detail by allowing many changes to the silk solutions to be tested rapidly with microliter scale sample sizes. Such transition mechanisms will lead to important generic insights into the folding patterns not only of silks but also of other fibrous protein (bio)polymers.
Vollrath, Fritz; Hawkins, Nick; Porter, David; Holland, Chris; Boulet-Audet, Maxime
Differential Scanning Fluorimetry provides high throughput data on silk protein transitions.
Here we present a set of measurements using Differential Scanning Fluorimetry (DSF) as an inexpensive, high throughput screening method to investigate the folding of silk protein molecules as they abandon their first native melt conformation, dehydrate and denature into their final solid filament conformation. Our first data and analyses comparing silks from spiders, mulberry and wild silkworms as well as reconstituted 'silk' fibroin show that DSF can provide valuable insights into details of silk denaturation processes that might be active during spinning. We conclude that this technique and technology offers a powerful and novel tool to analyse silk protein transitions in detail by allowing many changes to the silk solutions to be tested rapidly with microliter scale sample sizes. Such transition mechanisms will lead to important generic insights into the folding patterns not only of silks but also of other fibrous protein (bio)polymers. PMID:25004800
Vollrath, Fritz; Hawkins, Nick; Porter, David; Holland, Chris; Boulet-Audet, Maxime
Light can transform the secondary structure of silk protein
. ��Fibroin is the main component of silk and is expected to be used as a novel functional material in medicine and bioelectronics.\\u000a The main secondary structures of this protein are of the random-coil and the ?-sheet types. In this study, we carried out\\u000a laser-induced transformation of the secondary structure, from the random-coil type to the ?-sheettype, in solid fibroin
Y. Tsuboi; T. Ikejiri; S. Shiga; K. Yamada; A. Itaya
Stability of Silk and Collagen Protein Materials in Space
NASA Astrophysics Data System (ADS)
Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered.
Hu, Xiao; Raja, Waseem K.; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L.
Stability of silk and collagen protein materials in space.
Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered. PMID:24305951
Hu, Xiao; Raja, Waseem K; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L
Evidence for Diet Effects on the Composition of Silk Proteins Produced by Spiders
Silks are highly expressed, secreted proteins that represent a substantial metabolic cost to the insects and spiders that produce them. Female spiders in the superfamily Araneoidea (the orb-spinning spiders and their close relatives) spin six different kinds of silk (three fibroins and three fibrous protein glues) that differ in amino acid content and protein structure. In addition to this diversity
Catherine L. Craig; Christian Riekel; Marie E. Herberstein; Robert S. Weber; David Kaplan; Naomi E. Pierce
Novel nanocomposites from spider silk-silica fusion (chimeric) proteins
Silica skeletal architectures in diatoms are characterized by remarkable morphological and nanostructural details. Silk proteins from spiders and silkworms form strong and intricate self-assembling fibrous biomaterials in nature. We combined the features of silk with biosilica through the design, synthesis, and characterization of a novel family of chimeric proteins for subsequent use in model materials forming reactions. The domains from the major ampullate spidroin 1 (MaSp1) protein of Nephila clavipes spider dragline silk provide control over structural and morphological details because it can be self-assembled through diverse processing methods including film casting and fiber electrospinning. Biosilica nanostructures in diatoms are formed in aqueous ambient conditions at neutral pH and low temperatures. The R5 peptide derived from the silaffin protein of Cylindrotheca fusiformis induces and regulates silica precipitation in the chimeric protein designs under similar ambient conditions. Whereas mineralization reactions performed in the presence of R5 peptide alone form silica particles with a size distribution of 0.5�10 ?m in diameter, reactions performed in the presence of the new fusion proteins generate nanocomposite materials containing silica particles with a narrower size distribution of 0.5�2 ?m in diameter. Furthermore, we demonstrate that composite morphology and structure could be regulated by controlling processing conditions to produce films and fibers. These results suggest that the chimeric protein provides new options for processing and control over silica particle sizes, important benefits for biomedical and specialty materials, particularly in light of the all aqueous processing and the nanocomposite features of these new materials.
Wong Po Foo, Cheryl; Patwardhan, Siddharth V.; Belton, David J.; Kitchel, Brandon; Anastasiades, Daphne; Huang, Jia; Naik, Rajesh R.; Perry, Carole C.; Kaplan, David L.
Reducing blood glucose levels in TIDM mice with an orally administered extract of sericin from hIGF-I-transgenic silkworm cocoons.
In previous studies, we reported that the blood glucose levels of mice with type I diabetes mellitus (TIDM) was reduced with orally administered silk gland powder from silkworms transgenic for human insulin-like growth factor-I (hIGF-I). However, potential safety hazards could not be eliminated because the transgenic silk gland powder contained heterologous DNA, including the green fluorescent protein (gfp) and neomycin resistance (neo) genes. These shortcomings might be overcome if the recombinant hIGF-I were secreted into the sericin layer of the cocoon. In this study, silkworm eggs were transfected with a novel piggyBac transposon vector, pigA3GFP-serHS-hIGF-I-neo, containing the neo, gfp, and hIGF-I genes controlled by the sericin-1 (ser-1) promoter with the signal peptide DNA sequence of the fibrin heavy chain (Fib-H) and a helper plasmid containing the piggyBac transposase sequence under the control of the Bombyx mori actin 3 (A3) promoter, using sperm-mediated gene transfer to generate the transformed silkworms. The hIGF-I content estimated by enzyme-linked immunosorbent assay was approximately 162.7 ng/g. To estimate the biological activity of the expressed hIGF-I, streptozotocin-induced TIDM mice were orally administered sericin from the transgenic silkworm. The blood glucose levels of the mice were significantly reduced, suggesting that the extract from the transgenic hIGF-I silkworm cocoons can be used as an orally administered drug. PMID:24632065
Song, Zuowei; Zhang, Mengyao; Xue, Renyu; Cao, Guangli; Gong, Chengliang
Nanoscale control of silica particle formation via silk-silica fusion proteins for bone regeneration
The biomimetic design of silk/silica fusion proteins was carried out, combining the self assembling domains of spider dragline silk (Nephila clavipes) and silaffin derived R5 peptide of Cylindrotheca fusiformis that is responsible for silica mineralization. Genetic engineering was used to generate the protein-based biomaterials incorporating the physical properties of both components. With genetic control over the nanodomain sizes and chemistry, as well as modification of synthetic conditions for silica formation, controlled mineralized silk films with different silica morphologies and distributions were successfully generated; generating 3D porous networks, clustered silica nanoparticles (SNPs), or single SNPs. Silk serves as the organic scaffolding to control the material stability and multiprocessing makes silk/silica biomaterials suitable for different tissue regenerative applications. The influence of these new silk-silica composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on the silk/silica films. The presence of the silica in the silk films influenced osteogenic gene expression, with the upregulation of alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col 1) markers. Evidence for early bone formation as calcium deposits was observed on silk films with silica. These results indicate the potential utility of these new silk/silica systems towards bone regeneration.
Mieszawska, Aneta J.; Nadkarni, Lauren D.; Perry, Carole C.
High yield recombinant silk-like protein production in transgenic plants through protein targeting
DP1B is a synthetic analogue of spider dragline silk protein. It can be spun to form silk fiber. Previously, it had been expressed in transgenic plants, showing the general feasibility of the plant-based DP1B production. However, success of such a plant-based platform requires a great increase of DP1B productivity in plant cells to reduce production cost. This report describes a
Jianjun Yang; Leslie A. Barr; Stephen R. Fahnestock; Zhan-Bin Liu
National Technical Information Service (NTIS)
The aim of this project was to understand the structure-properties- performance relationship of honeybee silks and to use this information to guide design of second-generation honeybee silk materials. We demonstrated a single recombinant silk protein coul...
T. Sutherland
Mapping Domain Structures in Silks from Insects and Spiders Related to Protein Assembly
The exceptional solubility in vivo (20�30%, w\\/v) of the silk proteins of insects and spiders is dictated by both the need to produce solid fibres with a high packing fraction and the high mesogen concentration required for lyotropic liquid crystalline spinning. A further design requirement for silk proteins is a strong predominance of hydrophobic amino acid residues to provide for
Elisabetta Bini; David P Knight; David L Kaplan
A Novel Model System for Design of Biomaterials Based on Recombinant Analogs of Spider Silk Proteins
Spider dragline silk possesses impressive mechanical and biochemical properties. It is synthesized by a couple of major ampullate\\u000a glands in spiders and comprises of two major structural proteins�spidroins 1 and 2. The relationship between structure and\\u000a mechanical properties of spider silk is not well understood. Here, we modeled the complete process of the spider silk assembly\\u000a using two new recombinant
Vladimir G. Bogush; Olga S. Sokolova; Lyubov I. Davydova; Dmitri V. Klinov; Konstantin V. Sidoruk; Natalya G. Esipova; Tatyana V. Neretina; Igor A. Orchanskyi; Vsevolod Yu Makeev; Vladimir G. Tumanyan; Konstantin V. Shaitan; Vladimir G. Debabov; Mikhail P. Kirpichnikov
Electroresponsive aqueous silk protein as "smart" mechanical damping fluid.
Here we demonstrate the effectiveness of an electroresponsive aqueous silk protein polymer as a smart mechanical damping fluid. The aqueous polymer solution is liquid under ambient conditions, but is reversibly converted into a gel once subjected to an electric current, thereby increasing or decreasing in viscosity. This nontoxic, biodegradable, reversible, edible fluid also bonds to device surfaces and is demonstrated to reduce friction and provide striking wear protection. The friction and mechanical damping coefficients are shown to modulate with electric field exposure time and/or intensity. Damping coefficient can be modulated electrically, and then preserved without continued power for longer time scales than conventional "smart" fluid dampers. PMID:24750065
Jose, Rod R; Elia, Roberto; Tien, Lee W; Kaplan, David L
Purification and cytotoxcicity of tag-free bioengineered spider silk proteins3
Bioengineered spider silk-like proteins can serve as biomaterials for various biomedical applications. These proteins can be assembled in several morphological forms such as films, microcapsules, spheres, fibers, gels and scaffolds. However, crucial points for recombinant spider silks for human use are toxicity and immunogenicity. To assess this issue two bioengineered spider silk proteins composed of different numbers of repetitive motifs of the consensus repeats from spidroin-1 from Nephila clavipes (15X and 6X) were cloned and expressed in E. coli. The proteins were free of tag-sequence and were purified using two methods based on (i) thermal and (ii) organic acid resistance of the spider silks. The soluble spider silk proteins were not cytotoxic and did not activate macrophages over a wide range of concentrations, except when present at the highest concentration. Films made of the different silk variants supported the growth of the cells. Based on these data, and since the biodegradation rate of silk is very slow, the bioengineered spider silks are presumed safe biomaterials for biomedical applications.
Dams-Kozlowska, Hanna; Majer, Agnieszka; Tomasiewicz, Paulina; Lozinska, Jolanta; Kaplan, David L.; Mackiewicz, Andrzej
Purification and cytotoxicity of tag-free bioengineered spider silk proteins.
Bioengineered spider silk-like proteins can serve as biomaterials for various biomedical applications. These proteins can be assembled in several morphological forms such as films, microcapsules, spheres, fibers, gels, and scaffolds. However, crucial points for recombinant spider silks for human use are toxicity and immunogenicity. To assess this issue, two bioengineered spider silk proteins composed of different numbers of repetitive motifs of the consensus repeats from spidroin-1 from Nephila clavipes (15X and 6X) were cloned and expressed in Escherichia coli. The proteins were free of tag sequence and were purified using two methods based on (1) thermal and (2) organic acid resistance of the spider silks. The soluble spider silk proteins were not cytotoxic and did not activate macrophages over a wide range of concentrations, except when present at the highest concentration. Films made of the different silk variants supported the growth of the cells. Based on these data, and as the biodegradation rate of silk is very slow, the bioengineered spider silks are presumed safe biomaterials for biomedical applications. PMID:22865581
Dams-Kozlowska, Hanna; Majer, Agnieszka; Tomasiewicz, Paulina; Lozinska, Jolanta; Kaplan, David L; Mackiewicz, Andrzej
The effect of genetically engineered spider silk-dentin matrix protein 1 chimeric protein on hydroxyapatite nucleation
Spider silks exhibit remarkable mechanical properties while dentin matrix protein 1 provides controlled nucleation and hydroxyapatite growth. In the present work, these two attributes were combined via genetic engineering to form a chimera, a clone encoding consensus repeats from the major protein in the spider dragline silk of Nephila clavipes fused to the carboxyl terminal domain of dentin matrix protein
Jia Huang; Cheryl Wong; Anne George; David L. Kaplan
The biosynthesis of transfer RNA in insects. I. Increase of amino acid acceptor activity of specific tRNA's utilized for silk protein biosynthesis in the silk gland of Bombyx mori.
1) To detect the quantitative changes of amino acid acceptor activity of tRNA's from the posterior and middle silk glands of Bombyx mori at various ages, a relatively simple and rapid method was established using a mixture of radioactive amino acids in Chlorella hydrolysate. 2) The acceptor activities of silk gland tRNA for 15 amino acids tested seemed to be almost on the same level at the end of the 4th moult stage. During the 5th instar, however, characteristic increases were observed in glycine, alanine, and serine acceptor activities in both silk glands. 3) In the posterior silk gland, which produces fibroin, the acceptor activities for glycine and alanine increased more than that for serine. In the middle silk gland, which produces sericine, the acceptor activity for serine increased more than those for glycine and alanine. 4) In the light of observations on the increase of corresponding aminoacyl-tRNA synthetase activities in the silk glands, a functional adaptation of tRNA synthesis in the tissue is discussed. PMID:1228175
Majima, R; Kawakami, M; Shimura, K
Biological responses to spider silk-antibiotic fusion protein.
The development of a new generation of multifunctional biomaterials is a continual goal for the field of materials science. The in vivo functional behaviour of a new fusion protein that combines the mechanical properties of spider silk with the antimicrobial properties of hepcidin was addressed in this study. This new chimeric protein, termed 6mer + hepcidin, fuses spider dragline consensus sequences (6mer) and the antimicrobial peptide hepcidin, as we have recently described, with retention of bactericidal activity and low cytotoxicity. In the present study, mouse subcutaneous implants were studied to access the in vivo biological response to 6mer + hepcidin, which were compared with controls of silk alone (6mer), polylactic-glycolic acid (PLGA) films and empty defects. Along with visual observations, flow cytometry and histology analyses were used to determine the number and type of inflammatory cells at the implantation site. The results show a mild to low inflammatory reaction to the implanted materials and no apparent differences between the 6mer + hepcidin films and the other experimental controls, demonstrating that the new fusion protein has good in vivo biocompatibility, while maintaining antibiotic function. PMID:22514077
Gomes, S�lvia; Gallego-Llamas, Jabier; Leonor, Isabel B; Mano, Jo�o F; Reis, Rui L; Kaplan, David L
Processing and modification of films made from recombinant spider silk proteins
Protein films represent an interesting class of materials with various possibilities for applications. We investigated films\\u000a made of two different synthetic spider silk proteins derived from the garden spider�s (Araneus diadematus) two dragline silk\\u000a proteins ADF-3 and ADF-4. Protein films cast from hexafluoroisopropanol solutions displayed a predominantly ?-helical secondary\\u000a structure. Processing such films with potassium phosphate or methanol resulted in
D. Huemmerich; U. Slotta; T. Scheibel
Expression and Purification of a Spider Silk Protein: A New Strategy for Producing Repetitive Proteins
Synthetic genes were constructed based on the known sequence of the spider dragline silk protein MaSp 2. The genes had 8, 16, or 32 contiguous units of the consensus repeat sequence of the protein. These artificial genes were constructed using a strategy involving compatible but nonregenerable restriction sites, which allowed construction of very large inserts in a precisely controlled manner.
Randolph V. Lewis; Michael Hinman; Srinivas Kothakota; Maurille J. Fournier
Recombinant production and film properties of full-length hornet silk proteins.
Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1-4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160-330mg recombinant proteinl(-1). Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (?) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1-4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications. PMID:24862540
Kambe, Yusuke; Sutherland, Tara D; Kameda, Tsunenori
Integration of silk protein in organic and light-emitting transistors
We present the integration of a natural protein into electronic and optoelectronic devices by using silk fibroin as a thin film dielectric in an organic thin film field-effect transistor (OFET) ad an organic light emitting transistor device (OLET) structures. Both n- (perylene) and p-type (thiophene) silk-based OFETs are demonstrated. The measured electrical characteristics are in agreement with high-efficiency standard organic transistors, namely charge mobility of the order of 10-2 cm2/Vs and on/off ratio of 104. The silk-based optolectronic element is an advanced unipolar n-type OLET that yields a light emission of 100nW.
Capelli, R.; Amsden, J. J.; Generali, G.; Toffanin, S.; Benfenati, V.; Muccini, M.; Kaplan, D. L.; Omenetto, F. G.; Zamboni, R.
Optically Transparent Recombinant Silk-Elastinlike Protein Polymer Films
Recombinant protein polymers, evaluated extensively as biomaterials for applications in drug delivery and tissue engineering, are rarely reported as being optically transparent. Here we report the notable optical transparency of films composed of a genetically engineered silk-elastinlike protein polymer SELP-47K. SELP-47K films of 100 ?m in thickness display a transmittance of 93% in the wavelength range of 350�800 nm. While covalent cross-linking of SELP-47K via glutaraldehyde decreases its transmittance to 77% at the wavelength of 800 nm, noncovalent cross-linking using methanol slightly increases it to 95%. Non- and covalent cross-linking of SELP-47K films also influences their secondary structures and water contents. Cell viability and proliferation analyses further reveal the excellent cytocompatibility of both non- and covalently cross-linked SELP-47K films. The combination of high optical transparency and cytocompatibility of SELP-47K films, together with their previously reported outstanding mechanical properties, suggests that this protein polymer may be useful in unique, new biomedical applications.
Teng, Weibing; Huang, Yiding; Cappello, Joseph; Wu, Xiaoyi
Self-assembly of spider silk proteins is controlled by a pH-sensitive relay
Nature's high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to
Glareh Askarieh; My Hedhammar; Kerstin Nordling; Alejandra Saenz; Cristina Casals; Anna Rising; Jan Johansson; Stefan D. Knight
Genetically engineered silk-elastinlike protein polymers for controlled drug delivery
The silk-elastinlike class of genetically engineered protein polymers is composed of tandemly repeated silk-like (Gly�Ala�Gly�Ala�Gly�Ser) and elastin-like (Gly�Val�Gly�Val�Pro) amino acid blocks. The precision with which these polymers can be synthesized, as well as the ability to incorporate motifs that allow for gel-formation, stimuli-sensitivity, biodegradation, and biorecognition have stimulated interest in their use for controlled drug and gene delivery. This review
Zaki Megeed; Joseph Cappello; Hamidreza Ghandehari
Production and characterization of a silk-like hybrid protein, based on the polyalanine region of Samia cynthia ricini silk fibroin and a cell adhesive region derived from fibronectin
There are a variety of silkworms and silk fibroins produced by them. Silks have many inherent suitable properties for biomaterials. In this paper, a novel silk-like hybrid protein, [DGG(A)12GGAASTGRGDSPAAS]5, which consists of polyalanine region of silk fibroin from a wild silkworm, Samia cynthia ricini, and cell adhesive region including Arg-Gly-Asp (RGD) sequence, derived from fibronectin, was designed and produced. The
Tetsuo Asakura; Chikako Tanaka; Mingying Yang; Juming Yao; Masato Kurokawa
Bio-inspired Silicification of Silica-binding Peptide-Silk Protein Chimeras: Comparison of Chemically and Genetically Produced Proteins
Novel protein chimeras constituted of �silk� and a silica-binding peptide (KSLSRHDHIHHH) were synthesized by genetic or chemical approaches and their influence on silica-silk based chimera composite formation evaluated. Genetic chimeras were constructed from 6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipes spider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG]n) to which one silica binding peptide was fused at the N terminus. For the chemical chimera, 25 equivalents of the silica binding peptide were chemically coupled to natural Bombyx mori silk after modification of tyrosine groups by diazonium coupling and EDC/NHS activation of all acid groups. After silica formation under mild, biomaterial compatible conditions the effect of peptide addition on the properties of the silk and chimeric silk-silica composite materials was explored. The composite biomaterial properties could be related to the extent of silica condensation and to the higher number of silica binding sites in the chemical chimera as compared to the genetically derived variants. In all cases, the structure of the protein / chimera in solution dictated the type of composite structure that formed with the silica deposition process having little effect on the secondary structural composition of the silk based materials. Similarly to our study of genetic silk based chimeras containing the R5 peptide (SSKKSGSYSGSKGSKRRIL), the role of the chimeras (genetic and chemical) used in the present study resided more in aggregation and scaffolding than in the catalysis of condensation. The variables of peptide identity, silk construct (number of consensus repeats or silk source) and approach to synthesis (genetic or chemical) can be used to �tune� the properties of the composite materials formed and is a general approach which can be used to prepare a range of materials for biomedical and sensor based applications.
Canabady-Rochelle, Laetitia L.S.; Belton, David J.; Deschaume, Olivier; Currie, Heather A.; Kaplan, David L.; Perry, Carole C.
Sericin-carboxymethyl cellulose porous matrices as cellular wound dressing material.
In this study, porous three-dimensional (3D) hydrogel matrices are fabricated composed of silk cocoon protein sericin of non-mulberry silkworm Antheraea mylitta and carboxymethyl cellulose. The matrices are prepared via freeze-drying technique followed by dual cross-linking with glutaraldehyde and aluminum chloride. The microstructure of the hydrogel matrices is assessed using scanning electron microscopy and biophysical characterization are carried out using Fourier transform infrared spectroscopy and X-ray diffraction. The transforming growth factor ?1 release from the cross-linked matrices as a growth factor is evaluated by immunosorbent assay. Live dead assay and 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyl tetrazolium bromide assay show no cytotoxicity of blended matrices toward human keratinocytes. The matrices support the cell attachment and proliferation of human keratinocytes as observed through scanning electron microscope and confocal images. Gelatin zymography demonstrates the low levels of matrix metalloproteinase 2 (MMP-2) and insignificant amount of MMP-9 in the culture media of cell seeded matrices. Low inflammatory response of the matrices is indicated through tumor necrosis factor alpha release assay. The results indicate that the fabricated matrices constitute 3D cell-interactive environment for tissue engineering applications and its potential use as a future cellular biological wound dressing material. PMID:23853114
Nayak, Sunita; Kundu, S C
Native-sized spider silk proteins synthesized in planta via intein-based multimerization.
The synthesis of native-sized proteins is a pre-requisite for exploiting the potential of spider silk as a bio-based material. The unique properties of spider silk, such as extraordinary tensile strength and elasticity, result from the highly repetitive nature of spider silk protein motifs. The present report describes the combination of spider silk flagelliform protein (FLAG) production in the endoplasmic reticulum of tobacco plant leaf cells with an intein-based posttranslational protein fusion technology. The repeated ligation of FLAG monomers resulted in the formation of large multimers. This method avoids the need for highly repetitive transgenes, which may result in a higher genetic and transcriptional stability. Here we show, for the first time, the production of synthetic, high molecular weight spider silk proteins larger than 250�kDa based on the assembly of protein monomers via intein-mediated trans-splicing in planta. The resulting multimeric structures form microfibers, thereby demonstrating their great potential as a biomaterial. PMID:23001519
Hauptmann, Valeska; Weichert, Nicola; Menzel, Matthias; Knoch, Dominic; Paege, Norman; Scheller, J�rgen; Spohn, Uwe; Conrad, Udo; Gils, Mario
A comparison of the composition of silk proteins produced by spiders and insects
Proteins that are highly expressed and composed of amino acids that are costly to synthesize are likely to place a greater drain on an organism�s energy resources than proteins that are composed of ingested amino acids or ones that are metabolically simple to produce. Silks are highly expressed proteins produced by all spiders and many insects. We compared the metabolic
Catherine L. Craig; Michael Hsu; David Kaplan; N. E. Pierce
Transglutaminase mediated grafting of silk proteins onto wool fabrics leading to improved physical and mechanical properties
Transglutaminases have the ability to incorporate primary amines and to graft peptides (containing glutamine or lysine residues) into proteins. These properties enable transglutaminases to be used in the grafting of a range of compounds including peptides and\\/or proteins onto wool fibres, altering their functionality. In this paper we investigated the transglutaminase mediated grafting of silk proteins into wool and its
Jo�o Cortez; Anna Anghieri; Philip L. R. Bonner; Martin Griffin; Giuliano Freddi
Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus.
The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production. PMID:24477386
W�ltje, Michael; B�bel, Melanie; Rheinnecker, Michael; Tettamanti, Gianluca; Franzetti, Eleonora; Saviane, Alessio; Cappellozza, Silvia
Convergently-evolved structural anomalies in the coiled coil domains of insect silk proteins.
The use of coiled coil proteins as the basis of silk materials is an engineering solution that has evolved convergently in at least five insect lineages-the stinging hymenopterans (ants, bees, hornets), argid sawflies, fleas, lacewings, and praying mantises-and persisted throughout large radiations of these insect families. These coiled coil silk proteins share a characteristic distinct from other coiled coil proteins, in that they are fabricated into solid materials after accumulating as highly concentrated solutions within dedicated glands. Here, we relate the amino acid sequences of these proteins to the secondary and tertiary structural information available from biophysical methods such as X-ray scattering, nuclear magnetic resonance and Raman spectroscopy. We investigate conserved and convergently evolved features within these proteins and compare these to the features of classic coiled coil proteins including tropomyosin and leucine zippers. Our analysis finds that the coiled coil domains of insect silk proteins have several common structural anomalies including a high prevalence of alanine residues in core positions. These atypical features of the coiled coil fibrous proteins - which likely produce deviations from canonical coiled-coil structure - likely exist due to selection pressures related to the process of silk fabrication and the final function of the proteins. PMID:24434611
Sutherland, Tara D; Trueman, Holly E; Walker, Andrew A; Weisman, Sarah; Campbell, Peter M; Dong, Zhaoming; Huson, Mickey G; Woodhead, Andrea L; Church, Jeffrey S
Novel genes differentially expressed between posterior and median silk gland identified by SAGE-aided transcriptome analysis.
Serial analysis of gene expression (SAGE) profiles, from posterior and median cells of the silk gland of Bombyx mori, were analyzed and compared, so as to identify their respective distinguishing functions. The annotation of the SAGE libraries was performed with a B. mori reference tag collection, which was extracted from a novel set of Bombyx ESTs, sequenced from the 3' side. Most of the tags appeared at similar relative concentration within the two libraries, and corresponded with region-specific and highly abundant silk proteins. Strikingly, in addition to tags from silk protein mRNAs, 19 abundant tags were found (? 0.1%), in the median cell library, which were absent in the posterior cell tag collection. With the exception of tags from SP1 mRNA, no PSG specific tags were found in this subset class. The analysis of some of the MSG-specific transcripts, suggested that middle silk gland cells have diversified functions, in addition to their well characterized role in silk sericins synthesis and secretion. PMID:21078388
Royer, Corinne; Briolay, J�r�me; Garel, Annie; Brouilly, Patrick; Sasanuma, Shun-ichi; Sasanuma, Motoe; Shimomura, Michihiko; Keime, C�line; Gandrillon, Olivier; Huang, Yongping; Chavancy, G�rard; Mita, Kazuei; Couble, Pierre
Disulfide Bonds in Recombinant Repeat Units from an Aquatic Insect's Silk Protein.
National Technical Information Service (NTIS)
rCAS is a recombinant Constant And Subrepeat (rCAS) protein modelled after tandem core repeats found in a 1000-kDa silk protein synthesized by larvae of the midge, Chironomus tentans rCAS is encoded by a synthetic gene (synCAS) which is expressed in bacte...
S. T. Case S. V. Smith M. R. Bratton
The role of formic acid in solution stability and crystallization of silk protein polymer
In this paper, the regenerated silk fibroin (SF) solution dissolved in formic acid was used as a model protein to understand the role of formic acid in solution stability and crystallization of protein-based materials. The molecular decomposition of SF did not occur for the dissolution process in formic acid within 1�2 days of storage times. The ?-sheet crystallization of SF
In Chul Um; Hae Yong Kweon; Kwang Gill Lee; Young Hwan Park
Multifunctional adhesive silk fibroin with blending of RGD-bioconjugated mussel adhesive protein.
Silk has recently been exploited in various fields due to its superior mechanical properties. However, this material's lack of biological functions and relatively poor biodegradation have hindered its wide use in applications related to cells and tissues. Here, we improved the overall characteristics of silkworm silk fibroin (SF) by introduction of RGD peptide-fused recombinant mussel adhesive protein (MAP-RGD). Simple blending of MAP-RGD provided not only bulk-scale adhesive ability but also microscale adhesiveness to cells and various biomolecules. MAP-RGD-blended SF fibers supported enhanced adhesion, proliferation, and spreading of mammalian cells as well as the efficient attachment of biomolecules, including carbohydrate and protein. In addition, the hydrophilicity, swelling, and biodegradability of the MAP-RGD-blended SF material were improved without notable hampering of the original mechanical properties of SF. Therefore, the adhesive silk fibrous scaffold could be successfully used in diverse biomedical engineering applications. PMID:24601579
Yang, Yun Jung; Kwon, Yunkyeoung; Choi, Bong-Hyuk; Jung, Dooyup; Seo, Jeong Hyun; Lee, Ki Hoon; Cha, Hyung Joon
Designing recombinant spider silk proteins to control assembly
The consensus repeat sequence found in the dragline silk from the spider, Nephila clavipes, was redesigned to incorporate a redox trigger flanking the beta-sheet forming polyalanine sequences. The methionine redox trigger, in the oxidized state, was incorporated to prevent the formation of the beta sheets, while in the reduced state would not result in sterical limitations to beta sheet formation.
Stefan Winkler; Sandra Szela; Peter Avtges; Regina Valluzzi; Daniel A. Kirschner; David Kaplan
Microphase Separation Controlled Beta Sheet Crystallization Kinetics in Silk Fibroin Protein.
NASA Astrophysics Data System (ADS)
We investigate the mechanism of isothermal crystallization kinetics of beta-sheet crystals in silk multiblock fibrous proteins. The Avrami analysis kinetic theory, for studies of synthetic polymer crystal growth, is for the first time extended to investigate protein self-assembly in beta-sheet rich Bombyx mori silk fibroin samples, using time-resolved Fourier transform infrared spectroscopy, differential scanning calorimetry and synchrotron real-time wide-angle X-ray scattering. Results indicate formation of beta sheet crystals in silk proteins is different from the 3-D spherulitic crystal growth found in synthetic homopolymers. Observations by scanning electron microscopy support the view that the protein structures vary during the different stages of crystal growth, and show a microphase separation pattern after chymotrypsin enzyme biodegradation. We present a model to explain the crystallization of the multiblock silk fibroin protein, by analogy to synthetic block copolymers. This model could be widely applicable in other proteins with multiblock (i.e., crystallizable and non-crystallizable) domains.
Hu, Xiao; Lu, Qiang; Kaplan, David; Cebe, Peggy
Tunable Self-Assembly of Genetically Engineered Silk-Elastin-Like Protein Polymers
Silk-elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Due to the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. However, little is known about the fundamental self-assembly characteristics of these remarkable polymers. Here we propose a two-step self-assembly process of SELPs in aqueous solution for the first time and report the importance of the ratio of silk to elastin blocks in a SELP�s repeating unit on the assembly of the SELP. Through precise tuning of the ratio of silk to elastin, various structures including nanoparticles, hydrogels and nanofibers could be generated either reversibly or irreversibly. This assembly process might provide opportunities to generate innovative smart materials for biosensors, tissue engineering and drug delivery. Furthermore, the newly developed SELPs in this study may be potentially useful as biomaterials for controlled drug delivery and biomedical engineering.
Xia, Xiao-Xia; Xu, Qiaobing; Hu, Xiao; Qin, Guokui; Kaplan, David L.
Reinforcing Silk Scaffolds with Silk Particles
Silk fibroin is a useful protein polymer for biomaterials and tissue engineering. In this work, porogen leached scaffolds prepared from aqueous and HFIP silk solutions were reinforced through the addition of silk particles. This led to about 40 times increase in the specific compressive modulus and the yield strength of HFIP-based scaffolds. This increase in mechanical properties resulted from the high interfacial cohesion between the silk matrix and the reinforcing silk particles, due to partial solubility of the silk particles in HFIP. The porosity of scaffolds was reduced from ?90% (control) to ?75% for the HFIP systems containing 200% particle reinforcement, while maintaining pore interconnectivity. The presence of the particles slowed the enzymatic degradation of silk scaffolds.
Rajkhowa, Rangam; Gil, Eun Seok; Kluge, Jonathan; Numata, Keiji; Wang, Lijing; Kaplan, David L.
Integration of silk protein in organic and light-emitting transistors.
We present the integration of a natural protein into electronic and optoelectronic devices by using silk fibroin as a thin film dielectric in an organic thin film field-effect transistor (OFET) ad an organic light emitting transistor device (OLET) structures. Both n- (perylene) and p-type (thiophene) silk-based OFETs are demonstrated. The measured electrical characteristics are in agreement with high-efficiency standard organic transistors, namely charge mobility of the order of 10(-2) cm(2)/Vs and on/off ratio of 10(4). The silk-based optolectronic element is an advanced unipolar n-type OLET that yields a light emission of 100nW. PMID:22899899
Capelli, R; Amsden, J J; Generali, G; Toffanin, S; Benfenati, V; Muccini, M; Kaplan, D L; Omenetto, F G; Zamboni, R
Role of pH and charge on silk protein assembly in insects and spiders
Silk fibers possess impressive mechanical properties, dependant, in part, on the crystalline ?-sheets silk II conformation.\\u000a The transition to silk II from soluble silk I-like conformation in silk glands, is thought to originate in the spinning ducts\\u000a immediately before the silk is drawn down into a fiber. However the assembly process of these silk molecules into fibers,\\u000a whether in silkworms
C. Wong Po Foo; E. Bini; J. Hensman; D. P. Knight; R. V. Lewis; D. L. Kaplan
Silk fibroin protein from mulberry and non-mulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion
Silks fibers and films fabricated from fibroin protein of domesticated mulberry silkworm cocoon have been traditionally utilized\\u000a as sutures in surgery and recently as biomaterial films respectively. Here, we explore the possibility of application of silk\\u000a fibroin protein from non-mulberry silkworm cocoon as a potential biomaterial aid. In terms of direct inflammatory potential,\\u000a fibroin proteins from Antheraea mylitta and Bombyx
Chitrangada Acharya; Sudip K. Ghosh; S. C. Kundu
Disulfide Bonds in a Recombinant Protein Modeled After a Core Repeat in an Aquatic Insect's Silk Protein.
National Technical Information Service (NTIS)
We constructed a gene encoding rCAS, recombinant constant and subrepeat protein, modeled after tandem repeats found in the major silk proteins synthesized by aquatic larvae of the midge, Chironoinus ten tans. Bacterially synthesized rCAS was purified to n...
S. V. Smith J. J. Correia S. T. Case
Gene Delivery Mediated by Recombinant Silk Proteins Containing Cationic and Cell Binding Motifs
Silk proteins are biodegradable and biocompatible, and can also be tailored to contain additional features via genetic engineering, suggesting utility for gene delivery. In the present study, novel silk-based block copolymers were bioengineered both with poly(L-lysine) domains to interact with plasmid DNA (pDNA) and RGD, to enhance cell-binding and transfection efficiency. Ionic complexes of these silk-polylysine-RGD block copolymers with pDNA were prepared, characterized and utilized for gene delivery to HeLa cells and human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, zeta-potentialmeter, atomic force microscopy, and dynamic light scattering. Sizes and charges of the pDNA complexes were regulated by the polymer/nucleotide molar ratio. Samples with 30-lysine residues and 11 RGD sequences, prepared at the ratio of number of amines/phosphates from pDNA (N/P) of 2, had an average solution diameter of 186 nm and showed the highest transfection efficiency. The intracellular distribution of complexes of Cy5-labeled pDNA was investigated by confocal laser scanning microscopy. The Cy5-labeled pDNA was distributed near the cell membrane and around the nuclei, indicating that the pDNA was transferred near the nucleus. The results demonstrated the potential of bioengineered silk proteins with additional functional features as a new family of highly tailored gene delivery systems.
Numata, Keiji; Hamasaki, Juliana; Subramanian, Balajikarthick; Kaplan, David L
Structural changes of thin films from recombinant spider silk proteins upon post-treatment
NASA Astrophysics Data System (ADS)
Engineering of spider silk proteins offers the possibility to control their molecular sequence and thus their material properties. Spin coating was used to prepare films of engineered spider silk protein derived from the garden spider�s ( Araneus diadematus) dragline silk protein ADF-4. A conformational transition from ?-helix to ?-sheet-rich structures upon methanol treatment of the films was detected by external reflection IR spectroscopy. We present direct evidence for this structural transformation using grazing-incidence X-ray diffraction (GIXRD) and small-angle scattering (GISAXS). The protein film structure after the methanol treatment consists mainly of ?-sheet polyalanine crystals dispersed in an amorphous protein matrix. The GIXRD intensity profiles show Bragg peaks from ?-sheet polyalanine crystallites having an average size of 7.5 nm. The non-uniform and large crystal size distributions within the film were explained based on the protein composition. The effect of the chemical nature of the interface on the protein film structure was investigated as well.
Metwalli, E.; Slotta, U.; Darko, C.; Roth, S. V.; Scheibel, T.; Papadakis, C. M.
Synthetic spider dragline silk proteins and their production in Escherichia coli
Synthetic genes were designed to encode analogs of the two proteins of Nephila clavipes dragline silk, spidroins 1 and 2. The genes were constructed of tandem repeats of relatively long (more than 300?bp) DNA\\u000a sequences assembled from synthetic oligonucleotides, and encoded proteins of high molecular mass (65�163?kDa). Both analogs\\u000a were produced efficiently in Escherichia coli. The yield and homogeneity of
S. R. Fahnestock; S. L. Irwin
Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins
Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh. Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly ?-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable ?-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD). The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.
Lang, Gregor; Jokisch, Stephan; Scheibel, Thomas
Air filter devices including nonwoven meshes of electrospun recombinant spider silk proteins.
Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh. Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly ?-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable ?-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD). The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols. PMID:23685883
Lang, Gregor; Jokisch, Stephan; Scheibel, Thomas
The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening.
Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s(-1). The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders. PMID:24240554
Schwarze, Simone; Zwettler, Fabian U; Johnson, Christopher M; Neuweiler, Hannes
Swelling behavior of a genetically engineered silk-elastinlike protein polymer hydrogel
The influence of environmental conditions such as pH, temperature, and ionic strength on the equilibrium swelling ratio of physically crosslinked networks of a genetically engineered silk-elastinlike protein-based copolymer (SELP) with an amino acid repeat sequence of [(GVGVP)4GKGVP(GVGVP)3(GAGAGS)4]12 was investigated. The effects of gelation cure time and initial polymer concentration on the equilibrium swelling ratio and soluble fraction of the hydrogels
Adam A Dinerman; Joseph Cappello; Hamidreza Ghandehari; Stephen W Hoag
Solute diffusion in genetically engineered silk�elastinlike protein polymer hydrogels
The partitioning and diffusion behavior of theophylline, vitamin B12, and cytochrome c in physically crosslinked networks of a genetically engineered silk�elastinlike protein-based (SELP) copolymer with an amino acid sequence of [(GVGVP)4GKGVP(GVGVP)3(GAGAGS)4]12 was investigated. The effect of gelation kinetics on the equilibrium swelling ratio and normalized dimensions of loaded SELP hydrogel disks before and after release studies was also examined. Size
Adam A Dinerman; Joseph Cappello; Hamidreza Ghandehari; Stephen W Hoag
Cre-mediated targeted gene activation in the middle silk glands of transgenic silkworms (Bombyx mori).
Cre-mediated recombination is widely used to manipulate defined genes spatiotemporally in vivo. The present study evaluated the Cre/loxP system in Bombyx mori by establishing two transgenic lines. One line contained a Cre recombinase gene controlled by a sericin-1 gene (Ser1) promoter. The other line contained a loxP-Stop-loxP-DsRed cassette driven by the same Ser1 promoter. The precise deletion of the Stop fragment was found to be triggered by Cre-mediated site-specific excision, and led to the expression of DsRed fluorescence protein in the middle silk glands of all double-transgenic hybrids. This result was also confirmed by phenotypical analysis. Hence, the current study demonstrated the feasibility of Cre-mediated site-specific recombination in B. mori, and opened a new window for further refining genetic tools in silkworms. PMID:23264031
Duan, Jianping; Xu, Hanfu; Ma, Sanyuan; Guo, Huizhen; Wang, Feng; Zhao, Ping; Xia, Qingyou
Functional analysis of 30K proteins during silk gland degeneration by a caspase-dependent pathway in Bombyx.
The 30K proteins are involved with important functions in the growth and development of Bombyx mori. In this study, the synthesis and regulation of 30K proteins were examined during the degeneration of Bombyx silk glands. On day 3 of the fifth instar, the protein level of 30Kc19 was low, whereas the silk proteins were rapidly synthesized. However, synthesis and accumulation of the 30Kc19 protein significantly increased at the prepupal stage and on day 1 of the pupal stage. At this stage, the silk gland cells were filled with 30Kc19 and genomic DNA. Moreover, the transcript levels of the 30K-encoding genes, including 30Kc6, 30Kc12, 30Kc19 and 30Kc23 were up-regulated during the degeneration of the Bombyx silk glands. During the time that the levels of the 30Kc19 protein were significantly up-regulated, it is notable that the transcript levels of the BmAtg8, BmAtg6 and BmDronc genes dramatically increased to regulate the programmed cell death of this gland. On day 1 of the pupal stage, intense fragmentation of genomic DNA occurred in the silk gland cells, and the putative active form of caspase was detected in the cytoplasm, showing the complete degradation of the silk glands in one day. In conclusion, the 30K proteins are synthesized in high concentrations, while proteolysis mediates silk gland degeneration in Bombyx by a caspase-dependent pathway. We propose that the 30K proteins may be nutrients and energy vectors to be absorbed by the developing tissues of pupae or moths. PMID:23496335
Ji, M-M; Liu, A-Q; Gan, L-P; Xing, R; Wang, H; Sima, Y-H; Xu, S-Q
Harnessing disorder: onychophorans use highly unstructured proteins, not silks, for prey capture
Onychophora are ancient, carnivorous soft-bodied invertebrates which capture their prey in slime that originates from dedicated glands located on either side of the head. While the biochemical composition of the slime is known, its unusual nature and the mechanism of ensnaring thread formation have remained elusive. We have examined gene expression in the slime gland from an Australian onychophoran, Euperipatoides rowelli, and matched expressed sequence tags to separated proteins from the slime. The analysis revealed three categories of protein present: unique high-molecular-weight proline-rich proteins, and smaller concentrations of lectins and small peptides, the latter two likely to act as protease inhibitors and antimicrobial agents. The predominant proline-rich proteins (200 kDa+) are composed of tandem repeated motifs and distinguished by an unusually high proline and charged residue content. Unlike the highly structured proteins such as silks used for prey capture by spiders and insects, these proteins lack ordered secondary structure over their entire length. We propose that on expulsion of slime from the gland onto prey, evaporative water loss triggers a glass transition change in the protein solution, resulting in adhesive and enmeshing thread formation, assisted by cross-linking of complementary charged and hydrophobic regions of the protein. Euperipatoides rowelli has developed an entirely new method of capturing prey by harnessing disordered proteins rather than structured, silk-like proteins.
Haritos, Victoria S.; Niranjane, Ajay; Weisman, Sarah; Trueman, Holly E.; Sriskantha, Alagacone; Sutherland, Tara D.
Dielectric breakdown strength of regenerated silk fibroin films as a function of protein conformation.
Derived from Bombyx mori cocoons, regenerated silk fibroin (RSF) exhibits excellent biocompatibility, high toughness, and tailorable biodegradability. Additionally, RSF materials are flexible, optically clear, easily patterned with nanoscale features, and may be doped with a variety bioactive species. This unique combination of properties has led to increased interest in the use of RSF in sustainable and biocompatible electronic devices. In order to explore the applicability of this biopolymer to the development of future bioelectronics, the dielectric breakdown strength (Ebd) of RSF thin films was quantified as a function of protein conformation. The application of processing conditions that increased ?-sheet content (as determined by FTIR analysis) and produced films in the silk II structure resulted in RSF materials with improved Ebd with values reaching up to 400 V/?m. PMID:23987229
Dickerson, Matthew B; Fillery, Scott P; Koerner, Hilmar; Singh, Kristi M; Martinick, Katie; Drummy, Lawrence F; Durstock, Michael F; Vaia, Richard A; Omenetto, Fiorenzo G; Kaplan, David L; Naik, Rajesh R
Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield.
Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method. PMID:17525867
Zhang, Yuansong; Hu, Junhua; Miao, Yungen; Zhao, Aichun; Zhao, Tianfu; Wu, Dayang; Liang, Liefeng; Miikura, Ayumi; Shiomi, Kunihiro; Kajiura, Zenta; Nakagaki, Masao
Sericin Enhances the Bioperformance of Collagen-Based Matrices Preseeded with Human-Adipose Derived Stem Cells (hADSCs)
Current clinical strategies for adipose tissue engineering (ATE), including autologous fat implants or the use of synthetic surrogates, not only are failing in the long term, but also can�t face the latest requirements regarding the aesthetic restoration of the resulted imperfections. In this context, modern strategies in current ATE applications are based on the implantation of 3D cell-scaffold bioconstructs, designed for prospective achievement of in situ functional de novo tissue. Thus, in this paper, we reported for the first time the evaluation of a spongious 60% collagen and 40% sericin scaffold preseeded with human adipose-derived stem cells (hADSCs) in terms of biocompatibility and adipogenic potential in vitro. We showed that the addition of the sticky protein sericin in the composition of a classical collagen sponge enhanced the adhesion and also the proliferation rate of the seeded cells, thus improving the biocompatibility of the novel scaffold. In addition, sericin stimulated PPAR?2 overexpression, triggering a subsequent upregulated expression profile of FAS, aP2 and perilipin adipogenic markers. These features, together with the already known sericin stimulatory potential on cellular collagen production, promote collagen-sericin biomatrix as a good candidate for soft tissue reconstruction and wound healing applications.
Dinescu, Sorina; Galateanu, Bianca; Albu, Madalina; Cimpean, Anisoara; Dinischiotu, Anca; Costache, Marieta
Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders
The known silk fibroins and fibrous glues are thought to be encoded by members of the same gene family. All silk fibroins sequenced to date contain regions of long-range order (crystalline regions) and\\/or short-range order (non-crystalline regions). All of the sequenced fibroin silks (Flag or silk from flagelliform gland in spiders; Fhc or heavy chain fibroin silks produced by Lepidoptera
Catherine L. Craig; Christian Riekel
Protein Secondary Structure and Orientation in Silk as Revealed by Raman Spectromicroscopy
Taking advantage of recent advances in polarized Raman microspectroscopy, and based on a rational decomposition of the amide I band, the conformation and orientation of proteins have been determined for cocoon silks of the silkworms Bombyx mori and Samia cynthia ricini and dragline silks of the spiders Nephila clavipes and Nephila edulis. This study distinguished between band components due to ?-sheets, ?-turns, 31-helices, and unordered structure for the four fibers. For B. mori, the ?-sheet content is 50%, which matches the proportion of residues that form the GAGAGS fibroin motifs. For the Nephila dragline and S. c. ricini cocoon, the ?-sheet content (36�37% and 45%, respectively) is higher than the proportion of residues that belong to polyalanine blocks (18% and 42%, respectively), showing that adjacent GGA motifs are incorporated into the ?-sheets. Nephila spidroins contain fewer ?-sheets and more flexible secondary structures than silkworm fibroins. The amorphous polypeptide chains are preferentially aligned parallel to the fiber direction, although their level of orientation is much lower than that of ?-sheets. Overall, the results show that the four silks exhibit a common molecular organization, with mixtures of different amounts of ?-sheets and flexible structures, which are organized with specific orientation levels.
Lefevre, Thierry; Rousseau, Marie-Eve; Pezolet, Michel
Silk fibroin-based complex particles with bioactive encrustation for bone morphogenetic protein 2 delivery.
Application of bone morphogenetic protein 2 (BMP-2) currently faces its challenges, and its efficacy of delivery has to be improved. The proper dosage of the powerful bioactive molecule is still under discussion and needs to be investigated further. In this work, pure silk fibroin particles and particles with calcium carbonate encrustation (complex particles) are designed, developed, and functionalized by BMP-2. These are used to deliver the bioactive molecule to mesenchymal stem cells (MSCs) to induce osteogenic differentiation. Results are compared with those of control groups of BMP-2 carriers under the same condition. Silk fibroin-based particles with size and component variations are prepared by self-assembly, desolvation, and soft template formation to improve BMP-2 loading efficiency. Results show that the particles significantly enhance osteogenic differentiation of MSCs, which is evident in the high ALP enzyme activity as well as the increased level of expression of osteogenic genes. Specifically, the combination of calcium compound and BMP-2 in the silk fibroin-calcium carbonate complex particles synergistically enhances osteogenesis. Release tests and mathematical modeling are applied to describe BMP-2 dissolution profiles, and the release mechanism is based on diffusion and polymer chain relaxation. In summary, the particles show high efficacies of BMP-2 delivery, and introduction of the complex particle can progressively enhance osteogenesis. PMID:24224461
Shi, Pujiang; Abbah, Sunny A; Saran, Kushagra; Zhang, Yong; Li, Jun; Wong, Hee-Kit; Goh, James C H
21 CFR 878.5030 - Natural nonabsorbable silk surgical suture.
Code of Federal Regulations, 2010 CFR
...Natural nonabsorbable silk surgical suture. ...Natural nonabsorbable silk surgical suture is...composed of an organic protein called fibroin. This protein is derived from the domesticated...Natural nonabsorbable silk surgical suture is...
21 CFR 878.5030 - Natural nonabsorbable silk surgical suture.
Code of Federal Regulations, 2010 CFR
...Natural nonabsorbable silk surgical suture. ...Natural nonabsorbable silk surgical suture is...composed of an organic protein called fibroin. This protein is derived from the domesticated...Natural nonabsorbable silk surgical suture is...
NASA Astrophysics Data System (ADS)
The discovery of a novel silk production system in a marine amphipod provides insights into the wider potential of natural silks. The tube-building corophioid amphipod Crassicorophium bonellii produces from its legs fibrous, adhesive underwater threads that combine barnacle cement biology with aspects of spider silk thread extrusion spinning. We characterised the filamentous silk as a mixture of mucopolysaccharides and protein deriving from glands representing two distinct types. The carbohydrate and protein silk secretion is dominated by complex ?-sheet structures and a high content of charged amino acid residues. The filamentous secretion product exits the gland through a pore near the tip of the secretory leg after having moved through a duct, which subdivides into several small ductules all terminating in a spindle-shaped chamber. This chamber communicates with the exterior and may be considered the silk reservoir and processing/mixing space, in which the silk is mechanically and potentially chemically altered and becomes fibrous. We assert that further study of this probably independently evolved, marine arthropod silk processing and secretion system can provide not only important insights into the more complex arachnid and insect silks but also into crustacean adhesion cements.
Kronenberger, Katrin; Dicko, Cedric; Vollrath, Fritz
Physical Crosslinking Modulates Sustained Drug Release from Recombinant Silk-Elastinlike Protein Polymer for Ophthalmic Applications
We evaluated the drug release capability of optically transparent recombinant silk-elastinlike protein polymer, SELP-47K, films to sustainably deliver the common ocular antibiotic, ciprofloxacin. The ciprofloxacin release kinetics from drug-loaded SELP-47K films treated with ethanol or methanol vapor to induce different densities of physical crosslinking was investigated. Additionally, the drug-loaded protein films were embedded in a protein polymer coating to further prolong the release of the drug. Drug-loaded SELP-47K films released ciprofloxacin for up to 132 hours with near first-order release kinetics. Polymer coating of drug-loaded films prolonged drug release for up to 220 hours. The antimicrobial activity of ciprofloxacin released from the drug delivery matrices was not impaired by the film casting process or the ethanol or methanol treatments. The mechanism of drug release was elucidated by analyzing the physical properties of the film specimens, including equilibrium swelling, soluble fraction, surface roughness and hydrophobicity. Additionally, the conformation of the SELP-47K and its physical crosslinks in the films was analyzed by FTIR and Raman spectroscopy. A three-parameter physics based model accurately described the release rates observed for the various film and coating treatments and attributed the effects to the degree of physical crosslinking of the films and to an increasing affinity of the drug with the polymer network. Together, these results indicate that optically transparent silk-elastinlike protein films may be attractive material candidates for novel ophthalmic drug delivery devices.
Teng, Weibing; Cappello, Joseph; Wu, Xiaoyi
Autoclaving as a chemical-free process to stabilize recombinant silk-elastinlike protein polymer nanofibers
NASA Astrophysics Data System (ADS)
We report here that autoclaving is a chemical-free, physical crosslinking strategy capable of stabilizing electrospun recombinant silk-elastinlike protein (SELP) polymer nanofibers. Fourier transform infrared spectroscopy showed that the autoclaving of SELP nanofibers induced a conformational conversion of ?-turns and unordered structures to ordered ?-sheets. Tensile stress-strain analysis of the autoclaved SELP nanofibrous scaffolds in phosphate buffered saline at 37 �C revealed a Young's modulus of 1.02 +/- 0.28 MPa, an ultimate tensile strength of 0.34 +/- 0.04 MPa, and a strain at failure of 29% +/- 3%.
Qiu, Weiguo; Cappello, Joseph; Wu, Xiaoyi
Adenoviral Gene Delivery to Solid Tumors by Recombinant Silk�Elastinlike Protein Polymers
Purpose��The purpose of this study was to investigate the potential of silk�elastinlike protein polymers (SELPs) in controlling the\\u000a release rate of adenoviruses in vitro and in vivo while preserving their bioactivity.\\u000a \\u000a \\u000a \\u000a Materials and Methods��A hydrogel system composed of SELP\\/adenovirus mixture was prepared. The release of the adenovirus particles from the hydrogels\\u000a was quantified by Real Time-PCR and the bioactivity of
A. Hatefi; J. Cappello; H. Ghandehari
Studies on the quantitative and qualitative characters of cocoons and silk from methoprene and fenoxycarb treated Bombyx mori (L) larvae
th instar B. mori treated with selected doses of methoprene and fenoxycarb, quantitative parameters like cocoon weight, shell weight, shell percentage, filament length and denier followed by qualitative characters of the silk like non-breakable filament length, reelability, winding capacity, tenacity, elongation percentage, cohesiveness, sericin and fibroin contents were determined. The cocoon from 1.0 ? ? ? ?g methoprene and 3.0
D. M. Mamatha; H. P. P. Cohly; A. H. H. Raju; M. Rajeswara Rao
Characterization of silk fibroin modified surface: a proteomic view of cellular response proteins induced by biomaterials.
The purpose of this study was to develop the pathway of silk fibroin (SF) biopolymer surface induced cell membrane protein activation. Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer material using a mass spectrometry-based profiling system. The surface was covered by multiwalled carbon nanotubes (CNTs) and SF to increase the surface area, enhance the adhesion of biopolymer, and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNTs/SF electrodes of quartz crystal microbalance (QCM) greatly exceeded those on other surfaces. Moreover, analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approaches indicated that CD44 may be a key protein. Through this study, utilization of mass spectrometry-based proteomics in evaluation of cell adhesion on biopolymer was proposed. PMID:24818131
Yang, Ming-Hui; Yuan, Shyng-Shiou; Chung, Tze-Wen; Jong, Shiang-Bin; Lu, Chi-Yu; Tsai, Wan-Chi; Chen, Wen-Cheng; Lin, Po-Chiao; Chiang, Pei-Wen; Tyan, Yu-Chang
Characterization of Silk Fibroin Modified Surface: A Proteomic View of Cellular Response Proteins Induced by Biomaterials
The purpose of this study was to develop the pathway of silk fibroin (SF) biopolymer surface induced cell membrane protein activation. Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer material using a mass spectrometry-based profiling system. The surface was covered by multiwalled carbon nanotubes (CNTs) and SF to increase the surface area, enhance the adhesion of biopolymer, and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNTs/SF electrodes of quartz crystal microbalance (QCM) greatly exceeded those on other surfaces. Moreover, analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approaches indicated that CD44 may be a key protein. Through this study, utilization of mass spectrometry-based proteomics in evaluation of cell adhesion on biopolymer was proposed.
Yang, Ming-Hui; Yuan, Shyng-Shiou; Chung, Tze-Wen; Jong, Shiang-Bin; Lu, Chi-Yu; Tsai, Wan-Chi; Chen, Wen-Cheng; Lin, Po-Chiao; Chiang, Pei-Wen; Tyan, Yu-Chang
Identification and Characterization of Multiple Spidroin 1 Genes Encoding Major Ampullate Silk Proteins in Nephila clavipes
Spider dragline silk is primarily composed of proteins called major ampullate spidroins (MaSp) that consist of a large repeat array flanked by non-repetitive N- and C-terminal domains. Until recently, there has been little evidence for more than one gene encoding each of the two major spidroin silk proteins, MaSp1 and MaSp2. Here, we report the deduced N-terminal domain sequences for two distinct MaSp1 genes from Nephila clavipes (MaSp1A and MaSp1B) and for MaSp2. All three MaSp genes are co-expressed in the major ampullate gland. A search of the GenBank database also revealed two distinct MaSp1 C-terminal domain sequences. Sequencing confirmed that both MaSp1 genes are present in all seven Nephila clavipes spiders examined. The presence of nucleotide polymorphisms in these genes confirmed that MaSp1A and MaSp1B are distinct genetic loci and not merely alleles of the same gene. We have experimentally determined the transcription start sites for all three MaSp genes and established preliminary pairing between the two MaSp1 N- and C-terminal domains. Phylogenetic analysis of these new sequences and other published MaSp N- and C-terminal domain sequences illustrated that duplications of MaSp genes may be widespread among spider species.
Gaines, William A.; Marcotte, William R.
Conquering isoleucine auxotrophy of Escherichia coli BLR(DE3) to recombinantly produce spider silk proteins in minimal media.
Large-scale production of recombinant spider silk proteins is a long-term goal for their industrial use. Therefore, we have recently developed a process for bacterial production. Due to a highly repetitive gene sequence of spider silks, the host strain E. coli BLR(DE3) was employed since it shows no homologue recombination. Although perfectly suited for production in full media, the BLR strain does not grow in cost-effective minimal media, indicating a previously not reported L: -isoleucine auxotrophy. We provide evidence that mutated threonine deaminase is likely responsible for the detected auxotrophy of BLR. PMID:17611722
Schmidt, Martin; R�mer, Lin; Strehle, Martin; Scheibel, Thomas
Structure and applications of a temperature responsive recombinant protein hydrogel based on silk- and elastin-like amino acid motifs
NASA Astrophysics Data System (ADS)
Proteins form the main components of many natural materials, and they can be designed to offer tailored functionality and material properties. Silk elastin-like proteins (SELP)s come from a family of repeat sequence protein polymers based on Bombyx mori silk and mammalian elastin that are recombinantly expressed in E. coli. SELP gels are formed by heating the protein solutions in order to induce physical crosslinking of the silk ?-sheet regions, they contain approximately 80-90% water by weight and they can be used for encapsulation of enzymes or nanoparticles. For example, horseradish peroxidase demonstrates added resistance to drying and heat treatment when encapsulated in the gel matrix. During gel formation, small angle X-ray scattering shows intensity increases in two distinct regions of reciprocal space, one reversible with temperature and one irreversible. By fitting the scattering data to a unified power-law/Gunier model, morphological parameters are extracted. The thermally reversible intensity changes are attributed to a hydrophilic/hydrophobic transition in the elastin segments, while the irreversible intensity change is due to the crystalline regions formed by the silk blocks.
Drummy, Lawrence; Tomczak, Melanie; Macauliffe, Joseph; Vaia, Richard; Naik, Rajesh
Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells
Spider silks are protein-based ``biopolymer'' filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to ``biomimic'' the process of spider silk production by expressing in mammalian cells the dragline silk
Anthoula Lazaris; Steven Arcidiacono; Yue Huang; Jiang-Feng Zhou; Fran�ois Duguay; Nathalie Chretien; Elizabeth A. Welsh; Jason W. Soares; Costas N. Karatzas
The critical role of water in spider silk and its consequence for protein mechanics
NASA Astrophysics Data System (ADS)
Due to its remarkable mechanical and biological properties, there is considerable interest in understanding, and replicating, spider silk's stress-processing mechanisms and structure-function relationships. Here, we investigate the role of water in the nanoscale mechanics of the different regions in the spider silk fibre, and their relative contributions to stress processing. We propose that the inner core region, rich in spidroin II, retains water due to its inherent disorder, thereby providing a mechanism to dissipate energy as it breaks a sacrificial amide-water bond and gains order under strain, forming a stronger amide-amide bond. The spidroin I-rich outer core is more ordered under ambient conditions and is inherently stiffer and stronger, yet does not on its own provide high toughness. The markedly different interactions of the two proteins with water, and their distribution across the fibre, produce a stiffness differential and provide a balance between stiffness, strength and toughness under ambient conditions. Under wet conditions, this balance is destroyed as the stiff outer core material reverts to the behaviour of the inner core.
Brown, Cameron P.; MacLeod, Jennifer; Amenitsch, Heinz; Cacho-Nerin, Fernando; Gill, Harinderjit S.; Price, Andrew J.; Traversa, Enrico; Licoccia, Silvia; Rosei, Federico
Effect of degumming time on silkworm silk fibre for biodegradable polymer composites
NASA Astrophysics Data System (ADS)
Recently, many studies have been conducted on exploitation of natural materials for modern product development and bioengineering applications. Apart from plant-based materials (such as sisal, hemp, jute, bamboo and palm fibre), animal-based fibre is a kind of sustainable natural materials for making novel composites. Silkworm silk fibre extracted from cocoon has been well recognized as a promising material for bio-medical engineering applications because of its superior mechanical and bioresorbable properties. However, when producing silk fibre reinforced biodegradable/bioresorbable polymer composites, hydrophilic sericin has been found to cause poor interfacial bonding with most polymers and thus, it results in affecting the resultant properties of the composites. Besides, sericin layers on fibroin surface may also cause an adverse effect towards biocompatibility and hypersensitivity to silk for implant applications. Therefore, a proper pre-treatment should be done for sericin removal. Degumming is a surface modification process which allows a wide control of the silk fibre's properties, making the silk fibre possible to be used for the development and production of novel bio-composites with unique/specific mechanical and biodegradable properties. In this paper, a cleaner and environmentally friendly surface modification technique for tussah silk in polymer based composites is proposed. The effectiveness of different degumming parameters including degumming time and temperature on tussah silk is discussed through the analyses of their mechanical and morphological properties. Based on results obtained, it was found that the mechanical properties of tussah silk are affected by the degumming time due to the change of the fibre structure and fibroin alignment.
Ho, Mei-po; Wang, Hao; Lau, Kin-tak
The elaborate structure of spider silk
Biomaterials, having evolved over millions of years, often exceed man-made materials in their properties. Spider silk is one outstanding fibrous biomaterial which consists almost entirely of large proteins. Silk fibers have tensile strengths comparable to steel and some silks are nearly as elastic as rubber on a weight to weight basis. In combining these two properties, silks reveal a toughness that is two to three times that of synthetic fibers like Nylon or Kevlar. Spider silk is also antimicrobial, hypoallergenic and completely biodegradable. This article focuses on the structure-function relationship of the characterized highly repetitive spider silk spidroins and their conformational conversion from solution into fibers. Such knowedge is of crucial importance to understanding the intrinsic properties of spider silk and to get insight into the sophisticated assembly processes of silk proteins. This review further outlines recent progress in recombinant production of spider silk proteins and their assembly into distinct polymer materials as a basis for novel products.
Romer, Lin
Hydrogen bonding-assisted thermal conduction in ?-sheet crystals of spider silk protein.
Using atomistic simulations, we demonstrate that ?-sheet, an essential component of spider silk protein, has a thermal conductivity 1-2 orders of magnitude higher than that of some other protein structures reported in the literature. In contrast to several other nanostructured materials of similar bundled/layered structures (e.g. few-layer graphene and bundled carbon nanotubes), the ?-sheet is found to uniquely feature enhanced thermal conductivity with an increased number of constituting units, i.e. ?-strands. Phonon analysis identifies inter-?-strand hydrogen bonding as the main contributor to the intriguing phenomenon, which prominently influences the state of phonons in both low- and high-frequency regimes. A thermal resistance model further verifies the critical role of hydrogen bonding in thermal conduction through ?-sheet structures. PMID:24811747
Zhang, Lin; Chen, Teli; Ban, Heng; Liu, Ling
Shear-induced self-assembly of native silk proteins into fibrils studied by atomic force microscopy.
Noncontact mode atomic force microscopy was used to investigate native silk proteins prepared in different ways. Low protein concentrations revealed that single protein molecules exhibit a simple, round shape with apparent diameters of 20-25 nm. Shearing the native protein solutions after extraction from the gland and prior to drying led to a beads-on-a-string assembly at the nanometer scale. Protein concentration had a significant effect on the morphology of the protein assemblies. At higher protein concentrations, shear-induced alignment into nanofibrils was observed, while lower concentrations lead to the formation of much thinner fibrils with a width of about 8 nm. PMID:22352290
Greving, Imke; Cai, Minzhen; Vollrath, Fritz; Schniepp, Hannes C
Genetically programmable thermoresponsive plasmonic gold/silk-elastin protein core/shell nanoparticles.
The design and development of future molecular photonic/electronic systems pose the challenge of integrating functional molecular building blocks in a controlled, tunable, and reproducible manner. The modular nature and fidelity of the biosynthesis method provides a unique chemistry approach to one-pot synthesis of environmental factor-responsive chimeric proteins capable of energy conversion between the desired forms. In this work, facile tuning of dynamic thermal response in plasmonic nanoparticles was facilitated by genetic engineering of the structure, size, and self-assembly of the shell silk-elastin-like protein polymers (SELPs). Recombinant DNA techniques were implemented to synthesize a new family of SELPs, S4E8Gs, with amino acid repeats of [(GVGVP)4(GGGVP)(GVGVP)3(GAGAGS)4] and tunable molecular weight. The temperature-reversible conformational switching between the hydrophilic random coils and the hydrophobic ?-turns in the elastin blocks were programmed to between 50 and 60 �C by site-specific glycine mutation, as confirmed by variable-temperature proton NMR and circular dichroism (CD) spectroscopy, to trigger the nanoparticle aggregation. The dynamic self-aggregation/disaggregation of the Au-SELPs nanoparticles was regulated in size and pattern by the ?-sheet-forming, thermally stable silk blocks, as revealed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The thermally reversible, shell dimension dependent, interparticle plasmon coupling was investigated by both variable-temperature UV-vis spectroscopy and finite-difference time-domain (FDTD)-based simulations. Good agreement between the calculated and measured spectra sheds light on design and synthesis of responsive plasmonic nanostructures by independently tuning the refractive index and size of the SELPs through genetic engineering. PMID:24712906
Lin, Yinan; Xia, Xiaoxia; Wang, Ming; Wang, Qianrui; An, Bo; Tao, Hu; Xu, Qiaobing; Omenetto, Fiorenzo; Kaplan, David L
NASA Astrophysics Data System (ADS)
Periodic nanostructures provide the facility to control and manipulate the flow of light through their lattices. Three-dimensional photonic crystals enable the controlled design of structural colour, which can be varied by infiltrating the structure with different (typically liquid) fillers. Here, we report three-dimensional photonic crystals composed entirely of a purified natural protein (silk fibroin). The biocompatibility of this protein, as well as its favourable material properties and ease of biological functionalization, present opportunities for otherwise unattainable device applications such as bioresorbable integration of structural colour within living tissue or lattice functionalization by means of organic and inorganic material doping. We present a silk inverse opal that demonstrates a pseudo-photonic bandgap in the visible spectrum and show its associated structural colour beneath biological tissue. We also leverage silk's facile dopability to manufacture a gold nanoparticle silk inverse opal and demonstrate patterned heating mediated by enhancement of nanoparticle absorption at the band-edge frequency of the photonic crystal.
Kim, Sunghwan; Mitropoulos, Alexander N.; Spitzberg, Joshua D.; Tao, Hu; Kaplan, David L.; Omenetto, Fiorenzo G.
NSDL National Science Digital Library
Spider silk fibers are stronger than almost any other man-made fiber and they're also elastic. There are a lot of potential applications for the fiber, but how do you come up with enough raw material? With help from the National Science Foundation (NSF), researchers have figured out a way to put the spider's silk-making genes into goats in a way that they only make the protein in their milk.
A silk hydrogel-based delivery system of bone morphogenetic protein for the treatment of large bone defects.
The use of tissue grafting for the repair of large bone defects has numerous limitations including donor site morbidity and the risk of disease transmission. These limitations have prompted research efforts to investigate the effects of combining biomaterial scaffolds with biochemical cues to augment bone repair. The goal of this study was to use a critically-sized rat femoral segmental defect model to investigate the efficacy of a delivery system consisting of an electrospun polycaprolactone (PCL) nanofiber mesh tube with a silk fibroin hydrogel for local recombinant bone morphogenetic protein 2 (BMP-2) delivery. Bilateral 8 mm segmental femoral defects were formed in 13-week-old Sprague Dawley rats. Perforated electrospun PCL nanofiber mesh tubes were fitted into the adjacent native bone such that the lumen of the tubes contained the defect (Kolambkar et al., 2011b). Silk hydrogels with or without BMP-2 were injected into the defect. Bone regeneration was longitudinally assessed using 2D X-ray radiography and 3D microcomputed topography (?CT). Following sacrifice at 12 weeks after surgery, the extracted femurs were either subjected to biomechanical testing or assigned for histology. The results demonstrated that silk was an effective carrier for BMP-2. Compared to the delivery system without BMP-2, the delivery system that contained BMP-2 resulted in more bone formation (p<0.05) at 4, 8, 12 weeks after surgery. Biomechanical properties were also significantly improved in the presence of BMP-2 (p<0.05) and were comparable to age-matched intact femurs. Histological evaluation of the defect region indicated that the silk hydrogel has been completely degraded by the end of the study. Based on these results, we conclude that a BMP-2 delivery system consisting of an electrospun PCL nanofiber mesh tube with a silk hydrogel presents an effective strategy for functional repair of large bone defects. PMID:22658161
Diab, Tamim; Pritchard, Eleanor M; Uhrig, Brent A; Boerckel, Joel D; Kaplan, David L; Guldberg, Robert E
Transcript Profile Analyses of Maize Silks Reveal Effective Activation of Genes Involved in Microtubule-Based Movement, Ubiquitin-Dependent Protein Degradation, and Transport in the Pollination Process
Pollination is the first crucial step of sexual reproduction in flowering plants, and it requires communication and coordination between the pollen and the stigma. Maize (Zea mays) is a model monocot with extraordinarily long silks, and a fully sequenced genome, but little is known about the mechanism of its pollen�stigma interactions. In this study, the dynamic gene expression of silks at four different stages before and after pollination was analyzed. The expression profiles of immature silks (IMS), mature silks (MS), and silks at 20 minutes and 3 hours after pollination (20MAP and 3HAP, respectively) were compared. In total, we identified 6,337 differentially expressed genes in silks (SDEG) at the four stages. Among them, the expression of 172 genes were induced upon pollination, most of which participated in RNA binding, processing and transcription, signal transduction, and lipid metabolism processes. Genes in the SDEG dataset could be divided into 12 time-course clusters according to their expression patterns. Gene Ontology (GO) enrichment analysis revealed that many genes involved in microtubule-based movement, ubiquitin-mediated protein degradation, and transport were predominantly expressed at specific stages, indicating that they might play important roles in the pollination process of maize. These results add to current knowledge about the pollination process of grasses and provide a foundation for future studies on key genes involved in the pollen�silk interaction in maize.
Chen, Hao; Sun, Wei; Zhang, Xian Sheng
Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties
The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.
Teule, Florence; Miao, Yun-Gen; Sohn, Bong-Hee; Kim, Young-Soo; Hull, J. Joe; Fraser, Malcolm J.; Lewis, Randolph V.; Jarvis, Donald L.
Expression of a truncated ATHB17 protein in maize increases ear weight at silking.
ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the ?-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17?113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17?113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17?113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17?113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17?113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17?113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658
Rice, Elena A; Khandelwal, Abha; Creelman, Robert A; Griffith, Cara; Ahrens, Jeffrey E; Taylor, J Philip; Murphy, Lesley R; Manjunath, Siva; Thompson, Rebecca L; Lingard, Matthew J; Back, Stephanie L; Larue, Huachun; Brayton, Bonnie R; Burek, Amanda J; Tiwari, Shiv; Adam, Luc; Morrell, James A; Caldo, Rico A; Huai, Qing; Kouadio, Jean-Louis K; Kuehn, Rosemarie; Sant, Anagha M; Wingbermuehle, William J; Sala, Rodrigo; Foster, Matt; Kinser, Josh D; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E; Huang, Mingya G; Kuriakose, Saritha V; Skottke, Kyle; Repetti, Peter P; Reuber, T Lynne; Ruff, Thomas G; Petracek, Marie E; Loida, Paul J
Mechanical improvements to reinforced porous silk scaffolds.
Load-bearing porous biodegradable scaffolds are required to engineer functional tissues such as bone. Mechanical improvements to porogen leached scaffolds prepared from silk proteins were systematically studied through the addition of silk particles in combination with silk solution concentration, exploiting interfacial compatibility between the two components. Solvent solutions of silk up to 32 w/v % were successfully prepared in hexafluoroisopropanol (HFIP) for the study. The mechanical properties of the reinforced silk scaffolds correlated to the material density and matched by a power law relationship, independent of the ratio of silk particles to matrix. These results were similar to the relationships previously shown for cancellous bone. From these data we conclude that the increased mechanical properties were due to a densification effect and not due to the inclusion of stiffer silk particles into the softer silk matrix. A continuous interface between the silk matrix and the silk particles, as well as homogeneous distribution of the silk particles within the matrix was observed. Furthermore, we note that the roughness of the pore walls was controllable by varying the ratio of the particles matrix, providing a route to control topography. The rate of proteolytic hydrolysis of the scaffolds decreased with increase in mass of silk used in the matrix and with increasing silk particle content. PMID:21793193
Gil, Eun Seok; Kluge, Jonathan A; Rockwood, Danielle N; Rajkhowa, Rangam; Wang, Lijing; Wang, Xungai; Kaplan, David L
Mechanical Improvements to Reinforced Porous Silk Scaffolds
Load bearing porous biodegradable scaffolds are required to engineer functional tissues such as bone. Mechanical improvements to porogen leached scaffolds prepared from silk proteins were systematically studied through the addition of silk particles in combination with silk solution concentration, exploiting interfacial compatibility between the two components. Solvent solutions of silk up to 32 w/v% were successfully prepared in hexafluoroisopropanaol (HFIP) for the study. The mechanical properties of the reinforced silk scaffolds correlated to the material density and matched by a power law relationship, independent of the ratio of silk particles to matrix. These results were similar to the relationships previously shown for cancellous bone. The mechanism behind the increased mechanical properties was a densification effect, and not the effect of including stiffer silk particles into the softer silk continuous matrix. A continuous interface between the silk matrix and the silk particles, as well as homogeneous distribution of the silk particles within the matrix were observed. Furthermore, we note that the roughness of the pore walls was controllable by varying the ratio of particles matrix, providing a route to control topography. The rate of proteolytic hydrolysis of the scaffolds decreased with increase in mass of silk used in the matrix and with increasing silk particle content.
Gil, Eun Seok; Kluge, Jonathan A.; Rockwood, Danielle N.; Rajkhowa, Rangam; Wang, Lijing; Wang, Xungai; Kaplan, David L
Characterization of iron oxide nanoparticles in structural silk-elastinlike protein polymer
NASA Astrophysics Data System (ADS)
The structure of silk elastin-like protein (SELP) block copolymers containing Fe3O4 magnetic nanoparticles are investigated. These materials have potential applications for hyperthermia cancer therapy. SELPs undergo a gel transition at physiological temperatures, which can be used to localize delivery of nanoparticles at tumor sites. Vibrating sample magnetometry (VSM), transmission electron microscopy (TEM), and small angle neutron scattering (SANS) are used to characterize the nanoparticles and the SELP-nanoparticle nanocomposite system. A series of nanoparticles with three different nominal diameters, 30, 50 and 80 nm, were added to 4 and 8 wt.% SELP samples. Different functionalities on the nanoparticle surface affect their interactions with SELP. The 50 nm nanoparticles in SELP exhibit chaining (linear association of the nanoparticles), while the 30 nm nanoparticles are too small and settle out of the polymer mesh and the 80 nm nanoparticles tend to cluster without any regard for SELP structure. The SELP concentration does not have a major affect on nanoparticle behavior in the nanocomposites.
Shih, Jennifer
High Level Biosynthesis of a Silk-Elastin-like Protein in E. coli.
Silk-elastin-like proteins (SELPs) have enormous potential for use as customizable biomaterials in numerous biomedical and materials applications, yet success in harnessing this potential has been limited by the lack of a commercially viable industrially relevant production process. We have developed a scalable fed-batch production approach which enables a SELP volumetric productivity of 4.3 g L(-1) with E. coli BL21(DE3). This is the highest SELP productivity reported to date and is 50-fold higher than that reported by other groups. As compared to typical fed-batch processes, high preinduction growth rates and low inducer and oxygen concentrations are allowed whereas reduced postinduction feeding rates are preferred. Limiting factors were identified and productivity was found to be strongly influenced by a trade-off between the rate of production and plasmid stability. The process developed is robust, reproducible, and applicable to scale up to the industrial level and moves these biopolymers a step closer to the marketplace. PMID:24884240
Collins, Tony; Barroca, M�rio; Branca, Fernando; Padr�o, Jorge; Machado, Raul; Casal, Margarida
Hydrophobic and Hofmeister effects on the adhesion of spider silk proteins onto solid substrates: an AFM-based single-molecule study.
AFM-based single-molecule force spectroscopy has been used to study the effect of Hofmeister salts and protein hydrophobicity on the adhesion of recombinant spider silk proteins onto solid substrates. Therefore, a molecular probe consisting of a spider silk protein and an AFM tip has been developed, which (i) is a well-defined, small system that can be simulated by molecular dynamics simulations, (ii) allows access to the whole soluble concentration range for ions, and (iii) provides the distribution of desorption forces rather than just ensemble-averaged mean values. The measured desorption forces follow the Hofmeister series for anions (H2PO4-, Cl-, I-) with a stabilizing energy of more than 15 kBT for 5 M NaH2PO4. Moreover, this effect is influenced by the hydrophobicity of the spider silk protein, indicating that hydrophobic and Hofmeister effects are closely related. PMID:18041854
Geisler, Michael; Pirzer, Tobias; Ackerschott, Christian; Lud, Simon; Garrido, Jose; Scheibel, Thomas; Hugel, Thorsten
Effect of sodium chloride on the structure and stability of spider silk's N-terminal protein domain
A spider�s ability to store silk protein solutions at high concentration is believed to be related to the protein�s terminal domains. It has been suggested that a shift in salt concentration and pH can have a significant influence on the assembly process. Based on experimental data, a model has been proposed in which the N-terminal domain exists as a monomer during storage and assembles into a homodimer upon spinning. Here we perform a systematic computational study using atomistic, coarse-grained and well-tempered metadynamics simulation to understand how the NaCl concentration in the solution affects the N-terminal domain of the silk protein. Our results show that a high salt concentration, as found during storage, weakens key salt bridges between the monomers, inducing a loss in bond energy by 28.6% in a single salt bridge. As a result dimer formation is less likely as 35.5% less energy is required to unfold the dimer by mechanical force. Conversely, homodimer formation appears to be more likely at low salt concentrations as the salt bridge stays at the lower energy state. The link between salt concentration, structure and stability of the N-terminal domain provides a possible mechanism that prevents premature fiber formation during storage.
Gronau, Greta; Qin, Zhao; Buehler, Markus J.
Silk fibroin protein from mulberry and non-mulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion.
Silks fibers and films fabricated from fibroin protein of domesticated mulberry silkworm cocoon have been traditionally utilized as sutures in surgery and recently as biomaterial films respectively. Here, we explore the possibility of application of silk fibroin protein from non-mulberry silkworm cocoon as a potential biomaterial aid. In terms of direct inflammatory potential, fibroin proteins from Antheraea mylitta and Bombyx mori are immunologically inert and invoke minimal immune response. Stimulation of murine peritoneal macrophages and RAW 264.7 murine macrophages by these fibroin proteins both in solution and in the form of films assayed in terms of nitric oxide and TNFalpha production showed comparable stimulation as in collagen. Kinetics of adhesion of L929 murine fibroblasts, for biocompatibility evaluation, monitored every 4 h from seeding and studied over a period of 24 h, reveal A. mylitta fibroin film to be a better substrate in terms of rapid and easier cellularization. Cell viability studies by MTT assay and flow cytometric analyses indicate the ability of fibroin matrices to support cell growth and proliferation comparable to collagen for long-term culture. This matrix may have potential to serve in those injuries where rapid cellularization is essential. PMID:18322779
Acharya, Chitrangada; Ghosh, Sudip K; Kundu, S C
Electrostatic charges instigate 'concertina-like' mechanisms of molecular toughening in MaSp1 (spider silk) proteins.
According to a recent article authored by Ortega-Jimenez and Dudley [1], the capture success of spiders is in part due to electrostatic charges on the surfaces of insects that macroscopically deform the spider web and increase the chances of insect-web contact. In this brief communication, we further show that electrostatic charges instigate a molecular 'concertina-like' mechanism of deformation in MaSp1 protein, which effectively begins the toughening-up of dragline silk threads prior to insect-web contact. PMID:24907767
Pahlevan, Mahdi; Toivakka, Martti; Alam, Parvez
Sericins exhibit ROS-scavenging, anti-tyrosinase, anti-elastase, and in vitro immunomodulatory activities.
Some biological properties of Bombyx mori sericins from twenty strains were investigated, fourteen fed with artificial diet, two with fresh mulberry leaves and four with both diets. Sericin exhibited ROS-scavenging, anti-tyrosinase and anti-elastase properties, the strain significantly influenced these properties, while diet only influenced the anti-tyrosinase activity. Sericins were clustered into 5 groups and one sericin from each group was further studied: sericins showed anti-proliferative activity on in vitro stimulated peripheral blood mononuclear cells; some strains decreased in vitro secretion of IFN?, while no effects were observed on TNF? and IL10 release. Therefore, a mixture of sericins extracted from the most promising strains may be useful for dermatological and cosmetic use. PMID:23541552
Chlapanidas, Theodora; Farag�, Silvio; Lucconi, Giulia; Perteghella, Sara; Galuzzi, Marta; Mantelli, Melissa; Avanzini, Maria Antonietta; Tosca, Marta Cecilia; Marazzi, Mario; Vigo, Daniele; Torre, Maria Luisa; Faustini, Massimo
An independently evolved Dipteran silk with features common to Lepidopteran silks.
Male hilarine flies (Diptera: Empididae: Empidinae) present prospective mates with silk-wrapped gifts. The silk is produced by specialised cells located in the foreleg basitarsus of the fly. In this report, we describe 2.3 kbp of the silk gene from a hilarine fly (Hilara spp.) that was identified from highly expressed mRNA extracted from the prothoracic basitarsus of males. Using specific primers, we found that the silk gene is expressed in the basitarsi and not in any other part of the male fly. The silk gene from the basitarsi cDNA library matched an approximately 220 kDa protein from the silk-producing basitarsus. Although the predicted silk protein sequence was unlike any other protein sequence in available databases, the architecture and composition of the predicted protein had features in common with previously described silks. The convergent evolution of these features in the Hilarini silk and other silks emphasises their importance in the functional requirements of silk proteins. PMID:17785191
Sutherland, Tara D; Young, James H; Sriskantha, Alagacone; Weisman, Sarah; Okada, Shoko; Haritos, Victoria S
Identification of four small molecular mass proteins in the silk of Bombyx mori
This paper describes cDNAs of four small-size pro- teins that occur in the cocoon silk of Bombyx mori . Two of them (9.9 and 10.3 kDa), which have the N-terminal sequences and the spacing of a few amino acids at C- termini similar to the seroin of Galleria mellonella , are called seroin 1 and seroin 2. The corresponding genes
X. Nirmala; K. Mita; V. Vanisree; M. �urovec; F. Sehnal
Silk-elastinlike protein polymers improve the efficacy of adenovirus thymidine kinase enzyme prodrug therapy of head and neck tumors
Background Adenoviral directed enzyme prodrug therapy is a promising approach for head and neck cancer gene therapy. Challenges with this approach however are transient gene expression and dissemination of viruses to distant organs. Methods We used recombinant silk-elastinlike protein copolymer (SELP) matrices for intratumoral delivery of adenoviruses containing both thymidine kinase-1, and luciferase genes in a nude mice model of JHU-022 head and neck tumor. Hydrogels made from two SELP analogues (47K and 815K) with similar silk to elastinlike block ratios but different block lengths were studied for intratumoral viral delivery. Tumor bearing mice were followed up for tumor progression and luciferase gene expression concomitantly for five weeks. Polymer�s safety was evaluated through body weight change, blood count, liver and kidney functions in addition to gross and microscopic histological examination. Results SELP 815K analogues efficiently controlled the duration and extent of transfection in tumors for up to 5 weeks with no detectable spread to the liver. About five-fold greater reduction in tumor volume was obtained with matrix-mediated delivery compared to intra-tumoral injection of adenoviruses in saline. SELP matrix proved safe in all injected mice compared to control group. Conclusion SELP- controlled gene delivery approach could potentially improve the anticancer activity of virus-mediated gene therapy while limiting viral spread to normal organs.
Greish, Khaled; Frandsen, Jordan; Scharff, Stephanie; Gustafson, Joshua; Cappello, Joseph; Li, Daqing; O'Malley, Bert W.; Ghandehari, Hamidreza
Composition and Hierarchical Organisation of a Spider Silk
Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material.
Sponner, Alexander; Vater, Wolfram; Monajembashi, Shamci; Unger, Eberhard; Grosse, Frank; Weisshart, Klaus
Prediction of the structure of a silk-like protein in oligomeric states using explicit and implicit solvent models.
We perform Replica Exchange Molecular Dynamics (REMD) simulations on a silk-like protein design with amino-acid sequence [(Gly-Ala)3-Gly-Glu]5 to investigate the stability of a single protein, a dimer, a trimer and a tetramer made up of these proteins starting from ?-roll and ?-sheet structures in both explicit (TIP3P) and implicit (GBSA) solvent models. Our simulation results for the implicit solvent model agree with those for the explicit solvent model for simulation times up to the longest tested, being 30 ns per replica. From this we infer that the implicit solvent model that we use is reliable, allowing us to reach much longer time scales (up to 200 ns per replica). We find that the self-assembly of fibers of these proteins in solution must be a nucleated process, involving nuclei made up of at least three monomers. We also find that the conformation of the protein changes upon assembly, i.e., there is a transition from a disordered globular state to an ordered ?-sheet structure in the self-assembled state of aggregates containing more than two monomers. This indicates that autosteric effects must be important in the polymerization of this protein, reminiscent of what is observed for ?-amyloids. Our findings are consistent with recent experimental results on a protein with an amino acid sequence similar to that of the protein we study. PMID:24937549
Razzokov, Jamoliddin; Naderi, Saber; van der Schoot, Paul
Microdissection of black widow spider silk-producing glands.
Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk], tubuliform [synthesizes egg case silk], flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] and pyriform [produces attachment disc silk]. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments. PMID:21248709
Jeffery, Felicia; La Mattina, Coby; Tuton-Blasingame, Tiffany; Hsia, Yang; Gnesa, Eric; Zhao, Liang; Franz, Andreas; Vierra, Craig
PLGA nanofiber-coated silk microfibrous scaffold for connective tissue engineering.
A modified degumming technique, involving boiling in 0.25% Na2CO3 with addition of 1% sodium dodecyl sulphate and intermittent ultrasonic agitation, was developed for knitted silk scaffolds. Sericin was efficiently removed, while mechanical and structural properties of native silk fibroin were preserved. Biocompatible and mechanically robust hybrid nano-microscaffolds were fabricated by coating these degummed silk scaffolds with an intervening adhesive layer of silk solution followed by electrospun poly-lactic-co-glycolic acid (PLGA) nanofibers. Cell proliferation on the hybrid silk scaffolds was improved by seeding cells on both surfaces of the flat scaffolds. Rolling up and continued culture of the cell-seeded hybrid scaffolds yielded cylindrical constructs that permitted cell proliferation, extracellular matrix deposition, and generated ligament/tendon graft analogs. Although PLGA-based hybrid scaffolds have earlier been proposed for dense connective tissue engineering, rapid biodegradation of PLGA was a drawback. In contrast, the underlying strong and slowly-degrading microfibrous silk scaffold used in this study ensured that the hybrid scaffold maintained adequate mechanical properties for longer periods, which is vital for continued support to the injured ligament/tendon throughout its healing period. PMID:20665681
Sahoo, Sambit; Toh, Siew Lok; Goh, James Cho Hong
ERIC Educational Resources Information Center
Compares the attributes of the silk from spiders with those of the commercially harvested silk from silkworms. Discusses the evolution, design, and effectiveness of spider webs; the functional mechanics of the varieties of silk that can be produced by the same spider; and the composite, as well as molecular, structure of spider silk thread. (JJK)
Vollrath, Fritz
Pulsed laser deposition of silk protein: Effect of photosensitized-ablation on the secondary structure in thin deposited films
Silk fibroin is a simple protein expected to have functional applications in medicine and bioelectronics. The primary structure of this protein is quite simple, and the main secondary structures are {beta}-sheet crystals and amorphous random coils. In the present study, we investigated pulsed laser deposition (PLD) of fibroin with the {beta}-sheet structures as targets. The primary and secondary structures in films deposited were analyzed using infrared spectroscopy. Normal laser deposition at 351 nm using neat fibroin targets produced thin films of fibroin with a random coiled structure. Ablation was triggered by two-photonic excitation of the peptide chains, which resulted in the destruction of {beta}-sheet structure in PLD. In order to avoid the two-photonic excitation, we adopted a PLD method utilizing anthracene (5{endash}0.1 wt%) in a photosensitized reaction involving doped fibroin targets. Laser light (351 or 355 nm) was absorbed only by anthracene, which plays an important role converting photon energy to thermal energy with great ablation efficiency. Thin fibroin films deposited by this method had both random coil and {beta}-sheet structures. As the dopant concentration and laser fluence decreased, the ratio of {beta}-sheet domain to random coil increased in thin deposited films. {copyright} 2001 American Institute of Physics.
Tsuboi, Yasuyuki; Goto, Masaharu; Itaya, Akira
Transparent and flexible resistive switching memory devices with a very high ON/OFF ratio using gold nanoparticles embedded in a silk protein matrix
NASA Astrophysics Data System (ADS)
The growing demand for biomaterials for electrical and optical devices is motivated by the need to make building blocks for the next generation of printable bio-electronic devices. In this study, transparent and flexible resistive memory devices with a very high ON/OFF ratio incorporating gold nanoparticles into the Bombyx mori silk protein fibroin biopolymer are demonstrated. The novel electronic memory effect is based on filamentary switching, which leads to the occurrence of bistable states with an ON/OFF ratio larger than six orders of magnitude. The mechanism of this process is attributed to the formation of conductive filaments through silk fibroin and gold nanoparticles in the nanocomposite. The proposed hybrid bio-inorganic devices show promise for use in future flexible and transparent nanoelectronic systems.
Gogurla, Narendar; Mondal, Suvra P.; Sinha, Arun K.; Katiyar, Ajit K.; Banerjee, Writam; Kundu, Subhas C.; Ray, Samit K.
Modeling the Strength of ?-sheet Structures in Silk Crystals and Protein Molecules
NASA Astrophysics Data System (ADS)
The mechanical response of ?-sheet structures to a tensile force directed along the axis of one chain can be modeled as an array of elastic springs. The 3-D potential of H-bonds in ?-sheets gives a shear stiffness of 4.5Nm-1 and the chain repeat stiffness is 60Nm-1. Nanocrystals >3.5nm long with >=20 H-bonds/chain are the strong component of spider silk. They behave much like macro-scale objects, and two conditions must be met for pull-out failure: (1) the load on the most stressed H-bond exceeds the bond strength. (2) the energy of the system is lower after failure. (1) is the critical condition, and the predicted pull-out load is 3-4 times the H-bond strength. An energetically favorable `stick-slip' process is kinetically forbidden. Arrays within a single molecule such as titin have fewer bonds and can fail at low loads by the `stick-slip' process. The logarithmic rate dependence of failure load observed in AFM is 50pN/decade and the stick-slip prediction is 30pN/decade. Simulations at short times and high loads give slopes >10x higher, matching the prediction for failure at a single bond.
Grubb, David
Design, expression and solid-state NMR characterization of silk-like materials constructed from sequences of spider silk, Samia cynthia ricini and Bombyx mori silk fibroins.
Silk has a long history of use in medicine as sutures. To address the requirements of a mechanically robust and biocompatible material, basic research to clarify the role of repeated sequences in silk fibroin in its structures and properties seems important as well as the development of a processing technique suitable for the preparation of fibers with excellent mechanical properties. In this study, three silk-like protein analogs were constructed from two regions selected from among the crystalline region of Bombyx mori silk fibroin, (GAGSGA)(2), the crystalline region of Samia cynthia ricini silk fibroin, (Ala)(12), the crystalline region of spider dragline silk fibroin, (Ala)(6), and the Gly-rich region of spider silk fibroin, (GGA)(4). The silk-like protein analog constructed from the crystalline regions of the spider dragline silk and B. mori silk fibroins, (A(6)SCS)(8), that constructed from the crystalline regions of the S. c.ricini and B. mori silk fibroins, (A(12)SGS)(4), that constructed from and the crystalline region of S. c.ricini silk fibroin and the glycine-rich region of spider dragline silk fibroin, (A(12)SGS)(4),were expressed their molecular weights being about 36.0 kDa, 17.0 kDa and 17.5 kDa, respectively in E. coli by means of genetic engineering technologies. (A(12)SCS)(4) and (A(12)SGS)(4 )undergo a structural transition from alpha-helix to beta-sheet on a change in the solvent treatment from trifluoroacetic acid (TFA) to formic acid (FA). However, (A(6)SCS)(8) takes on the beta-sheet structure predominantly on TFA treatment and FA treatment. Structural analysis was performed on model peptides selected from spider dragline and S. c.ricini silks by means of (13)C CP/MAS NMR. PMID:16002994
Yang, Mingying; Asakura, Tetsuo
Electrospun chitosan/sericin composite nanofibers with antibacterial property as potential wound dressings.
Chitosan and sericin are natural and low cost biomaterials. Both biomaterials displayed good compatibility to human tissues and antibacterial properties for biomedical application. In this study, we have successfully fabricated chitosan/sericin composite nanofibers by electrospinning. The obtained composite nanofibers were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectrometer (FT-IR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA) studies. The composite nanofibers had good morphology with diameter between 240nm and 380nm. In vitro methyl thiazolyl tetrazolium (MTT) assays demonstrated that the chitosan/sericin composite nanofibers were biocompatible and could promote the cell proliferation. Furthermore, the composite nanofibers showed good bactericidal activity against both of Gram-positive and Gram-negative bacteria. Thus, the chitosan/sericin composite nanofibers are promising for wound dressing applications. PMID:24769088
Zhao, Rui; Li, Xiang; Sun, Bolun; Zhang, Ying; Zhang, Dawei; Tang, Zhaohui; Chen, Xuesi; Wang, Ce
Hypoxia and Amino Acid Supplementation Synergistically Promote the Osteogenesis of Human Mesenchymal Stem Cells on Silk Protein Scaffolds
Tailoring tissue engineering strategies to match patient- and tissue-specific bone regeneration needs offers to improve clinical outcomes. As a step toward this goal, osteogenic outcomes and metabolic parameters were assessed when varying inputs into the bone formation process. Silk protein scaffolds seeded with human mesenchymal stem cells in osteogenic differentiation media were used to study in vitro osteogenesis under varied conditions of amino acid (lysine and proline) concentration and oxygen level. The cells were assessed to probe how the microenvironment impacted metabolic pathways and thus osteogenesis. The most favorable osteogenesis outcomes were found in the presence of low (5%) oxygen combined with high lysine and proline concentrations during in vitro cultivation. This same set of culture conditions also showed the highest glucose consumption, lactate synthesis, and certain amino acid consumption rates. On the basis of these results and known pathways, a holistic metabolic model was derived which shows that lysine and proline supplements as well as low (5%) oxygen levels regulate collagen matrix synthesis and thereby rates of osteogenesis. This study establishes early steps toward a foundation for patient- and tissue-specific matches between metabolism, repair site, and tissue engineering approaches toward optimized bone regeneration.
Sengupta, Sejuti; Park, Sang-Hyug; Patel, Atur; Carn, Julia; Lee, Kyongbum
Silk fibroin rods for sustained delivery of breast cancer therapeutics.
A silk-protein based reservoir rod was developed for zero-order and long-term sustained drug delivery applications. Silk reservoir rod formulations were processed in three steps. First, a regenerated silk fibroin solution, rich in random-coil content was transformed into a tubular silk film with controllable dimensions, uniform film morphology and a structure rich in silk II, ?-sheet content via "film-spinning." Second, the drug powder was loaded into swollen silk tubes followed by tube end clamping. Last, clamped silk tube ends were sealed completely via dip coating. Anastrozole, an FDA approved active ingredient for the treatment of breast cancer, was used as a model drug to investigate viability of the silk reservoir rod technology for sustained drug delivery. The in�vitro and in�vivo pharmacokinetic data (in a female Sprague-Dawley rat model) analyzed via liquid chromatography-tandem mass spectroscopy indicated zero-order release for 91 days. Both in�vitro and in�vivo anastrozole release rates could be controlled simply by varying silk rod dimensions. The swelling behavior of silk films and zero-order anastrozole release kinetics indicated practically immediate film hydration and formation of a linear anastrozole concentration gradient along the silk film thickness. The dependence of anastrozole release rate on the overall silk rod dimensions was in good agreement with an essentially diffusion-controlled sustained release from a reservoir cylindrical geometry. In�vivo results highlighted a strong in�vitro-in�vivo pharmacokinetic correlation and a desirable biocompatibility profile of silk reservoir rods. During a 6-month implantation in rats, the apparent silk molecular weight values decreased gradually, while rod dry mass and ?-sheet crystal content values remained essentially constant, providing a suitable timeframe for controlled, long-term sustained delivery applications. Overall, the silk reservoir rod may be a viable candidate for sustained delivery of breast cancer therapeutics. PMID:25009069
Yucel, Tuna; Lovett, Michael L; Giangregorio, Raffaella; Coonahan, Erin; Kaplan, David L
Production of Bombyx mori Silk Fibroin Incorporated with Unnatural Amino Acids.
Silk fibroin incorporated with unnatural amino acids was produced by in vivo feeding of p-chloro-, p-bromo-, and p-azido-substituted analogues of l-phenylalanine (Phe) to transgenic silkworms (Bombyx mori) that expressed a mutant of phenylalanyl-tRNA synthetase with expanded substrate recognition capabilities in silk glands. Cutting down the content of Phe in the diet was effective for increasing the incorporation of Phe analogues but simultaneously caused a decrease of fibroin production. The azide groups incorporated in fibroin were active as chemical handles for click chemistry in both the solubilized and the solid (fibrous) states. The azides survived degumming in the boiling alkaline solution that is required for complete removal of the sericin layer, demonstrating that AzPhe-incorporated silk fibroin could be a versatile platform to produce "clickable" silk materials in various forms. This study indicates the huge potential of UAA mutagenesis as a novel methodology to alter the characteristics of B. mori silk. PMID:24884258
Teramoto, Hidetoshi; Kojima, Katsura
Controlling silk fibroin particle features for drug delivery
Silk proteins are a promising material for drug delivery due to their aqueous processability, biocompatibility, and biodegradability. A simple aqueous preparation method for silk fibroin particles with controllable size, secondary structure and zeta potential is reported. The particles were produced by salting out a silk fibroin solution with potassium phosphate. The effect of ionic strength and pH of potassium phosphate solution on the yield and morphology of the particles was determined. Secondary structure and zeta potential of the silk particles could be controlled by pH. Particles produced by salting out with 1.25 M potassium phosphate pH 6 showed a dominating silk II (crystalline) structure whereas particles produced at pH 9 were mainly composed of silk I (less crystalline). The results show that silk I rich particles possess chemical and physical stability and secondary structure which remained unchanged during post treatments even upon exposure to 100% ethanol or methanol. A model is presented to explain the process of particle formation based on intra- and intermolecular interactions of the silk domains, influenced by pH and kosmotrope salts. The reported silk fibroin particles can be loaded with small molecule model drugs, such as alcian blue, rhodamine B, and crystal violet, by simple absorption based on electrostatic interactions. In vitro release of these compounds from the silk particles depends on charge � charge interactions between the compounds and the silk. With crystal violet we demonstrated that the release kinetics are dependent on the secondary structure of the particles.
Lammel, Andreas; Hu, Xiao; Park, Sang-Hyug; Kaplan, David L.; Scheibel, Thomas
Rapid communication: Computational simulation and analysis of a candidate for the design of a novel silk-based biopolymer.
This work theoretically investigates the mechanical properties of a novel silk-derived biopolymer as polymerized in silico from sericin and elastin-like monomers. Molecular Dynamics simulations and Steered Molecular Dynamics were the principal computational methods used, the latter of which applies an external force onto the system and thereby enables an observation of its response to stress. The models explored herein are single-molecule approximations, and primarily serve as tools in a rational design process for the preliminary assessment of properties in a new material candidate. � 2014 Wiley Periodicals, Inc. Biopolymers 101: 915-923, 2014. PMID:24723330
Golas, Ewa I; Czaplewski, Cezary
In vivo biological responses to silk proteins functionalized with bone sialoprotein.
Recombinant 6mer?+?BSP protein, combining six repeats of the consensus sequence for Nephila clavipes dragline (6mer) and bone sialoprotein sequence (BSP), shows good support for cell viability and induces the nucleation of hydroxyapatite and tricalcium phosphate during osteoblast in vitro culture. The present study is conducted to characterize this bioengineered protein-based biomaterial further for in vivo behavior related to biocompatibility. 6mer?+?BSP protein films are implanted in subcutaneous pouches in the back of mice and responses are evaluated by flow cytometry and histology. The results show no major differences between the inflammatory responses induced by 6mer?+?BSP films and the responses observed for the controls. Thus, this new chimeric protein could represent an alternative for bone regeneration applications. PMID:23359587
Gomes, S�lvia; Gallego-Llamas, Jabier; Leonor, Isabel B; Mano, Jo�o F; Reis, Rui L; Kaplan, David L
Amino Acid Composition-Dependent Elasticity of SPIDER Silk
NASA Astrophysics Data System (ADS)
Spider silk exhibits excellent mechanical features in both toughness and extensibility. In recent years considerable investigations have focused on it. The understanding of spider silk protein is important for the development bionic silk. In this paper, we study by Monte Carlo simulation the force-of-extension property of spider silk proteins as a function of the residue composition for major and minor ampullate glands of typical Araneid orb weaver as well as that for one artificial spider silk. The results are also compared with those from a designed protein whose amino acid composition is uniform. The results clearly show that the major and minor ampullate gland proteins are much tougher than the designed protein, whereas the artificial protein, as a model of a nature spider silk, does have good mechanical properties. Our simulation reveals that mechanical property of a spider silk protein is dependent on its amino acid composition and that an excellent result of natural evolution is manifest in the composition of the spider silk protein.
Zhang, Yong; Dai, Luru; Ou-Yang, Zhong-Can
Synthesis and characterization of water-soluble silk peptides and recombinant silk protein containing polyalanine, the integrin binding site, and two glutamic acids at each terminal site as a possible candidate for use in bone repair materials.
The recombinant proteins [EE(A)12EETGRGDSPAAS]n (n = 5,10) were prepared as a potential scaffold material for bone repair. The construct was based on Antheraea perni silk fibroin to which cells adhere well and combined poly(alanine), the integrin binding site TGRGDSPA, and a pair of glutamic acids (E2) at both the N- and C-terminal sites to render the construct water-soluble and with the hope that it might enhance mineralization with hydroxyapatite. Initially, two peptides E2(A)nE2TGRGDSPAE2(A)nE2 (n = 6, 12) were prepared by solid state synthesis to examine the effect of size on conformation and on cell binding. The larger peptide bound osteoblasts more readily and had a higher helix content than the smaller one. Titration of the side chain COO(-) to COOH of the E2 and D units in the peptide was monitored by solution NMR. On the basis of these results, we produced the related recombinant His tagged protein [EE(A)12EETGRGDSPAAS]n (n = 5,10) by expression in Escherichia coli . The solution NMR spectra of the recombinant protein indicated that the poly(alanine) regions are helical, and one E2 unit is helical and the other is a random coil. A molecular dynamics simulation of the protein supports these conclusions from NMR. We showed that the recombinant protein, especially, [EE(A)12EETGRGDSPAAS]10 has some of the properties required for bone tissue engineering scaffold including insolubility, and evidence of enhanced cell binding through focal adhesions, and enhanced osteogenic expression of osteoblast-like cells bound to it, and has potential for use as a bone repair material. PMID:24004453
Asakura, Tetsuo; Suzuki, Yu; Nagano, Aya; Knight, David; Kamiya, Masakatsu; Demura, Makoto
Materials Fabrication from Bombyx mori Silk Fibroin
Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years, and it can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent processing methods can be used to generate silk biomaterials for a range of applications. In this protocol we include methods to extract silk from B. mori cocoons in order to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, and for drug delivery.
Rockwood, Danielle N.; Preda, Rucsanda C.; Yucel, Tuna; Wang, Xiaoqin; Lovett, Michael L.; Kaplan, David L.
Silk nanospheres and microspheres from silk/pva blend films for drug delivery.
Silk fibroin protein-based micro- and nanospheres provide new options for drug delivery due to their biocompatibility, biodegradability and their tunable drug loading and release properties. In the present study, we report a new aqueous-based preparation method for silk spheres with controllable sphere size and shape. The preparation was based on phase separation between silk fibroin and polyvinyl alcohol (PVA) at a weight ratio of 1/1 and 1/4. Water-insoluble silk spheres were easily obtained from the blend in a three step process: (1) air-drying the blend solution into a film, (2) film dissolution in water and (3) removal of residual PVA by subsequent centrifugation. In both cases, the spheres had approximately 30% beta-sheet content and less than 5% residual PVA. Spindle-shaped silk particles, as opposed to the spherical particles formed above, were obtained by stretching the blend films before dissolving in water. Compared to the 1/1 ratio sample, the silk spheres prepared from the 1/4 ratio sample showed a more homogeneous size distribution ranging from 300 nm up to 20 microm. Further studies showed that sphere size and polydispersity could be controlled either by changing the concentration of silk and PVA or by applying ultrasonication on the blend solution. Drug loading was achieved by mixing model drugs in the original silk solution. The distribution and loading efficiency of the drug molecules in silk spheres depended on their hydrophobicity and charge, resulting in different drug release profiles. The entire fabrication procedure could be completed within one day. The only chemical used in the preparation except water was PVA, an FDA-approved ingredient in drug formulations. Silk micro- and nanospheres reported have potential as drug delivery carriers in a variety of biomedical applications. PMID:19945157
Wang, Xiaoqin; Yucel, Tuna; Lu, Qiang; Hu, Xiao; Kaplan, David L
Silk Nanospheres and Microspheres from Silk/PVA Blend Films for Drug Delivery
Silk fibroin protein-based micro- and nanospheres provide new options for drug delivery due to their biocompatibility, biodegradability and their tunable drug loading and release properties. In the present study, we report a new aqueous-based preparation method for silk spheres with controllable sphere size and shape. The preparation was based on phase separation between silk fibroin and polyvinyl alcohol (PVA) at a weight ratio of 1/1 and 1/4. Water-insoluble silk spheres were easily obtained from the blend in a three step process: (1) air-drying the blend solution into a film, (2) film dissolution in water and (3) removal of residual PVA by subsequent centrifugation. In both cases, the spheres had approximately 30% beta-sheet content and less than 5% residual PVA. Spindle-shaped silk particles, as opposed to the spherical particles formed above, were obtained by stretching the blend films before dissolving in water. Compared to the 1/1 ratio sample, the silk spheres prepared from the 1/4 ratio sample showed a more homogeneous size distribution ranging from 300 nm up to 20 ?m. Further studies showed that sphere size and polydispersity could be controlled either by changing the concentration of silk and PVA or by applying ultrasonication on the blend solution. Drug loading was achieved by mixing model drugs in the original silk solution. The distribution and loading efficiency of the drug molecules in silk spheres depended on their hydrophobicity and charge, resulting in different drug release profiles. The entire fabrication procedure could be completed within one day. The only chemical used in the preparation except water was PVA, an FDA-approved ingredient in drug formulations. Silk micro- and nanospheres reported have potential as drug delivery carriers in a variety of biomedical applications.
Wang, Xiaoqin; Yucel, Tuna; Lu, Qiang; Hu, Xiao; Kaplan, David L.
Mechanism of silk processing in insects and spiders.
Silk spinning by insects and spiders leads to the formation of fibres that exhibit high strength and toughness. The lack of understanding of the protein processing in silk glands has prevented the recapitulation of these properties in vitro from reconstituted or genetically engineered silks. Here we report the identification of emulsion formation and micellar structures from aqueous solutions of reconstituted silkworm silk fibroin as a first step in the process to control water and protein-protein interactions. The sizes (100-200 nm diameter) of these structures could be predicted from hydrophobicity plots of silk protein primary sequence. These micelles subsequently aggregated into larger 'globules' and gel-like states as the concentration of silk fibroin increased, while maintaining solubility owing to the hydrophilic regions of the protein interspersed among the larger hydrophobic regions. Upon physical shearing or stretching structural transitions, increased birefringence and morphological alignment were demonstrated, indicating that this process mimics the behaviour of similar native silk proteins in vivo. Final morphological features of these silk materials are similar to those observed in native silkworm fibres. PMID:12944968
Jin, Hyoung-Joon; Kaplan, David L
Structure-function-property-design interplay in biopolymers: spider silk.
Spider silks have been a focus of research for almost two decades due to their outstanding mechanical and biophysical properties. Recent advances in genetic engineering have led to the synthesis of recombinant spider silks, thus helping to unravel a fundamental understanding of structure-function-property relationships. The relationships between molecular composition, secondary structures and mechanical properties found in different types of spider silks are described, along with a discussion of artificial spinning of these proteins and their bioapplications, including the role of silks in biomineralization and fabrication of biomaterials with controlled properties. PMID:23962644
Tokareva, Olena; Jacobsen, Matthew; Buehler, Markus; Wong, Joyce; Kaplan, David L
Silk-based delivery systems of bioactive molecules
Silks are biodegradable, biocompatible, self-assemblying proteins that can also be tailored via genetic engineering to contain specific chemical features, offering utility for drug and gene delivery. Silkworm silk has been used in biomedical sutures for decades and has recently achieved Food and Drug Administration approval for expanded biomaterials device utility. With the diversity and control of size, structure and chemistry, modified or recombinant silk proteins can be designed and utilized in various biomedical application, such as for the delivery of bioactive molecules. This review focuses on the biosynthesis and applications of silk-based multi-block copolymer systems and related silk protein drug delivery systems. The utility of these systems for the delivery of small molecule drugs, proteins and genes are reviewed.
Numata, Keiji; Kaplan, David L
Decrease in corneal damage due to benzalkonium chloride by the addition of sericin into timolol maleate eye drops.
We investigated the protective effects of sericin on corneal damage due to benzalkonium chloride (BAC) used as a preservative in commercially available timolol maleate eye drops using rat debrided corneal epithelium and a human cornea epithelial cell line (HCE-T). Corneal wounds were monitored using a fundus camera TRC-50X equipped with a digital camera; eye drops were instilled into the rat eyes five times a day after corneal epithelial abrasion. The viability of HCE-T cells was calculated by TetraColor One; and Escherichia coli (ATCC 8739) were used to measure antimicrobial activity. The reducing effects on transcorneal penetration and intraocular pressure (IOP) of the eye drops were determined using rabbits. The corneal wound healing rate and rate constants (kH) as well as cell viability were higher following treatment with 0.005% BAC solution containing 0.1% sericin than in the case of treatment with BAC solution alone; the antimicrobial activity was approximately the same for BAC solutions with and without sericin. In addition, the kH for rat eyes instilled with commercially available timolol maleate eye drops containing 0.1% sericin was significantly higher than that of eyes instilled with timolol maleate eye drops without sericin, and the addition of sericin did not affect the corneal penetration or IOP reducing effect of commercially available timolol maleate eye drops. A preservative system comprising BAC and sericin may provide effective therapy for glaucoma patients requiring long-term anti-glaucoma agents. PMID:23470443
Nagai, Noriaki; Ito, Yoshimasa; Okamoto, Norio; Shimomura, Yoshikazu
Sequential origin in the high performance properties of orb spider dragline silk
NASA Astrophysics Data System (ADS)
Major ampullate (MA) dragline silk supports spider orb webs, combining strength and extensibility in the toughest biomaterial. MA silk evolved ~376 MYA and identifying how evolutionary changes in proteins influenced silk mechanics is crucial for biomimetics, but is hindered by high spinning plasticity. We use supercontraction to remove that variation and characterize MA silk across the spider phylogeny. We show that mechanical performance is conserved within, but divergent among, major lineages, evolving in correlation with discrete changes in proteins. Early MA silk tensile strength improved rapidly with the origin of GGX amino acid motifs and increased repetitiveness. Tensile strength then maximized in basal entelegyne spiders, ~230 MYA. Toughness subsequently improved through increased extensibility within orb spiders, coupled with the origin of a novel protein (MaSp2). Key changes in MA silk proteins therefore correlate with the sequential evolution high performance orb spider silk and could aid design of biomimetic fibers.
Blackledge, Todd A.; P�rez-Rigueiro, Jos�; Plaza, Gustavo R.; Perea, Bel�n; Navarro, Andr�s; Guinea, Gustavo V.; Elices, Manuel
Sequential origin in the high performance properties of orb spider dragline silk
Major ampullate (MA) dragline silk supports spider orb webs, combining strength and extensibility in the toughest biomaterial. MA silk evolved ~376?MYA and identifying how evolutionary changes in proteins influenced silk mechanics is crucial for biomimetics, but is hindered by high spinning plasticity. We use supercontraction to remove that variation and characterize MA silk across the spider phylogeny. We show that mechanical performance is conserved within, but divergent among, major lineages, evolving in correlation with discrete changes in proteins. Early MA silk tensile strength improved rapidly with the origin of GGX amino acid motifs and increased repetitiveness. Tensile strength then maximized in basal entelegyne spiders, ~230?MYA. Toughness subsequently improved through increased extensibility within orb spiders, coupled with the origin of a novel protein (MaSp2). Key changes in MA silk proteins therefore correlate with the sequential evolution high performance orb spider silk and could aid design of biomimetic fibers.
Blackledge, Todd A.; Perez-Rigueiro, Jose; Plaza, Gustavo R.; Perea, Belen; Navarro, Andres; Guinea, Gustavo V.; Elices, Manuel
Water-insoluble Silk Films with Silk I Structure
Water-insoluble regenerated silk materials are normally produced by increasing the {beta}-sheet content (silk II). In the present study water-insoluble silk films were prepared by controlling the very slow drying of Bombyx mori silk solutions, resulting in the formation of stable films with a predominant silk I instead of silk II structure. Wide angle X-ray scattering indicated that the silk films stabilized by slow drying were mainly composed of silk I rather than silk II, while water- and methanol-annealed silk films had a higher silk II content. The silk films prepared by slow drying had a globule-like structure at the core surrounded by nano-filaments. The core region was composed of silk I and silk II, surrounded by hydrophilic nano-filaments containing random turns and {alpha}-helix secondary structures. The insoluble silk films prepared by slow drying had unique thermal, mechanical and degradative properties. Differential scanning calorimetry results revealed that silk I crystals had stable thermal properties up to 250 C, without crystallization above the T{sub g}, but degraded at lower temperatures than silk II structure. Compared with water- and methanol-annealed films the films prepared by slow drying had better mechanical ductility and were more rapidly enzymatically degraded, reflecting the differences in secondary structure achieved via differences in post processing of the cast silk films. Importantly, the silk I structure, a key intermediate secondary structure for the formation of mechanically robust natural silk fibers, was successfully generated by the present approach of very slow drying, mimicking the natural process. The results also point to a new mode of generating new types of silk biomaterials with enhanced mechanical properties and increased degradation rates, while maintaining water insolubility, along with a low {beta}-sheet content.
Lu, Q.; Hu, X; Wang, X; Kluge, J; Lu, S; Cebe, P; Kaplan, D
Silk Fibroin Films Crystallized by Multiwalled Carbon Nanotubes
NASA Astrophysics Data System (ADS)
Silk films prepared from regenerated silk fibroin are normally stabilized by ?-sheet formation through the use of solvents (methanol, water etc.). Herein, we report a new method of preparing water-stable films without a ?-sheet conformation from regenerated silk fibroin solutions by incorporating a small amount (0.2 wt%) of multiwalled carbon nanotubes (MWCNTs). To extend the biomaterial utility of silk proteins, forming water-stable silk-based materials with enhanced mechanical properties is essential. Scanning electron microscopy and transmission electron microscopy were used to observe the morphology of the MWCNT-incorporated silk films. The wide-angle X-ray diffraction provided clear evidence of the crystallization of the silk fibroin induced by MWCNT in the composite films without any additional annealing processing. The tensile modulus and strength of the composite films were improved by 108% and 51%, respectively, by the incorporation of 0.2 wt% of MWCNTs, as compared with those of the pure silk films. The method described in this study will provide an alternative means of crystallizing silk fibroin films without using an organic solvent or blending with any other polymers, which may be important in biomedical applications.
Kim, H.-S.; Park, W.-I.; Kim, Y.; Jin, H.-J.
Antimicrobial functionalized genetically engineered spider silk
Genetically engineered fusion proteins offer potential as multifunctional biomaterials for medical use. Fusion or chimeric proteins can be formed using recombinant DNA technology by combining nucleotide sequences encoding different peptides or proteins that are otherwise not found together in nature. In the present study, three new fusion proteins were designed, cloned and expressed and assessed for function, by combining the consensus sequence of dragline spider silk with three different antimicrobial peptides. The human antimicrobial peptides human neutrophil defensin 2 (HNP-2), human neutrophil defensins 4 (HNP-4) and hepcidin were fused to spider silk through bioengineering. The spider silk domain maintained its self-assembly features, a key aspect of these new polymeric protein biomaterials, allowing the formation of ?-sheets to lock in structures via physical interactions without the need for chemical cross-linking. These new functional silk proteins were assessed for antimicrobial activity against Gram - Escherichia coli and Gram + Staphylococcus aureus and microbicidal activity was demonstrated. Dynamic light scattering was used to assess protein aggregation to clarify the antimicrobial patterns observed. Attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and circular dichroism (CD) were used to assess the secondary structure of the new recombinant proteins. In vitro cell studies with a human osteosarcoma cell line (SaOs-2) demonstrated the compatibility of these new proteins with mammalian cells.
Gomes, Silvia; Leonor, Isabel B.; Mano, Joao F.; Reis, Rui L.; Kaplan, David L.
NSDL National Science Digital Library
Silk Road Seattle is a "collaborative public education project using the 'Silk Road' theme to explore cultural interaction across Eurasia from the beginning of the Common Era (A.D.) to the Sixteenth Century." The Silk Road is a term understood to have been the overland trade route from China to the Mediterranean, opened first in the 2nd century and coming to an end between the 15th and 17th centuries. Traditional discussions of the Silk Road, however, recognize that there were branches that went into South Asia, or extended from Central Asia north of the Caspian Sea to the Black Sea. Sponsored primarily by the Simpson Center for the Humanities at the University of Washington and directed by Professors Daniel C. Waugh, Joel Walker, and Cynthea Bogel, this Web site contains a vast range of Silk Road materials. These materials include a list of texts that may be used in teaching and learning about the Silk Road, a section on cities and architecture along the Silk Road, information on traditional culture in Central Asia, a page of teaching and learning guides that list and annotate Silk Road materials, and a virtual art exhibit. This site is still in a constructive stage; therefore, every link is not yet accessible. In short, this site may be of value for a range of audiences, including school children, teachers, college and graduate students, and independent adult learners.
ERIC Educational Resources Information Center
Painting on silk has a magic all its own. Versions of painting on silk can be found throughout the world from Japan and Europe to the United States. Themes for the paintings can be most any type of design or imagery. Applying the liquid dyes is exciting, as the vivid liquid colors flow and blend into the fabric. The process captures students'�
Padrick, Deborah
Extraction conditions of Antheraea mylitta sericin with high yields and minimum molecular weight degradation.
Although the technique for extracting the Bombyx mori sericin has been extensively known, the extraction of sericin from wild-silkworm cocoons is not yet standardized. The aim of this study was to find the optimal conditions for the extraction of sericin from Antheraea mylitta cocoons, with high yields and minimum degradation. We attempted to apply various protocols for the extraction of the A. mylitta sericin (AmS). Among these, we found that the extraction of AmS with a sodium carbonate solution exhibited the highest yield except the conventional soap-alkali extraction. To find the optimal conditions for the AmS extraction with the sodium carbonate, we changed the concentration of sodium carbonate and the treatment time. With an increase in the sodium carbonate concentration and the extraction time, the yield of AmS increased, but the molecular weight (MW) of AmS decreased. Considering the yield, molecular weight distribution (MWD) and amino acid composition of AmS, we suggest that the optimal conditions for the AmS extraction require treatment with 0.02 M sodium carbonate and boiling for 60 min. PMID:23026092
Yun, Haesung; Oh, Hanjin; Kim, Moo Kon; Kwak, Hyo Won; Lee, Jeong Yun; Um, In Chul; Vootla, Shyam Kumar; Lee, Ki Hoon
Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay
Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications. To develop a novel affinity silk material, we generated a transgenic silkworm that spins silk protein containing the fibroin L-chain linked with the single-chain variable fragment (scFv) as a fusion protein. Previously, the scFv-conjugated �affinity� silk powder specifically immunoprecipitated its target protein, Wiskott-Aldrich syndrome protein. To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates. Enzyme-linked immunosorbent assay demonstrated specific detection of Wiskott-Aldrich syndrome protein, both as a recombinant protein and in its native form extracted from mouse macrophages. These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system.
Sato, Mitsuru; Kojima, Katsura; Sakuma, Chisato; Murakami, Maria; Tamada, Yasushi; Kitani, Hiroshi
Distinctive influence of two hexafluoro solvents on the structural stabilization of Bombyx mori silk fibroin protein and its derived peptides: 13C NMR and CD studies.
Employing high-resolution (13)C solution NMR and circular dichroism (CD) spectroscopic techniques, the distinctive influence of two intimately related hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and hexafluoroacetone trihydrate (HFA), on the structural characteristics of Bombyx mori (B. mori) silk fibroin, the chymotrypsin precipitate (C(p)) fraction, and two synthetic peptides, (AGSGAG)(5) and (AG)(15), is described. The observed (13)C solution NMR and CD spectra of these polypeptides in HFIP and HFA revealed a distinctive influence on their conformational characteristics. The (13)C NMR spectra, as analyzed from the unique chemical shifts of C(alpha) and C(beta) resonances of constituent residues revealed that fibroin largely assumes helical conformation(s) in both solvents. However, the peak shifts were greater for the samples in HFIP, indicating that the types of helical structure(s) may be different from the one populated in HFA. Similar structural tendencies of these polypeptides were reflected in CD spectra. The observed CD patterns, i.e., a strong positive band at approximately 190 nm and negative bands at approximately 206 and 222 nm, have been attributed to the preponderance of helical structures. Of the two prevalent helical structures, alpha-helix and 3(10)-helix, the evidence emerged for the fibroin protein in favor of 3(10)-helical structure stabilization in HFIP and its significant disruption in HFA, as deduced from the characteristic R1 (=[theta](190)/[theta](202)) and R2 (=[theta](222)/[theta](206)) ratios, determined from the CD data. Conversely, the native polypeptides and synthetic peptide fragments derived from highly crystalline regions of the silk fibroin protein sustained predominantly an unordered structure in HFA solvent. PMID:16398492
Ha, Sung-Won; Asakura, Tetsuo; Kishore, Raghuvansh
Silk material modification and microfabrication strategies for applications in biosensors and drug delivery
NASA Astrophysics Data System (ADS)
Silk has proven to be a promising biomaterial for the development of a novel generation of biomedical devices due to the material's intrinsic properties of biocompatibility and biodegradability. To further the development of silk-based devices, augmented functionality can be provided to silk by means of microfabrication and material functionalization. In this dissertation, we set out to explore possibilities of silk-based biomedical device development with particular attention to different fabrication strategies that can be leveraged for this purpose, taking inspiration from conventional lithography, contact imprinting and chemical modification of the silk biomaterial. In particular, we have produced a novel silk-based drug delivery device, in the form of microneedles. For this purpose we have developed micromolding strategies which allow the fabrication of high aspect ratio silk structures. Furthermore, we have produced a THz split ring resonator based sensor device on silk films. To realize this device, we have developed a convenient fabrication method, allowing transfer of previously microfabricated metal and oxide microstructures to the silk film surface. This method has proven useful for hard masking and patterning silk films with reactive ion etching. Moreover, we have explored patterning semiconductor and glass substrates with silk films. For this purpose we have modified a standard microfabrication method -lift off- to be amendable to silk. Furthermore, combining silk based fabrication and material functionalization has lead to microfluidics pH sensing applications. Finally we have explored the possibility of utilizing silk for injectable and biodegradable glucose sensors. For this purpose, we have doped silk hydrogels with a fluorescence based protein biosensor. The advanced silk fabrication and material synthesis strategies and the resulting novel devices presented here could potentially lead to a new class of biomedical applications.
Tsioris, Konstantinos
A silk fibroin based hepatocarcinoma model and the assessment of the drug response in hyaluronan-binding protein 1 overexpressed HepG2 cells.
Microenvironment around tumor cells plays an important role in its malignancy or invasiveness. Hyaluronan (HA), a major component of extracellular matrix is found to be elevated in most of cancerous niche/microenvironment and performs regulatory role in the progression of tumors and metastasis. Overexpression of the hyaladherin, hyaluronan-binding protein 1 (HABP1) in the hepatocarcinoma cells (HepG2) termed as HepR21 leads to enhanced cell proliferation with increased HA 'pool' associated with HA 'cables' indicating elevated tumorous potential under 2D culture conditions. For in vitro experimentation, scaffold based three dimensional niche modeling may have greater acceptance than conventional 2D culture condition. Thus, we have examined the influence of intrinsic properties of non-mulberry tropical tasar silk fibroin on the HepR21 cells in order to develop a 3D hepatocarcinoma construction to act as model. The scaffold of tasar silk fibroin of Antheraea mylitta when efficiently loaded with transformed hepatocarcinoma cells, HepR21; exhibits enhanced adhesiveness, viability, metabolic activity, proliferation and enlarged cellular morphology in 3D compared to its parent cell line HepG2, supporting the earlier observation made in 2D system. In addition, formation of multicellular aggregates, the indicator of tumor progression is also revealed in silk based 3D culture conditions. Further, the use of 4-MU (a hyaluronan synthase inhibitor) on HepR21 cells reduces the HA level and downregulates the expression of growth promoting factors like pAKT and PKC; while upregulating the expression of the tumor suppressor p53. Thus, 4-MU efficiently reduces the tumor potency associated with increased HA pool as well as HA cables and the effect of 4-MU doubling up as an anticancer agent in 2D and 3D are also comparable. The in vitro 3D multicellular model demonstrates the insight of hepatocarcinoma progression and offers the predictability of cellular response to transfection efficacy, drug treatment and therapeutic intervention. PMID:24016853
Kundu, Banani; Saha, Paramita; Datta, Kasturi; Kundu, Subhas C
Carbondioxide gating in silk cocoon.
Silk is the generic name given to the fibrous proteins spun by a number of arthropods. During metamorphosis, the larva of the silk producing arthropods excrete silk-fiber from its mouth and spun it around the body to form a protective structure called cocoon. An adult moth emerges out from the cocoon after the dormant phase (pupal phase) varying from 2 weeks to 9 months. It is intriguing how CO(2)/O(2) and ambient temperature are regulated inside the cocoon during the development of the pupa. Here we show that the cocoon membrane is asymmetric, it allows preferential gating of CO(2) from inside to outside and it regulates a physiological temperature inside the cocoon irrespective of the surrounding environment temperature. We demonstrate that under simulating CO(2) rich external environment, the CO(2) does not diffuse inside the cocoon. Whereas, when CO(2) was injected inside the cocoon, it diffuses out in 20 s, indicating gating of CO(2) from inside to outside the membrane. Removal of the calcium oxalate hydrate crystals which are naturally present on the outer surface of the cocoon affected the complete blockade of CO(2) flow from outside to inside suggesting its role to trap most of the CO(2) as hydrogen bonded bicarbonate on the surface. The weaved silk of the cocoon worked as the second barrier to prevent residual CO(2) passage. Furthermore, we show that under two extreme natural temperature regime of 5 and 50 �C, a temperature of 25 and 34 �C respectively were maintained inside the cocoons. Our results demonstrate, how CO(2) gating and thermoregulation helps in maintaining an ambient atmosphere inside the cocoon for the growth of pupa. Such natural architectural control of gas and temperature regulation could be helpful in developing energy saving structures and gas filters. PMID:22791361
Roy, Manas; Meena, Sunil Kumar; Kusurkar, Tejas Sanjeev; Singh, Sushil Kumar; Sethy, Niroj Kumar; Bhargava, Kalpana; Sarkar, Sabyasachi; Das, Mainak
Safety Evaluation of Silk Protein Film (A Novel Wound Healing Agent) in Terms of Acute Dermal Toxicity, Acute Dermal Irritation and Skin Sensitization
Acute dermal toxicity study was conducted in rats. The parameters studied were body weight, serum biochemistry and gross pathology. The animals were also observed for clinical signs and mortality after the application of test film. The dermal irritation potential of silk protein film was examined using Draize test. In the initial test, three test patches were applied sequentially for 3 min, 1 and 4 hours, respectively, and skin reaction was graded. The irritant or negative response was confirmed using two additional animals, each with one patch, for an exposure period of 4 hours. The responses were scored at 1, 24, 48 and 72 hours after the patch removal. Skin sensitization study was conducted according to Buehler test in guinea pigs, in which on day 0, 7 and 14, the animals were exposed to test material for 6 hours (Induction phase) and on day 28, the animals were exposed for a period of 24 hours (Challenge phase). The skin was observed and recorded at 24 and 48 hours after the patch removal. In acute dermal toxicity study, the rats dermally treated with silk film did not show any abnormal clinical signs and the body weight, biochemical parameters and gross pathological observations were not significantly different from the control group. In acute dermal irritation study, the treated rabbits showed no signs of erythema, edema and eschar, and the scoring was given as �0� for all time points of observations according to Draize scoring system. In skin sensitization study, there were no skin reactions 24 and 48 hours after the removal of challenge patch, which was scored �0� based on Magnusson/Kligman grading scale.
Padol, Amol R.; Jayakumar, K.; Shridhar, N. B.; Narayana Swamy, H. D.; Narayana Swamy, M.; Mohan, K.
A conserved spider silk domain acts as a molecular switch that controls fibre assembly
A huge variety of proteins are able to form fibrillar structures, especially at high protein concentrations. Hence, it is surprising that spider silk proteins can be stored in a soluble form at high concentrations and transformed into extremely stable fibres on demand. Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich polar elements forming a
Franz Hagn; Lukas Eisoldt; John G. Hardy; Charlotte Vendrely; Murray Coles; Thomas Scheibel; Horst Kessler
Clay-Enriched Silk Biomaterials for Bone Formation
The formation of silk protein/clay composite biomaterials for bone tissue formation is described. Silk fibroin serves as an organic scaffolding material offering mechanical stability suitable for bone specific uses. Clay montmorillonite (Cloisite � Na+) and sodium silicate are sources of osteoinductive silica-rich inorganic species, analogous to bioactive bioglass-like bone repair biomaterial systems. Different clay particle-silk composite biomaterial films were compared to silk films doped with sodium silicate as controls for support of human bone marrow derived mesenchymal stem cells (hMSCs) in osteogenic culture. The cells adhered and proliferated on the silk/clay composites over two weeks. Quantitative real-time RT-PCR analysis revealed increased transcript levels for alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col I) osteogenic markers in the cells cultured on the silk/clay films in comparison to the controls. Early evidence for bone formation based on collagen deposition at the cell-biomaterial interface was also found, with more collagen observed for the silk films with higher contents of clay particles. The data suggest that the silk/clay composite systems may be useful for further study toward bone regenerative needs.
Mieszawska, Aneta J.; Llamas, Jabier Gallego; Vaiana, Christopher A.; Kadakia, Madhavi P.; Naik, Rajesh R.; Kaplan, David L.
Lyophilized Silk Fibroin Hydrogels for the Sustained Local Delivery of Therapeutic Monoclonal Antibodies
The development of sustained delivery systems compatible with protein therapeutics continues to be a significant unmet need. A lyophilized silk fibroin hydrogel matrix (lyogel) for the sustained release of pharmaceutically relevant monoclonal antibodies is described. Sonication of silk fibroin prior to antibody incorporation avoids exposing the antibody to the sol-gel transition inducing shear stress. Fourier Transform Infrared (FTIR) analysis showed no change in silk structural composition between hydrogel and lyogel or with increasing silk fibroin concentration. Antibody release from hydrogels occurred rapidly over 10 days regardless of silk concentration. Upon lyophilization, sustained antibody release was observed over 38 days from lyogels containing 6.2% (w/w) silk fibroin and above. In 3.2% (w/w) silk lyogels, antibody release was comparable to hydrogels. Swelling properties of lyogels followed a similar threshold behavior. Lyogels at 3.2% (w/w) silk recovered approximately 90% of their fluid mass upon rehydration, while approximately 50% fluid recovery was observed at 6.2% (w/w) silk and above. Antibody release was primarily governed by hydrophobic/hydrophilic silk-antibody interactions and secondarily altered by the hydration resistance of the lyogel. Hydration resistance was controlled by altering ?-sheet (crystalline) density of the matrix. The antibody released from lyogels maintained biological activity. Silk lyogels offer an advantage as a delivery matrix over other hydrogel materials for the slow release of the loaded protein, making lyogels suitable for long-term sustained release applications.
Guziewicz, Nicholas; Best, Annie; Perez-Ramirez, Bernardo; Kaplan, David L.
Lyophilized silk fibroin hydrogels for the sustained local delivery of therapeutic monoclonal antibodies.
The development of sustained delivery systems compatible with protein therapeutics continues to be a significant unmet need. A lyophilized silk fibroin hydrogel matrix (lyogel) for the sustained release of pharmaceutically relevant monoclonal antibodies is described. Sonication of silk fibroin prior to antibody incorporation avoids exposing the antibody to the sol-gel transition inducing shear stress. Fourier Transform Infrared (FTIR) analysis showed no change in silk structural composition between hydrogel and lyogel or with increasing silk fibroin concentration. Antibody release from hydrogels occurred rapidly over 10 days regardless of silk concentration. Upon lyophilization, sustained antibody release was observed over 38 days from lyogels containing 6.2% (w/w) silk fibroin and above. In 3.2% (w/w) silk lyogels, antibody release was comparable to hydrogels. Swelling properties of lyogels followed a similar threshold behavior. Lyogels at 3.2% (w/w) silk recovered approximately 90% of their fluid mass upon rehydration, while approximately 50% fluid recovery was observed at 6.2% (w/w) silk and above. Antibody release was primarily governed by hydrophobic/hydrophilic silk-antibody interactions and secondarily altered by the hydration resistance of the lyogel. Hydration resistance was controlled by altering ?-sheet (crystalline) density of the matrix. The antibody released from lyogels maintained biological activity. Silk lyogels offer an advantage as a delivery matrix over other hydrogel materials for the slow release of the loaded protein, making lyogels suitable for long-term sustained release applications. PMID:21216004
Guziewicz, Nicholas; Best, Annie; Perez-Ramirez, Bernardo; Kaplan, David L
Quantifying osteogenic cell degradation of silk biomaterials.
The degradation of silk protein films by human mesenchymal stem cells (hMSCs), osteoblasts and osteoclasts, cells involved in osteogenic functions in normal and diseased bone, was assessed in vitro. The involvement of specific matrix metalloproteinases (MMPs) and integrin signaling in the degradation process was determined. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to quantitatively compare degradation by the different cell types using surface patterned silk films. Osteoblasts and osteoclasts demonstrated significant degradation of the silk films in vitro in comparison to the hMSCs and the film controls without cells. The osteoclasts degraded the silk films the most and also generated the highest level of MMPs 1 and 2. The osteoblasts upregulated integrins ?5 and ?1, while the osteoclasts upregulated integrins ?2 and ?1. There was significant contrast in responses on the silk matrices between osteogenic cells versus undifferentiated hMSCs to illustrate in vitro the role of cell type on matrix remodeling. These are important issues in matching biomaterial matrix features and studies in vitro to remodeling in vivo, in both normal and disease tissue systems. Cell populations and niche factors impact tissue regeneration, wound healing, physiological state, and the ability to better understand the role of different cell types is critical to overall regenerative outcomes. PMID:21105641
Sengupta, Sejuti; Park, Sang-Hyug; Seok, Gil Eun; Patel, Atur; Numata, Keiji; Lu, Chia-Li; Kaplan, David L
Quantifying Osteogenic Cell Degradation of Silk Biomaterials
The degradation of silk protein films by human mesenchymal stem cells (hMSCs), osteoblasts and osteoclasts, cells involved in osteogenic functions in normal and diseased bone, was assessed in vitro. The involvement of specific matrix metalloproteinases (MMPs) and integrin signaling in the degradation process was determined. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to quantitatively compare degradation by the different cell types using surface patterned silk films. Osteoblasts and osteoclasts demonstrated significant degradation of the silk films in vitro in comparison to the hMSCs and the film controls without cells. The osteoclasts degraded the silk films the most and also generated the highest level of MMPs 1 and 2. The osteoblasts upregulated integrins ?5 and ?1 while the osteoclasts upregulated integrins ?2 and ?1. There was significant contrast in responses on the silk matrices between osteogenic cells vs undifferentiated hMSCs to illustrate in vitro the role of cell type on matrix remodeling. These are important issues in matching biomaterial matrix features and studies in vitro to remodeling in vivo, in both normal and disease tissue systems. Cell populations and niche factors impact tissue regeneration, wound healing and physiological state and the ability to better understand the role of different cell types is critical to overall regenerative outcomes.
Sengupta, Sejuti; Park, Sang-Hyug; Seok, Gil Eun; Patel, Atur; Numata, Keiji; Lu, Chia-Li; Kaplan, David L.
Atomistic model of the spider silk nanostructure
NASA Astrophysics Data System (ADS)
Spider silk is an ultrastrong and extensible self-assembling biopolymer that outperforms the mechanical characteristics of many synthetic materials including steel. Here we report atomic-level structures that represent aggregates of MaSp1 proteins from the N. Clavipes silk sequence based on a bottom-up computational approach using replica exchange molecular dynamics. We discover that poly-alanine regions predominantly form distinct and orderly beta-sheet crystal domains while disorderly structures are formed by poly-glycine repeats, resembling 31-helices. These could be the molecular source of the large semicrystalline fraction observed in silks, and also form the basis of the so-called ``prestretched'' molecular configuration. Our structures are validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots, alpha-carbon atomic distances, as well as secondary structure content.
Keten, Sinan; Buehler, Markus J.
Silk film biomaterials for cornea tissue engineering
Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. Silk protein films were used in a biomimetic approach to replicate corneal stromal tissue architecture. The films were 2 ?m thick to emulate corneal collagen lamellae dimensions, and were surface patterned to guide cell alignment. To enhance trans-lamellar diffusion of nutrients and to promote cell-cell interaction, pores with 0.5 to 5.0 ?m diameters were introduced into the silk films. Human and rabbit corneal fibroblast proliferation, alignment and corneal extracellular matrix expression on these films in both 2D and 3D cultures was demonstrated. The mechanical properties, optical clarity and surface patterned features of these films, combined with their ability to support corneal cell functions suggest this new biomaterial system offers important potential benefits for corneal tissue regeneration.
Lawrence, Brian D.; Marchant, Jeffrey K.; Pindrus, Mariya; Omenetto, Fiorenzo; Kaplan, David L.
Greatly Increased Toughness of Infiltrated Spider Silk
NASA Astrophysics Data System (ADS)
In nature, tiny amounts of inorganic impurities, such as metals, are incorporated in the protein structures of some biomaterials and lead to unusual mechanical properties of those materials. A desire to produce these biomimicking new materials has stimulated materials scientists, and diverse approaches have been attempted. In contrast, research to improve the mechanical properties of biomaterials themselves by direct metal incorporation into inner protein structures has rarely been tried because of the difficulty of developing a method that can infiltrate metals into biomaterials, resulting in a metal-incorporated protein matrix. We demonstrated that metals can be intentionally infiltrated into inner protein structures of biomaterials through multiple pulsed vapor-phase infiltration performed with equipment conventionally used for atomic layer deposition (ALD). We infiltrated zinc (Zn), titanium (Ti), or aluminum (Al), combined with water from corresponding ALD precursors, into spider dragline silks and observed greatly improved toughness of the resulting silks. The presence of the infiltrated metals such as Al or Ti was verified by energy-dispersive x-ray (EDX) and nuclear magnetic resonance spectra measured inside the treated silks. This result of enhanced toughness of spider silk could potentially serve as a model for a more general approach to enhance the strength and toughness of other biomaterials.
Lee, Seung-Mo; Pippel, Eckhard; G�sele, Ulrich; Dresbach, Christian; Qin, Yong; Chandran, C. Vinod; Br�uniger, Thomas; Hause, Gerd; Knez, Mato
New Materials Based on Spider Silk.
National Technical Information Service (NTIS)
In this project, we investigated the dragline silk proteins ADF-3 and ADF-4 of the spider Araneus diadematus. Adf-3 and Adf-4 cDNA can each be expressed directly in insect cells using the Baculovirus-expression-System. Recombinant ADF-4 is insolubly produ...
C. Vendrely T. Scheibel
Enzymatic mineralization of silk scaffolds.
The present study focuses on the alkaline phosphatase (ALP) mediated formation of apatitic minerals on porous silk fibroin protein (SFP) scaffolds. Porous SFP scaffolds impregnated with different concentrations of ALP are homogeneously mineralized under physiological conditions. The mineral structure is apatite while the structures differ as a function of the ALP concentration. Cellular adhesion, proliferation, and colonization of osteogenic MC3T3 cells improve on the mineralized SFP scaffolds. These findings suggest a simple process to generate mineralized scaffolds that can be used to enhanced bone tissue engineering-related utility. PMID:24610728
Samal, Sangram K; Dash, Mamoni; Declercq, Heidi A; Gheysens, Tom; Dendooven, Jolien; Voort, Pascal Van Der; Cornelissen, Ria; Dubruel, Peter; Kaplan, David L
Effect of sequence features on assembly of spider silk block copolymers.
Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions. PMID:24613991
Tokareva, Olena S; Lin, Shangchao; Jacobsen, Matthew M; Huang, Wenwen; Rizzo, Daniel; Li, David; Simon, Marc; Staii, Cristian; Cebe, Peggy; Wong, Joyce Y; Buehler, Markus J; Kaplan, David L
Unravelling the biodiversity of nanoscale signatures of spider silk fibres
NASA Astrophysics Data System (ADS)
Living organisms are masters at designing outstanding self-assembled nanostructures through a hierarchical organization of modular proteins. Protein-based biopolymers improved and selected by the driving forces of molecular evolution are among the most impressive archetypes of nanomaterials. One of these biomacromolecules is the myriad of compound fibroins of spider silks, which combine surprisingly high tensile strength with great elasticity. However, no consensus on the nano-organization of spider silk fibres has been reached. Here we explore the biodiversity of spider silk fibres, focusing on nanoscale characterization with high-resolution atomic force microscopy. Our results reveal an evolution of the nanoroughness, nanostiffness, nanoviscoelastic, nanotribological and nanoelectric organization of microfibres, even when they share similar sizes and shapes. These features are related to unique aspects of their molecular structures. The results show that combined nanoscale analyses of spider silks may enable the screening of appropriate motifs for bioengineering synthetic fibres from recombinant proteins.
Silva, Luciano P.; Rech, Elibio L.
Spider silk: understanding the structure-function relationship of a natural fiber.
Spider silk is of great interest because of its extraordinary physical properties, such as strength and toughness. Here we discuss how these physical properties relate to the way in which spiders have utilized this material in prey capture, forcing its evolution to a high-performance fiber. Female spiders can produce up to seven different types of silk, and all these have different physical properties, which relate to their various functions. The variation in properties are due to underlying differences in the proteins making up these silks. As our understanding of spider silk has increased in the recent years, it has been possible to produce recombinant versions of the respective proteins. Recombinant proteins open up the potential to produce synthetic silk fibers with properties similar to those of the natural spider silk threads. PMID:21999996
Humenik, Martin; Scheibel, Thomas; Smith, Andrew
Inhibitory effect of corn silk on skin pigmentation.
In this study, the inhibitory effect of corn silk on melanin production was evaluated. This study was performed to investigate the inhibitory effect of corn silk on melanin production in Melan-A cells by measuring melanin production and protein expression. The corn silk extract applied on Melan-A cells at a concentration of 100 ppm decreased melanin production by 37.2% without cytotoxicity. This was a better result than arbutin, a positive whitening agent, which exhibited a 26.8% melanin production inhibitory effect at the same concentration. The corn silk extract did not suppress tyrosinase activity but greatly reduced the expression of tyrosinase in Melan-A cells. In addition, corn silk extract was applied to the human face with hyperpigmentation, and skin color was measured to examine the degree of skin pigment reduction. The application of corn silk extract on faces with hyperpigmentation significantly reduced skin pigmentation without abnormal reactions. Based on the results above, corn silk has good prospects for use as a material for suppressing skin pigmentation. PMID:24595276
Choi, Sang Yoon; Lee, Yeonmi; Kim, Sung Soo; Ju, Hyun Min; Baek, Ji Hwoon; Park, Chul-Soo; Lee, Dong-Hyuk
Evidence for antimicrobial activity associated with common house spider silk
Background Spider silk is one of the most versatile materials in nature with great strength and flexibility. Native and synthetically produced silk has been used in a wide range of applications including the construction of artificial tendons and as substrates for human cell growth. In the literature there are anecdotal reports that suggest that native spider silk may also have antimicrobial properties. Findings In this study we compared the growth of a Gram positive and a Gram negative bacterium in the presence and absence of silk produced by the common house spider Tegenaria domestica. We demonstrate that native web silk of Tegenaria domestica can inhibit the growth of the Gram positive bacterium, Bacillus subtilis. No significant inhibition of growth was detected against the Gram negative bacterium, Escherichia coli. The antimicrobial effect against B. subtilis appears to be short lived thus the active agent potentially acts in a bacteriostatic rather than bactericidal manner. Treatment of the silk with Proteinase K appears to reduce the ability to inhibit bacterial growth. This is consistent with the active agent including a protein element that is denatured or cleaved by treatment. Tegenaria silk does not appear to inhibit the growth of mammalian cells in vitro thus there is the potential for therapeutic applications.
Increasing silk fibre strength through heterogeneity of bundled fibrils
Can naturally arising disorder in biological materials be beneficial? Materials scientists are continuously attempting to replicate the exemplary performance of materials such as spider silk, with detailed techniques and assembly procedures. At the same time, a spider does not precisely machine silk�imaging indicates that its fibrils are heterogeneous and irregular in cross section. While past investigations either focused on the building material (e.g. the molecular scale protein sequence and behaviour) or on the ultimate structural component (e.g. silk threads and spider webs), the bundled structure of fibrils that compose spider threads has been frequently overlooked. Herein, I exploit a molecular dynamics-based coarse-grain model to construct a fully three-dimensional fibril bundle, with a length on the order of micrometres. I probe the mechanical behaviour of bundled silk fibrils with variable density of heterogenic protrusions or globules, ranging from ideally homogeneous to a saturated distribution. Subject to stretching, the model indicates that cooperativity is enhanced by contact through low-force deformation and shear �locking� between globules, increasing shear stress transfer by up to 200 per cent. In effect, introduction of a random and disordered structure can serve to improve mechanical performance. Moreover, addition of globules allows a tuning of free volume, and thus the wettability of silk (with implications for supercontraction). These findings support the ability of silk to maintain near-molecular-level strength at the scale of silk threads, and the mechanism could be easily adopted as a strategy for synthetic fibres.
Cranford, Steven W.
Recombinant spider silk genetically functionalized with affinity domains.
Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule. PMID:24678858
Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-�ke; Hedhammar, My
Spider genomes provide insight into composition and evolution of venom and silk.
Spiders are ecologically important predators with complex venom and extraordinarily tough silk that enables capture of large prey. Here we present the assembled genome of the social velvet spider and a draft assembly of the tarantula genome that represent two major taxonomic groups of spiders. The spider genomes are large with short exons and long introns, reminiscent of mammalian genomes. Phylogenetic analyses place spiders and ticks as sister groups supporting polyphyly of the Acari. Complex sets of venom and silk genes/proteins are identified. We find that venom genes evolved by sequential duplication, and that the toxic effect of venom is most likely activated by proteases present in the venom. The set of silk genes reveals a highly dynamic gene evolution, new types of silk genes and proteins, and a novel use of aciniform silk. These insights create new opportunities for pharmacological applications of venom and biomaterial applications of silk. PMID:24801114
Sanggaard, Kristian W; Bechsgaard, Jesper S; Fang, Xiaodong; Duan, Jinjie; Dyrlund, Thomas F; Gupta, Vikas; Jiang, Xuanting; Cheng, Ling; Fan, Dingding; Feng, Yue; Han, Lijuan; Huang, Zhiyong; Wu, Zongze; Liao, Li; Settepani, Virginia; Th�gersen, Ida B; Vanthournout, Bram; Wang, Tobias; Zhu, Yabing; Funch, Peter; Enghild, Jan J; Schauser, Leif; Andersen, Stig U; Villesen, Palle; Schierup, Mikkel H; Bilde, Trine; Wang, Jun
Isolation of a Clone Encoding a Second Dragline Silk Fibroin. (Reannouncement with New Availability Information).
National Technical Information Service (NTIS)
Spider dragline silk is a unique protein fiber possessing both high tensile strength and high elasticity. A partial cDNA clone for one dragline silk protein (Spidroin 1) was previously isolated. However, the predicted amino acid sequence could not account...
M. B. Hinnman R. V. Lewis
Structural Properties of Silk Electro-Gels
NASA Astrophysics Data System (ADS)
The interest in Bombyx Mori silk emerges from its biocompatibility and its structural superiority to synthetic polymers. Our particular interest lies in understanding the capabilities of silk electro-gels because of their reversibility and tunable adhesion. We create an electro-gel by applying a DC electric potential across a reconstituted silk fibroin solution derived directly from Bombyx Mori cocoons. This process leads to the intermolecular self-assembly of fibroin proteins into a weak gel. In this talk we will present our results on the effects of applied shear on electro-gels. We quantify the structural properties while dynamically imaging shear induced fiber formation; known as fibrillogenesis. It is observed that the mechanical properties and microstructure of these materials are highly dependent on shear history. We will also discuss the role of surface modification, through micro-patterning, on the observed gel structure. Our results provide an understanding of both the viscoelastiticity and microstucture of reconstituted silks that are being utilized as tissue scaffolds.
Tabatabai, A. P.; Urbach, J. S.; Blair, D. L.; Kaplan, D. L.
Hierarchical Chain Model of Spider Capture Silk Elasticity
NASA Astrophysics Data System (ADS)
Spider capture silk is a biomaterial with both high strength and high elasticity, but the structural design principle underlying these remarkable properties is still unknown. It was revealed recently by atomic force microscopy that an exponential force-extension relationship holds both for capture silk mesostructures and for intact capture silk fibers [N. Becker et al.,
Zhou, Haijun; Zhang, Yang
Sericin cream reduces pruritus in hemodialysis patients: a randomized, double-blind, placebo-controlled experimental study
Background Uremic pruritus (UP) is a significant complication in ESRD patients and substantially impairs their quality of life. UP is considered to be a skin manifestation of chronic inflammation. Because sericin can suppress the release of pro-inflammatory cytokines, the purpose of this study was to investigate the short-term safety and efficacy of sericin cream for treating UP in hemodialysis patients. Methods This study used a double-blind design to investigate the effects of random topical administration of sericin cream and cream base (placebo) on either the right or left extremities of hemodialysis patients for 6?weeks. Skin hydration, irritation and pigmentation were evaluated every 2?weeks using Skin Diagnostic SD27. The visual analog scale for itching was also evaluated every 2?weeks, and the Kidney Disease Quality of Life Short Form was performed on the day of each patient�s enrollment and after 6?weeks of treatment. Results Fifty dialysis patients were enrolled, 47 of which completed the study. The hydration of the skin of the patients� extremities increased significantly after administration of sericin cream; significant differences were found between sericin treatment and control after 6?weeks of treatment (p?=?0.041 for arms and p?=?0.022 for legs, respectively). Moreover, a significant difference was also found in skin irritation between the two treatments (p?=?0.013 for arms and p?=?0.027 for legs, respectively). At the end of the study, the skin pigmentation level was significantly reduced on both the arms (p?=?0.032) and legs (p?=?0.021) of the sericin-treated side compared with the side treated with cream base. The mean itching score decreased significantly from moderate to severe at the time of enrollment to mild pruritus after 6?weeks of treatment (p?=?0.002). A better quality of life was found in all domains tested although statistically significant differences before and after treatment was found only in the patients� pain scores, the effect of kidney disease on daily life, sleep quality and symptoms or problems related to kidney disease. Conclusions We conclude that sericin cream has a high potential for reducing UP in hemodialysis patients. The trial registration number of this study is ISRCTN16019033; its public title is �sericin cream reduces pruritus in hemodialysis patients�.
Functional silk: colored and luminescent.
Silkworm silk is among the most widely used natural fibers for textile and biomedical applications due to its extraordinary mechanical properties and superior biocompatibility. A number of physical and chemical processes have also been developed to reconstruct silk into various forms or to artificially produce silk-like materials. In addition to the direct use and the delicate replication of silk's natural structure and properties, there is a growing interest to introduce more new functionalities into silk while maintaining its advantageous intrinsic properties. In this review we assess various methods and their merits to produce functional silk, specifically those with color and luminescence, through post-processing steps as well as biological approaches. There is a highlight on intrinsically colored and luminescent silk produced directly from silkworms for a wide range of applications, and a discussion on the suitable molecular properties for being incorporated effectively into silk while it is being produced in the silk gland. With these understanding, a new generation of silk containing various functional materials (e.g., drugs, antibiotics and stimuli-sensitive dyes) would be produced for novel applications such as cancer therapy with controlled release feature, wound dressing with monitoring/sensing feature, tissue engineering scaffolds with antibacterial, anticoagulant or anti-inflammatory feature, and many others. PMID:22302383
Tansil, Natalia C; Koh, Leng Duei; Han, Ming-Yong
Existing and proposed mechanisms for digital money all require large overhead to transfer money between parties. This overhead makes them unsuitable for extremely low cost activities, such as delivering and routing packets. The digital silk road is a proposed money system with extremely low transaction cost built into the communication protocols. The money introduced by this system is much more
Norman Hardy; Eric Dean Tribble
Osteogenic and adipogenic differentiation of rat bone marrow cells on non-mulberry and mulberry silk gland fibroin 3D scaffolds
This study investigates the potential of 3D silk scaffolds fabricated using tropical tasar non-mulberry, Antheraea mylitta and mulberry, Bombyx mori silk gland fibroin proteins as substrate for osteogenic and adipogenic differentiation of rat bone marrow cells (BMCs). The scaffolds are mechanically robust and show homogenous pore distribution with high porosity and interconnected pore walls. Low immunogenicity of fabricated silk scaffolds
Biman B. Mandal; Subhas C. Kundu
Flexibility regeneration of silk fibroin in vitro
Although natural silk fibers have excellent strength and flexibility, the regenerated silk materials generally become brittle in the dry state. How to reconstruct the flexibility for silk fibroin has bewildered scientists for many years. In the present study, the flexible regenerated silk fibroin films were achieved by simulating the natural forming and spinning process. Silk fibroin films composed of silk I structure were firstly prepared by slow drying process. Then the silk fibroin films were stretched in the wet state, following the structural transition from silk I to silk II. The difference between the flexible film and different brittle regenerated films was investigated to reveal the critical factors in regulating the flexibility of regenerated silk materials. Compared to the methanol-treated silk films, although having similar silk II structure and water content, the flexible silk films contained more bound water rather than free water, implying the great influence of bound water on the flexibility. Then, further studies revealed that the distribution of bound water was also a critical factor in improving silk flexibility in the dry state, which could be regulated by the nano-assembly of silk fibroin. Importantly, the results further elucidate the relation between mechanical properties and silk fibroin structures, pointing to a new mode of generating new types of silk materials with enhanced mechanical properties in the dry state, which would facilitate the fabrication and application of regenerated silk fibroin materials in different fields.
Zhang, Cencen; Song, Dawei; Lu, Qiang; Hu, Xiao; Kaplan, David L; Zhu, Hesun
Electrospun Silk Biomaterial Scaffolds for Regenerative Medicine
Electrospinning is a versatile technique that enables the development of nanofiber-based biomaterial scaffolds. Scaffolds can be generated that are useful for tissue engineering and regenerative medicine since they mimic the nanoscale properties of certain fibrous components of the native extracellular matrix in tissues. Silk is a natural protein with excellent biocompatibility, remarkable mechanical properties as well as tailorable degradability. Integrating these protein polymer advantages with electrospinning results in scaffolds with combined biochemical, topographical and mechanical cues with versatility for a range of biomaterial, cell and tissue studies and applications. This review covers research related to electrospinning of silk, including process parameters, post treatment of the spun fibers, functionalization of nanofibers, and the potential applications for these material systems in regenerative medicine. Research challenges and future trends are also discussed.
Zhang, Xiaohui; Reagan, Michaela R; Kaplan, David L.
Spider silk reduces insect herbivory
The role of predators in food webs extends beyond their ability to kill and consume prey. Such trait-mediated effects occur when signals of the predator influence the behaviour of other animals. Because all spiders are silk-producing carnivores, we hypothesized that silk alone would signal other arthropods and enhance non-lethal effects of spiders. We quantified the herbivory inflicted by two beetle species on green bean plants (Phaseolus vulgaris) in the presence of silkworm silk and spider silk along with no silk controls. Single leaflets were treated and enclosed with herbivores in the laboratory and field. Another set of leaflets were treated and left to experience natural herbivory in the field. Entire plants in the field were treated with silk and enclosed with herbivores or left exposed to herbivory. In all cases, the lowest levels of herbivory occurred with spider silk treatments and, in general, silkworm silk produced intermediate levels of leaf damage. These results suggest that silk may be a mechanism for the trait-mediated impacts of spiders and that it might contribute to integrated pest management programmes.
Rypstra, Ann L.; Buddle, Christopher M.
Relationships between supercontraction and mechanical properties of spider silk
NASA Astrophysics Data System (ADS)
Typical spider dragline silk tends to outperform other natural fibres and most man-made filaments. However, even small changes in spinning conditions can have large effects on the mechanical properties of a silk fibre as well as on its water uptake. Absorbed water leads to significant shrinkage in an unrestrained dragline fibre and reversibly converts the material into a rubber. This process is known as supercontraction and may be a functional adaptation for the silk's role in the spider's web. Supercontraction is thought to be controlled by specific motifs in the silk proteins and to be induced by the entropy-driven recoiling of molecular chains. In analogy, in man-made fibres thermal shrinkage induces changes in mechanical properties attributable to the entropy-driven disorientation of `unfrozen' molecular chains (as in polyethylene terephthalate) or the `broken' intermolecular hydrogen bonds (as in nylons). Here we show for Nephila major-ampullate silk how in a biological fibre the spinning conditions affect the interplay between shrinkage and mechanical characteristics. This interaction reveals design principles linking the exceptional properties of silk to its molecular orientation.
Liu, Yi; Shao, Zhengzhong; Vollrath, Fritz
The effects of corn silk on glycaemic metabolism
Background Corn silk contains proteins, vitamins, carbohydrates, Ca, K, Mg and Na salts, fixed and volatile oils, steroids such as sitosterol and stigmasterol, alkaloids, saponins, tannins, and flavonoids. Base on folk remedies, corn silk has been used as an oral antidiabetic agent in China for decades. However, the hypoglycemic activity of it has not yet been understood in terms of modern pharmacological concepts. The purpose of this study is to investigate the effects of corn silk on glycaemic metabolism. Methods Alloxan and adrenalin induced hyperglycemic mice were used in the study. The effects of corn silk on blood glucose, glycohemoglobin (HbA1c), insulin secretion, damaged pancreatic ?-cells, hepatic glycogen and gluconeogenesis in hyperglycemic mice were studied respectively. Results After the mice were orally administered with corn silk extract, the blood glucose and the HbA1c were significantly decreased in alloxan-induced hyperglycemic mice (p < 0.05, p < 0.01, respectively), while the level of insulin secretionn was markedly elevated in alloxa-induced hyperglycemic mice (p < 0.05). The alloxan-damaged pancreatic ?-cells of the mice were partly recovered gradually after the mice were administered with corn silk extract 15 days later. Also, the body weight of the alloxan-induced hyperglycemic mice was increased gradually. However, ascension of blood glucose induced by adrenalin and gluconeogenesis induced by L-alanine were not inhibited by corn silk extract treatment (p > 0.05). Although corn silk extract increased the level of hepatic glycogen in the alloxan-induced hyperglycemic mice, there was no significant difference between them and that of the control group(p > 0.05). Conclusion Corn silk extract markedly reduced hyperglycemia in alloxan-induced diabetic mice. The action of corn silk extract on glycaemic metabolism is not via increasing glycogen and inhibiting gluconeogenesis but through increasing insulin level as well as recovering the injured ?-cells. The results suggest that corn silk extract may be used as a hypoglycemic food or medicine for hyperglycemic people in terms of this modern pharmacological study.
Ingrowth of Human Mesenchymal Stem Cells into Porous Silk Particle Reinforced Silk Composite Scaffolds: An In Vitro Study
Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous 3D silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (nonreinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to six weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microCT (?CT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per ?g of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1 and 1:2, respectively. In addition, ?CT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for nonreinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes.
Rockwood, Danielle N.; Gil, Eun Seok; Park, Sang-Hyug; Kluge, Jonathan A.; Grayson, Warren; Bhumiratana, Sarindr; Rajkhowa, Rangam; Wang, Xungai; Kim, Sung Jun; Vunjak-Novakovic, Gordana; Kaplan, David L
Properties and potential medical applications of silk fibers produced by Rothischildia lebeau.
Rothischildia lebeau which belongs to the Saturniidae family of silk-producing insects secretes protein fibers with properties between that of the Bombyx mori and the common wild silks. Traditionally, wild silks produced by insects such as Antheraea mylitta are considerably coarser and have inferior tensile properties than the domesticated and most commonly used silk produced by B. mori. Recently, it has been demonstrated that some of the wild silks have unique properties and preferable for medical applications. Wild silks are comparatively easier to rear, produce larger cocoons, and could have unique properties. In this research, the structure and properties of the silk fibers produced by R. lebeau were studied and the potential of using the fibers for medical applications was investigated. Fibers produced by R. lebeau had average tensile strength of 3.3?g/den, similar to that of wild silks but lower than that of the B. mori silk. R. lebeau fibers were biocompatible and showed potential to be useful for tissue engineering and other medical applications. PMID:23594071
Reddy, Narendra; Jiang, Qiuran; Yang, Yiqi
Tuning chemical and physical cross-links in silk electrogels for morphological analysis and mechanical reinforcement.
Electrochemically controlled, reversible assembly of biopolymers into hydrogel structures is a promising technique for on-demand cell or drug encapsulation and release systems. An electrochemically sol-gel transition has been demonstrated in regenerated Bombyx mori silk fibroin, offering a controllable way to generate biocompatible and reversible adhesives and other biomedical materials. Despite the involvement of an electrochemically triggered electrophoretic migration of the silk molecules, the mechanism of the reversible electrogelation remains unclear. It is, however, known that the freshly prepared silk electrogels (e-gels) adopt a predominantly random coil conformation, indicating a lack of cross-linking as well as thermal, mechanical, and morphological stabilities. In the present work, the tuning of covalent and physical ?-sheet cross-links in silk hydrogels was studied for programming the structural properties. Scanning electron microscopy (SEM) revealed delicate morphology, including locally aligned fibrillar structures, in silk e-gels, preserved by combining glutaraldehyde-cross-linking and ethanol dehydration. Fourier transform infrared (FTIR) spectroscopic analysis of either electrogelled, vortex-induced or spontaneously formed silk hydrogels showed that the secondary structure of silk e-gels was tunable between non-?-sheet-dominated and ?-sheet-dominated states. Dynamic oscillatory rheology confirmed the mechanical reinforcement of silk e-gels provided by controlled chemical and physical cross-links. The selective incorporation of either chemical or physical or both cross-links into the electrochemically responsive, originally unstructured silk e-gel should help in the design for electrochemically responsive protein polymers. PMID:23859710
Lin, Yinan; Xia, Xiaoxia; Shang, Ke; Elia, Roberto; Huang, Wenwen; Cebe, Peggy; Leisk, Gary; Omenetto, Fiorenzo; Kaplan, David L
Silk constructs for delivery of muskuloskeletal therapeutics
Silk fibroin (SF) is a biopolymer with distinguishing features from many other bio- as well as synthetic polymers. From a biomechanical and drug delivery perspective, SF combines remarkable versatility for scaffolding (solid implants, hydrogels, threads, solutions), with advanced mechanical properties and good stabilization and controlled delivery of entrapped protein and small molecule drugs, respectively. It is this combination of mechanical and pharmaceutical features which render SF so exciting for biomedical applications. his pattern along with the versatility of this biopolymer have been translated into progress for musculoskeletal applications. We review the use and potential of silk fibroin for systemic and localized delivery of therapeutics in diseases affecting the musculoskeletal system. We also present future directions for this biopolymer as well as the necessary research and development steps for their achievement.
Meinel, Lorenz; Kaplan, David L.
Materials: Surprising strength of silkworm silk
Commercial silkworm silk is presumed to be much weaker and less extensible than spider dragline silk, which has been hailed as a 'super-fibre'. But we show here that the mechanical properties of silkworm silks can approach those of spider dragline silk when reeled under controlled conditions. We suggest that silkworms might be able to produce threads that compare well with
Zhengzhong Shao; Fritz Vollrath
Enhancing the interface in silk-polypyrrole composites through chemical modification of silk fibroin.
To produce conductive, biocompatible, and mechanically robust materials for use in bioelectrical applications, we have developed a new strategy to selectively incorporate poly(pyrrole) (Ppy) into constructs made from silk fibroin. Here, we demonstrate that covalent attachment of negatively charged, hydrophilic sulfonic acid groups to the silk protein can selectively promote pyrrole absorption and polymerization within the modified films to form a conductive, interpenetrating network of Ppy and silk that is incapable of delamination. To further increase the conductivity and long-term stability of the Ppy network, a variety of small molecule sulfonic acid dopants were utilized and the properties of these silk-conducting polymer composites were monitored over time. The composites were evaluated using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), optical microscopy, energy-dispersive X-ray (EDX) spectroscopy, cyclic voltammetry, a 4-point resistivity probe and mechanical testing. In addition, the performance was evaluated following exposure to several biologically relevant enzymes. Using this strategy, we were able to produce mechanically robust polymer electrodes with stable electrochemical performance and sheet resistivities on the order of 1 � 10(2) ?/sq (conductivity ?1 S/cm). PMID:23320759
Romero, Isabella S; Schurr, Morgan L; Lally, Jack V; Kotlik, Mitchell Z; Murphy, Amanda R
Evidence of ?-helical coiled coils and ?-sheets in hornet silk.
?-Helical coiled coil and ?-sheet complexes are essential structural building elements of silk proteins produced by different species of the Hymenoptera. Beside X-ray scattering at wide and small angles we applied cryo-electron diffraction and microscopy to demonstrate the presence and the details of such structures in silk of the giant hornet Vespa mandarinia japonica. Our studies on the assembly of the fibrous silk proteins and their internal organization in relation to the primary chain structure suggest a 172 � pitch supercoil consisting of four intertwined alanine-rich ?-helical strands. The axial periodicity may adopt even multiples of the pitch value. Coiled coil motifs form the largest portion of the hornet silk structure and are aligned nearly parallel to the cocoon fiber axis in the same way as the membrane-like parts of the cocoon are molecularly orientated in the spinning direction. Supercoils were found to be associated with ?-crystals, predominantly localized in the l-serine-rich chain sequences terminating each of the four predominant silk proteins. Such ?-sheet blocks are considered resulting from transformation of random coil molecular sequences due to the action of elongational forces during the spinning process. PMID:24345346
Kameda, Tsunenori; Nemoto, Takashi; Ogawa, Tetsuya; Tosaka, Masatoshi; Kurata, Hiroki; Schaper, Andreas K
Thermovoltaic properties of hornet silk.
In silk from the larval silk caps of the Oriental hornet Vespa orientalis (Hymenoptera, Vespinae), temperature-dependent changes in the electric voltage have been recorded, with rise in the voltage occurring mainly upon rise in the temperature between 10-36 degrees C. The peak voltage was measured between 32-38 degrees C and attained 240-360 mV, but with further increase in temperature, the voltage decreased, dropping to 0 mV at about 45-50 degrees C. Upon second measurement (of same silk specimen), the voltage peak usually occurred later (by 8-9 degrees C) and at higher temperature than in the first measurement. Continuous measurements during warming up to 30 degrees C followed by cooling down to 15 degrees C yielded an hysteresis between the warming "line" and the cooling "line", the former often straight and the latter usually curved. Maintaining the silk specimen at a fixed temperature for a prolonged period (hours) initially causes the voltage to rise, then remain steady, and finally drop. Boiling the silk caps in tap water for 7-10 min exerts some changes in the silk properties, mainly a decrease in voltage level. The general behavior of the silk suggests that it is a polymer endowed with the qualities of an organic semiconductor. The various properties of the larval silk are discussed in great detail. PMID:17029238
Volynchik, S; Plotkin, M; Ermakov, N Y; Bergman, D J; Ishay, J S
Synthetic spider silk production on a laboratory scale.
As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks. PMID:22847722
Hsia, Yang; Gnesa, Eric; Pacheco, Ryan; Kohler, Kristin; Jeffery, Felicia; Vierra, Craig
Synthetic Spider Silk Production on a Laboratory Scale
As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.
Hsia, Yang; Gnesa, Eric; Pacheco, Ryan; Kohler, Kristin; Jeffery, Felicia; Vierra, Craig
Silk fibroin as a vehicle for drug delivery applications
Silk fibroin (SF), a naturally occurring protein polymer, has several unique properties making it a favorable matrix for the incorporation and delivery of a range of therapeutic agents. SF is biocompatible, slowly biodegradable, and endowed with excellent mechanical properties and processability. Novel manufacturing techniques including mild all-aqueous processes have expanded its range of application even to sensitive protein and nucleic
Esther Wenk; Hans P. Merkle; Lorenz Meinel
Shear-induced rigidity in spider silk glands
NASA Astrophysics Data System (ADS)
We measure the elastic stiffnesses of the concentrated viscous protein solution of the dehydrated Nephila clavipes major ampullate gland with Brillouin light scattering. The glandular material shows no rigidity but possesses a tensile stiffness similar to that of spider silk. We show, however, that with application of a simple static shear, the mechanical properties of the spider gland protein mixture can be altered irreversibly, lowering symmetry and enabling shear waves to be supported, thus, giving rise to rigidity and yielding elastic properties similar to those of the naturally spun (i.e., dynamically sheared) silk.
Koski, Kristie J.; McKiernan, Keri; Akhenblit, Paul; Yarger, Jeffery L.
Accelerated in vitro Degradation of Optically Clear Low ?-sheet Silk Films by Enzyme-Mediated Pretreatment
Purpose To design patterned, transparent silk films with fast degradation rates for the purpose of tissue engineering corneal stroma, Methods ?-sheet (crystalline) content of silk films was decreased significantly by using a short water annealing time. Additionally, a protocol combining short water annealing time with enzymatic pretreatment of silk films with protease XIV was developed. Results Low ?-sheet content (17�18%) and enzymatic pre-treatment provided film stability in aqueous environments and accelerated degradation of the silk films in the presence of human corneal fibroblasts in vitro. The results demonstrate a direct relationship between reduced ?-sheet content and enzymatic pre-treatment and overall degradation rate of the protein films. Conclusions The novel protocol developed here provides new approaches to modulate the regeneration rate of silk biomaterials for corneal tissue regeneration needs. Translational relevance Patterned silk protein films possess desirable characteristics for corneal tissue engineering, including optical transparency, biocompatibility, cell alignment and tunable mechanical properties, but current fabrication protocols do not provide adequate degradation rates to match the regeneration properties of the human cornea. This novel processing protocol makes silk films more suitable for the construction of human corneal stroma tissue and a promising way to tune silk film degradation properties to match corneal tissue regeneration.
Shang, Ke; Rnjak-Kovacina, Jelena; Lin, Yinan; Hayden, Rebecca S.; Hu, Tao; Kaplan, David L.
Uncovering spider silk nanocrystalline variations that facilitate wind-induced mechanical property changes.
Spider major ampullate (MA) silk varies in mechanical properties when spun in different environments. Amino acid compositional changes induced by variations in MaSp1 and MaSp2 expression, and various biochemical and physiological glandular processes induce silk property variability. Quantifying the contributions of these mechanisms on silk variability may facilitate the development of silk biomimetics. Wind is a medium that induces variations in MA silk mechanics. We exposed the spider Cyclosa mulmeinensis to wind and measured the amino acid composition, tensile mechanics, and crystalline structure of its MA silk using HPLC, tensile tests, and X-ray diffraction. We found the mechanical properties of MA silks from spiders exposed to wind to differ from unexposed spiders. The amino acid compositions did not differ, but X-ray diffraction found a lower crystal density and greater ?-sheet alignment relative to the fiber axis in the silks of spiders exposed to wind. We found no evidence that the mechanical property variations were a product of profound changes to the alignment of the protein within the amorphous region. We conclude that variations in the density and alignment of the crystalline ?-sheets, probably accompanied by some alignment change in the amorphous region as a result of "stretching" during spinning of the silk, probably explains the mechanical property variations that we found across treatment subgroups. As C. mulmeinensis MA silk increases both in strength and elasticity when the spiders are exposed to wind, bioengineers may consider it as a model for the development of high-performance silk biomimetics. PMID:23947397
Blamires, Sean J; Wu, Chao-Chia; Wu, Chung-Lin; Sheu, Hwo-Shuenn; Tso, I-Min
Micromolar biosensing of nitric oxide using myoglobin immobilized in a synthetic silk film.
In this work we investigate the use of coiled-coil silk proteins, produced in recombinant Escherichia coli, as a new material for immobilizing biosensors. Myoglobin was embedded in transparent honeybee silk protein films. Immobilized myoglobin maintained a high affinity for nitric oxide (KD NO=52�M) and good sensitivity with a limit of detection of 5�M. The immobilized myoglobin-silk protein film was stable and could be stored as a dry film at room temperature for at least 60 days. The effect of immobilization on the structure of myoglobin was fully investigated using UV/visible, Fourier Transform Infrared and Raman spectroscopy, which indicated a weakening in the strength of the iron-histidine bond. This study demonstrates that recombinant coiled-coil silk proteins provide a safe and environmentally friendly alternative to sol-gels for stabilizing heme proteins for use as optical biosensors. PMID:25014754
Rapson, Trevor D; Church, Jeffrey S; Trueman, Holly E; Dacres, Helen; Sutherland, Tara D; Trowell, Stephen C
Identification and dynamics of polyglycine II nanocrystals in Argiope trifasciata flagelliform silk
NASA Astrophysics Data System (ADS)
Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine ?-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the -GGX- and -GPG- motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract.
Perea, G. B.; Riekel, C.; Guinea, G. V.; Madurga, R.; Daza, R.; Burghammer, M.; Hayashi, C.; Elices, M.; Plaza, G. R.; P�rez-Rigueiro, J.
An emerging functional natural silk biomaterial from the only domesticated non-mulberry silkworm Samia ricini.
Mulberry silk fibroin is a widely used biomaterial and recent work on non-mulberry silk fibroin also suggests it may have similar uses. We expect silk fibroin from the only domesticated non-mulberry eri silkworm, Samia ricini, to possess useful properties as a biomaterial. Eri silk gland fibroin is a heterodimeric protein of approximately 450?kDa. Cytocompatibility evaluation with fibroblasts and osteoblast-like cells shows good cell attachment, viability and proliferation. The matrices, which have high thermal stability and good swellability, are also haemocompatible. Eri silk production is cost effective as no agronomic practices are required for their host plant cultivation. This fibroin provide new opportunities as an alternative natural functional biomaterial in various biomedical applications. PMID:23733347
Pal, Shilpa; Kundu, Joydip; Talukdar, Sarmistha; Thomas, Tintu; Kundu, Subhas C
Identification and dynamics of polyglycine II nanocrystals in Argiope trifasciata flagelliform silk
Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine ?-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the �GGX� and �GPG� motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract.
Perea, G. B.; Riekel, C.; Guinea, G. V.; Madurga, R.; Daza, R.; Burghammer, M.; Hayashi, C.; Elices, M.; Plaza, G. R.; Perez-Rigueiro, J.
Identification and dynamics of polyglycine II nanocrystals in Argiope trifasciata flagelliform silk.
Spider silks combine a significant number of desirable characteristics in one material, including large tensile strength and strain at breaking, biocompatibility, and the possibility of tailoring their properties. Major ampullate gland silk (MAS) is the most studied silk and their properties are explained by a double lattice of hydrogen bonds and elastomeric protein chains linked to polyalanine ?-nanocrystals. However, many basic details regarding the relationship between composition, microstructure and properties in silks are still lacking. Here we show that this relationship can be traced in flagelliform silk (Flag) spun by Argiope trifasciata spiders after identifying a phase consisting of polyglycine II nanocrystals. The presence of this phase is consistent with the dominant presence of the -GGX- and -GPG- motifs in its sequence. In contrast to the passive role assigned to polyalanine nanocrystals in MAS, polyglycine II nanocrystals can undergo growing/collapse processes that contribute to increase toughness and justify the ability of Flag to supercontract. PMID:24162473
Perea, G B; Riekel, C; Guinea, G V; Madurga, R; Daza, R; Burghammer, M; Hayashi, C; Elices, M; Plaza, G R; P�rez-Rigueiro, J
Physical characterization of functionalized spider silk: electronic and sensing properties
NASA Astrophysics Data System (ADS)
This work explores functional, fundamental and applied aspects of naturally harvested spider silk fibers. Natural silk is a protein polymer where different amino acids control the physical properties of fibroin bundles, producing, for example, combinations of ?-sheet (crystalline) and amorphous (helical) structural regions. This complexity presents opportunities for functional modification to obtain new types of material properties. Electrical conductivity is the starting point of this investigation, where the insulating nature of neat silk under ambient conditions is described first. Modification of the conductivity by humidity, exposure to polar solvents, iodine doping, pyrolization and deposition of a thin metallic film are explored next. The conductivity increases exponentially with relative humidity and/or solvent, whereas only an incremental increase occurs after iodine doping. In contrast, iodine doping, optimal at 70?�C, has a strong effect on the morphology of silk bundles (increasing their size), on the process of pyrolization (suppressing mass loss rates) and on the resulting carbonized fiber structure (that becomes more robust against bending and strain). The effects of iodine doping and other functional parameters (vacuum and thin film coating) motivated an investigation with magic angle spinning nuclear magnetic resonance (MAS-NMR) to monitor doping-induced changes in the amino acid-protein backbone signature. MAS-NMR revealed a moderate effect of iodine on the helical and ?-sheet structures, and a lesser effect of gold sputtering. The effects of iodine doping were further probed by Fourier transform infrared (FTIR) spectroscopy, revealing a partial transformation of ?-sheet-to-amorphous constituency. A model is proposed, based on the findings from the MAS-NMR and FTIR, which involves iodine-induced changes in the silk fibroin bundle environment that can account for the altered physical properties. Finally, proof-of-concept applications of functionalized spider silk are presented for thermoelectric (Seebeck) effects and incandescence in iodine-doped pyrolized silk fibers, and metallic conductivity and flexibility of micron-sized gold-sputtered silk fibers. In the latter case, we demonstrate the application of gold-sputtered neat spider silk to make four-terminal, flexible, ohmic contacts to organic superconductor samples.
Steven, Eden; Park, Jin Gyu; Paravastu, Anant; Branco Lopes, Elsa; Brooks, James S.; Englander, Ongi; Siegrist, Theo; Kaner, Papatya; Alamo, Rufina G.
Physical Characterization of Functionalized Silk Material for Electronic Application and Devices
NASA Astrophysics Data System (ADS)
Naturally harvested spider silk fibers are investigated for their physical properties under ambient, humidified, iodine-doped, pyrolized, sputtered gold and carbon nanotube coated conditions. The functional properties include: humidity activated conductivity; enhanced flexibility and carbon yield of pyrolized iodized silk fibers; full metallic conductivity and flexibility of micron-sized gold-sputtered silk fibers; and high strain sensitivity of carbon nanotube coated silk fibers. Magic angle spinning nuclear magnetic resonance (MAS-NMR) and Fourier transform infrared spectroscopy (FTIR) are used to explore the nature of ambient and functionalized spider silk fiber, and significant changes in amino acid-protein backbone signature are correlated with gold sputtering, and iodine-doped conditions. The application of gold-sputtered neat spider silk fibers for making four terminal flexible, clean, ohmic contacts to organic superconductor samples and carbon nanotube coated silk fibers for heart pulse monitoring sensor are demonstrated. The role of silk thin film in organic thin film transistor will be briefly discussed.
Steven, Eden; Jobiliong, Eric; Park, Jin Gyu; Paravastu, Anant; Davidson, Michael; Baird, Michelle; Alamo, Rufina; Kaner, Papatya; Brooks, James; Siegrist, Theo
All-water-based electron-beam lithography using silk as a resist
NASA Astrophysics Data System (ADS)
Traditional nanofabrication techniques often require complex lithographic steps and the use of toxic chemicals. To move from the laboratory scale to large scales, nanofabrication should be carried out using alternative procedures that are simple, inexpensive and use non-toxic solvents. Recent efforts have focused on nanoimprinting and the use of organic resists (such as quantum dot-polymer hybrids, DNA and poly(ethylene glycol)), which still require, for the most part, noxious chemicals for processing. Significant advances have been achieved using `green' resists that can be developed with water, but so far these approaches have suffered from low electron sensitivity, line edge roughness and scalability constraints. Here, we present the use of silk as a natural and biofunctional resist for electron-beam lithography. The process is entirely water-based, starting with the silk aqueous solution and ending with simple development of the exposed silk film in water. Because of its polymorphic crystalline structure, silk can be used either as a positive or negative resist through interactions with an electron beam. Moreover, silk can be easily modified, thereby enabling a variety of `functional resists', including biologically active versions. As a proof of principle of the viability of all-water-based silk electron-beam lithography (EBL), we fabricate nanoscale photonic lattices using both neat silk and silk doped with quantum dots, green fluorescent proteins (GFPs) or horseradish peroxidase (HRP).
Kim, Sunghwan; Marelli, Benedetto; Brenckle, Mark A.; Mitropoulos, Alexander N.; Gil, Eun-Seok; Tsioris, Konstantinos; Tao, Hu; Kaplan, David L.; Omenetto, Fiorenzo G.
Smart assembly of polymer fibers: lessons from major ampullate spider silk
NASA Astrophysics Data System (ADS)
Studies of major ampullate silk (MAS), especially the secretions and fibers produced by the spider Nephila clavipes (golden orb weaver), have yielded several results of potential value to the materials scientist/engineer. There are lessons to be learned about synthesis, processing and microstructural design of high-tensile polymer fibers. The 'smart' aspect of silk production in nature concerns the ability of the spider to rapidly process a concentrated, viscous aqueous solution of silk protein (stored in the gland) into water-insoluble fiber on demand. This process centers on the assembly of a shear-sensitive supramolecular liquid crystalline phase by aggregation of the solubilized globular protein molecules.
Viney, Christopher
Neural responses to electrical stimulation on patterned silk films.
Peripheral nerve injury is a critical issue for patients with trauma. Following injury, incomplete axon regeneration or misguided axon innervation into tissue will result in loss of sensory and motor functions. The objective of this study was to examine axon outgrowth and axon alignment in response to surface patterning and electrical stimulation. To accomplish our objective, metal electrodes with dimensions of 1.5 mm � 4 cm, were sputter coated onto micropatterned silk protein films, with surface grooves 3.5 ?m wide � 500 nm deep. P19 neurons were seeded on the patterned electronic silk films and stimulated at 120 mV, 1 kHz, for 45 min each day for 7 days. Responses were compared with neurons on flat electronic silk films, patterned silk films without stimulation, and flat silk films without stimulation. Significant alignment was found on the patterned film groups compared with the flat film groups. Axon outgrowth was greater (p < 0.05) on electronic films on days 5 and 7 compared with the unstimulated groups. In conclusion, electrical stimulation, at 120 mV, 1 kHz, for 45 min daily, in addition to surface patterning, of 3.5 ?m wide � 500 nm deep grooves, offered control of nerve axon outgrowth and alignment. PMID:23401351
Hronik-Tupaj, Marie; Raja, Waseem Khan; Tang-Schomer, Min; Omenetto, Fiorenzo G; Kaplan, David L
Sporicidal/bactericidal textiles via the chlorination of silk.
Bacterial spores, such as those of the Bacillus genus, are extremely resilient, being able to germinate into metabolically active cells after withstanding harsh environmental conditions or aggressive chemical treatments. The toughness of the bacterial spore in combination with the use of spores, such as those of Bacillus anthracis, as a biological warfare agent necessitates the development of new antimicrobial textiles. In this work, a route to the production of fabrics that kill bacterial spores and cells within minutes of exposure is described. Utilizing this facile process, unmodified silk cloth is reacted with a diluted bleach solution, rinsed with water, and dried. The chlorination of silk was explored under basic (pH 11) and slightly acidic (pH 5) conditions. Chloramine-silk textiles prepared in acidified bleach solutions were found to have superior breaking strength and higher oxidative Cl contents than those prepared under caustic conditions. Silk cloth chlorinated for ?1 h at pH 5 was determined to induce >99.99996% reduction in the colony forming units of Escherichia coli, as well as Bacillus thuringiensis Al Hakam (B. anthracis simulant) spores and cells within 10 min of contact. The processing conditions presented for silk fabric in this study are highly expeditionary, allowing for the on-site production of protein-based antimicrobial materials from a variety of agriculturally produced feed-stocks. PMID:22352921
Dickerson, Matthew B; Lyon, Wanda; Gruner, William E; Mirau, Peter A; Slocik, Joseph M; Naik, Rajesh R
Functionalized silk-based biomaterials for bone formation.
Silks are being reassessed as biomaterial scaffolds due to their unique mechanical properties, opportunities for genetic tailoring of structure and thus function, and recent studies clarifying biocompatibility. We report on the covalent decoration of silk films with integrin recognition sequences (RGD) as well as parathyroid hormone (PTH, 1-34 amino acids) and a modified PTH 1-34 (mPTH) involved in the induction of bone formation. Osteoblast-like cell (Saos-2) responses to the decorated silk films indicate that the proteins serve as suitable bone-inducing matrices. Osteoblast-like cell adhesion was significantly increased on RGD and PTH compared to plastic, mPTH, and the control peptide RAD. At 2 weeks of culture, message levels of alkaline phosphatase were similar on all substrates, but by 4 weeks, alkaline phosphatase mRNA was greatest on RGD. At 2 weeks of culture, alpha 1(I) procollagen mRNA was elevated on silk, RGD, RAD, and PTH, and hardly detectable on mPTH and plastic. However, by 4 weeks RGD demonstrated the highest level compared to the other substrates. Osteocalcin message levels detected by RT-PCR were greatest on RGD at both time points. Calcification was also significantly elevated on RGD compared to the other substrates with an increase in number and size of the mineralized nodules in culture. Thus, RGD covalently decorated silk appears to stimulate osteoblast-based mineralization in vitro. PMID:11077413
Sofia, S; McCarthy, M B; Gronowicz, G; Kaplan, D L
Carbon nanotubes on a spider silk scaffold.
Understanding the compatibility between spider silk and conducting materials is essential to advance the use of spider silk in electronic applications. Spider silk is tough, but becomes soft when exposed to water. Here we report a strong affinity of amine-functionalised multi-walled carbon nanotubes for spider silk, with coating assisted by a water and mechanical shear method. The nanotubes adhere uniformly and bond to the silk fibre surface to produce tough, custom-shaped, flexible and electrically conducting fibres after drying and contraction. The conductivity of coated silk fibres is reversibly sensitive to strain and humidity, leading to proof-of-concept sensor and actuator demonstrations. PMID:24022336
Steven, Eden; Saleh, Wasan R; Lebedev, Victor; Acquah, Steve F A; Laukhin, Vladimir; Alamo, Rufina G; Brooks, James S
Carbon nanotubes on a spider silk scaffold
Understanding the compatibility between spider silk and conducting materials is essential to advance the use of spider silk in electronic applications. Spider silk is tough, but becomes soft when exposed to water. Here we report a strong affinity of amine-functionalised multi-walled carbon nanotubes for spider silk, with coating assisted by a water and mechanical shear method. The nanotubes adhere uniformly and bond to the silk fibre surface to produce tough, custom-shaped, flexible and electrically conducting fibres after drying and contraction. The conductivity of coated silk fibres is reversibly sensitive to strain and humidity, leading to proof-of-concept sensor and actuator demonstrations.
Steven, Eden; Saleh, Wasan R.; Lebedev, Victor; Acquah, Steve F. A.; Laukhin, Vladimir; Alamo, Rufina G.; Brooks, James S.
Total X-Ray Scattering of Spider Dragline Silk
NASA Astrophysics Data System (ADS)
Total x-ray scattering measurements of spider dragline silk fibers from Nephila clavipes, Argiope aurantia, and Latrodectus hesperus all yield similar structure factors, with only small variations between the different species. Wide-angle x-ray scattering from fibers orientated perpendicular to the beam shows a high degree of anisotropy, and differential pair distribution functions obtained by integrating over wedges of the equatorial and meridian planes indicate that, on average, the majority (95%) of the atom-atom correlations do not extend beyond 1 nm. Futhermore, the atom-atom correlations between 1 and 3 nm are not associated with the most intense diffraction peaks at Q=1-2�-1. Disordered molecular orientations along the fiber axis are consistent with proteins in similar structural arrangements to those in the equatorial plane, which may be associated with the silk�s greater flexibility in this direction.
Benmore, C. J.; Izdebski, T.; Yarger, J. L.
Anisotropic growth of hydroxyapatite on the silk fibroin films
NASA Astrophysics Data System (ADS)
Bombyx mori silk fibroin is of practical interest for its excellent intrinsic properties utilizable in the biotechnological and biomedical fields. Here, the silk fibroin films were pretreated with different methods and then used as the template for the hydroxyapatite (HA) crystal growth. To study the effect of silk films' surface structure on the protein biomineralization, the films were immersed into 1.5 times simulated body fluid (1.5 � SBF) to induce the HA deposition at 37 �C. The results showed that an anisotropic growth of HA crystals was observed on the different films as judging from XRD, TEM and HRTEM data. This was thought that the positions and density of carboxyl groups, C dbnd O and amino groups on the surface of SF films may be different, which play the key effect on HA crystal growth.
Li, Yucheng; Cai, Yurong; Kong, Xiangdong; Yao, Juming
Processing of ?-glucosidase-silk fibroin nanoparticle bioconjugates and their characteristics.
Silk fibroin derived from Bombyx mori is a biomacromolecular protein with excellent biocompatibility. The aim of this work was to develop silk fibroin nanoparticles (SFNs) derived from the fibrous protein, which is a novel vector for enzyme modification in food processing. Silk fibroin was dissolved in highly concentrated CaCl2 and subjected to lengthy desalting in water. The resulting liquid silk, which contained water-soluble polypeptides with molecular mass ranging from 10 to 200 kDa, and ?-glucosidase were added rapidly into acetone. The ?-glucosidase molecules were embedded into silk fibroin nanoparticles, forming ?-glucosidase-silk fibroin nanoparticles (?G-SFNs) with a diameter of 50-150 nm. The enzyme activity of the ?G-SFN bioconjugates was determined with p-nitrophenyl-?-D-glucoside as the substrate, and the optimum conditions for the preparation of ?G-SFNs were investigated. The enzyme activity recovery of ?G-SFNs was 59.2 % compared to the free enzyme (specific activity was 1 U mg(-1)). The kinetic parameters of the ?G-SFNs and the free ?-glucosidase were the same. The ?G-SFNs had good operational stability and could be used repeatedly. These results confirmed that silk protein nanoparticles were good carriers as bioconjugates for the modification of enzymes with potential value for research and development. The method used in this study has potential applications in food processing and the production of flavour agents. PMID:24671567
Cao, Ting-Ting; Zhou, Zhen-Zhen; Zhang, Yu-Qing
Cell culture's spider silk road.
A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk. PMID:24924388
Perkel, Jeffrey
A Study of the Mechanical Behavior of Spider Silks.
National Technical Information Service (NTIS)
A preliminary physical and chemical examination of a selected group of spider silks was made. The program's ultimate goal was the synthesis of 'super tenacity' protein fibers, and the work reported here was designed to: Confirm the existence of very stron...
J. C. Zemlin
Sonication-Induced Gelation of Silk Fibroin for Cell Encapsulation
Purified native silk fibroin forms ?-sheet-rich, physically crosslinked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of ?-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical crosslinks responsible for gel stabilization. K+ at physiological concentrations and low pH promoted gelation which was not observed in the presence of Ca2+. The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4, 8, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5 to 2 hrs. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol-gel transitions, such as for cell encapsulation.
Wang, Xiaoqin; Kluge, Jon; Leisk, Gary G.; Kaplan, David L.
Sonication-induced gelation of silk fibroin for cell encapsulation.
Purified native silk fibroin forms beta-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of beta-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical cross-links responsible for gel stabilization. K(+) at physiological concentrations and low pH promoted gelation, which was not observed in the presence of Ca(2+). The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5-2 h. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol-gel transitions, such as for cell encapsulation. PMID:18031805
Wang, Xiaoqin; Kluge, Jonathan A; Leisk, Gary G; Kaplan, David L
Structure to function: Spider silk and human collagen
NASA Astrophysics Data System (ADS)
Nature has the ability to assemble a variety of simple molecules into complex functional structures with diverse properties. Collagens, silks and muscles fibers are some examples of fibrous proteins with self-assembling properties. One of the great challenges facing Science is to mimic these designs in Nature to find a way to construct molecules that are capable of organizing into functional supra-structures by self-assembly. In order to do so, a construction kit consisting of molecular building blocks along with a complete understanding on how to form functional materials is required. In this current research, the focus is on spider silk and collagen as fibrous protein-based biopolymers that can shed light on how to generate nanostructures through the complex process of self-assembly. Spider silk in fiber form offers a unique combination of high elasticity, toughness, and mechanical strength, along with biological compatibility and biodegrability. Spider silk is an example of a natural block copolymer, in which hydrophobic and hydrophilic blocks are linked together generating polymers that organize into functional materials with extraordinary properties. Since silks resemble synthetic block copolymer systems, we adopted the principles of block copolymer design from the synthetic polymer literature to build block copolymers based on spider silk sequences. Moreover, we consider spider silk to be an important model with which to study the relationships between structure and properties in our system. Thus, the first part of this work was dedicated to a novel family of spider silk block copolymers, where we generated a new family of functional spider silk-like block copolymers through recombinant DNA technology. To provide fundamental insight into relationships between peptide primary sequence, block composition, and block length and observed morphological and structural features, we used these bioengineered spider silk block copolymers to study secondary structure, morphological features and assembly. Aside from fundamental perspectives, we anticipate that these results will provide a blueprint for the design of precise materials for a range of potential applications such as controlled release devices, functional coatings, components of tissue regeneration materials and environmentally friendly polymers in future studies. In the second part of this work, human collagen type I was studied as another representative of the family of fibrous proteins. Collagen type I is the most abundant extracellular matrix protein in the human body, providing the basis for tissue structure and directing cellular functions. Collagen has a complex structural hierarchy, organized at different length scales, including the characteristic triple helical feature. In the present study we assessed the relationship between collagen structure (native vs. denatured) and sensitivity to UV radiation with a focus on changes in the primary structure, conformation, microstructure and material properties. Free radical reactions are involved in collagen degradation and a mechanism for UV-induced collagen degradation related to structure was proposed. The results from this study demonstrated the role of collagen supramolecular organization (triple helix) in the context of the effects of electromagnetic radiation on extracellular matrices. Owing to the fact that both silks and collagens are proteins that have found widespread interest for biomaterial related needs, we anticipate that the current studies will serve as a foundation for future biomaterial designs with controlled properties. Furthermore, fundamental insight into self-assembly and environmentally-2mediated degradation, will build a foundation for fundamental understanding of the remodeling and functions of these types of fibrous proteins in vivo and in vitro. This type of insight is essential for many areas of scientific inquiry, from drug delivery, to scaffolds for tissue engineering, and to the stability of materials in space.
Rabotyagova, Olena S.
Preparation and characterization of silk fibroin as a biomaterial with potential for drug delivery
Background Degummed silk fibroin from Bombyx mori (silkworm) has potential carrier capabilities for drug delivery in humans; however, the processing methods have yet to be comparatively analyzed to determine the differential effects on the silk protein properties, including crystalline structure and activity. Methods In this study, we treated degummed silk with four kinds of calcium-alcohol solutions, and performed secondary structure measurements and enzyme activity test to distinguish the differences between the regenerated fibroins and degummed silk fibroin. Results Gel electrophoresis analysis revealed that Ca(NO3)2-methanol, Ca(NO3)2-ethanol, or CaCl2-methanol treatments produced more lower molecular weights of silk fibroin than CaCl2-ethanol. X-ray diffraction and Fourier-transform infrared spectroscopy showed that CaCl2-ethanol produced a crystalline structure with more silk I (?-form, type II ?-turn), while the other treatments produced more silk II (?-form, anti-parallel ?-pleated sheet). Solid-State 13C cross polarization and magic angle spinning-nuclear magnetic resonance measurements suggested that regenerated fibroins from CaCl2-ethanol were nearly identical to degummed silk fibroin, while the other treatments produced fibroins with significantly different chemical shifts. Finally, enzyme activity test indicated that silk fibroins from CaCl2-ethanol had higher activity when linked to a known chemotherapeutic drug, L-asparaginase, than the fibroins from other treatments. Conclusions Collectively, these results suggest that the CaCl2-ethanol processing method produces silk fibroin with biomaterial properties that are appropriate for drug delivery.
Spider Silk Spun and Integrated into Composites.
National Technical Information Service (NTIS)
We gained significant new insights into the role of hydration for silks with our data allowing us to model in considerable detail the interaction between silk molecules and water. Our fibre and feedstock experiments demonstrated that the hierachical morph...
F. Vollrath
Mechanical Response of Silk Crystalline Units from Force-Distribution Analysis
The outstanding mechanical toughness of silk fibers is thought to be caused by embedded crystalline units acting as cross links of silk proteins in the fiber. Here, we examine the robustness of these highly ordered ?-sheet structures by molecular dynamics simulations and finite element analysis. Structural parameters and stress-strain relationships of four different models, from spider and Bombyx mori silk peptides, in antiparallel and parallel arrangement, were determined and found to be in good agreement with x-ray diffraction data. Rupture forces exceed those of any previously examined globular protein many times over, with spider silk (poly-alanine) slightly outperforming Bombyx mori silk ((Gly-Ala)n). All-atom force distribution analysis reveals both intrasheet hydrogen-bonding and intersheet side-chain interactions to contribute to stability to similar extent. In combination with finite element analysis of simplified ?-sheet skeletons, we could ascribe the distinct force distribution pattern of the antiparallel and parallel silk crystalline units to the difference in hydrogen-bond geometry, featuring an in-line or zigzag arrangement, respectively. Hydrogen-bond strength was higher in antiparallel models, and ultimately resulted in higher stiffness of the crystal, compensating the effect of the mechanically disadvantageous in-line hydrogen-bond geometry. Atomistic and coarse-grained force distribution patterns can thus explain differences in mechanical response of silk crystals, opening up the road to predict full fiber mechanics.
Xiao, Senbo; Stacklies, Wolfram; Cetinkaya, Murat; Markert, Bernd; Grater, Frauke
Nephila clavipes Flagelliform Silk-like GGX Motifs Contribute to Extensibility and Spacer Motifs Contribute to Strength in Synthetic Spider Silk Fibers
Flagelliform spider silk is the most extensible silk fiber produced by orb weaver spiders, though not as strong as the dragline silk of the spider. The motifs found in the core of the Nephila clavipes flagelliform Flag protein are: GGX, spacer, and GPGGX. Flag does not contain the polyalanine motif known to provide the strength of dragline silk. To investigate the source of flagelliform fiber strength, four recombinant proteins were produced containing variations of the three core motifs of the Nephila clavipes flagelliform Flag protein that produces this type of fiber. The as-spun fibers were processed in 80% aqueous isopropanol using a standardized process for all four fiber types, which produced improved mechanical properties. Mechanical testing of the recombinant proteins determined that the GGX motif contributes extensibility and the spacer motif contributes strength to the recombinant fibers. Recombinant protein fibers containing the spacer motif were stronger than the proteins constructed without the spacer that contained only the GGX motif or the combination of the GGX and GPGGX motifs. The mechanical and structural X-ray diffraction analysis of the recombinant fibers provide data that suggests a functional role of the spacer motif that produces tensile strength though the spacer motif is not clearly defined structurally. These results indicate that the spacer is likely a primary contributor of strength with the GGX motif supplying mobility to the protein network of native N. clavipes flagelliform silk fibers.
Adrianos, Sherry L.; Teule, Florence; Hinman, Michael B.; Jones, Justin A.; Weber, Warner S.; Yarger, Jeffery L.; Lewis, Randolph V.
Discrimination of cultivated silk and wild silk by conventional instrumental analyses.
In Japan, recent trends have seen wild silk preferred over cultivated silk because of its texture. Some cases of fraud have occurred where cultivated silk garments are sold as wild silk. Samples from these cases, morphological observation using light microscope and polarized microscope have been conducted in forensic science laboratories. Sometimes scanning electron microscopy was also carried out. However, the morphology of silk shows quite wide variation, which makes it difficult to discriminate wild and cultivated silks by this method. In this report, silk discrimination was investigated using conventional instrumental analyses commonly available in forensic laboratories, such as Fourier-transfer infrared spectrometry (FT-IR), pyrolysis-gas chromatography/mass spectrometry (pyr-GC/MS) and differential thermal analysis (DTA). By FT-IR, cultivated and wild silk gave similar infrared spectra, but wild silk had a characteristic peak at 965 cm(-1) from the deformation vibration of the carbon-carbon double bond of the indole ring. Comparison of the pyrograms of cultivated and wild silk showed that wild silk had large indole and skatole peaks that cultivated silk did not, and these peaks might arise from tryptophan. The results of thermogravimetry/DTA showed that the endothermic peak was about 40 �C higher for wild silk than for cultivated silk. Using a combination of these results, cultivated and wild silk could be discriminated by common forensic instrumental techniques. PMID:23742990
Matsuyama, Yuji; Nagatani, Yoshiaki; Goto, Toshiyuki; Suzuki, Shinichi
SILK: Scout Paths in the Linux Kernel
SILK stands for Scout In the Linux Kernel, and is a port of the Scout operating system to run as a Linux kernel mod- ule. SILK forms a replacement networking subsystem for standard Linux 2.4 kernels. Linux applications create and use Scout paths via the Linux socket interface with virtually no modifications to the applications themselves. SILK pro- vides Linux
Andy Bavier; Thiemo Voigt; Mike Wawrzoniak; Larry Peterson; Per Gunningberg
Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing
We present a simple and effective method to obtain refined control of the molecular structure of silk biomaterials through physical temperature-controlled water vapor annealing (TCWVA). The silk materials can be prepared with control of crystallinity, from a low content using conditions at 4�C (alpha-helix dominated silk I structure), to highest content of ~60% crystallinity at 100�C (beta-sheet dominated silk II structure). This new physical approach covers the range of structures previously reported to govern crystallization during the fabrication of silk materials, yet offers a simpler, green chemistry, approach with tight control of reproducibility. The transition kinetics, thermal, mechanical, and biodegradation properties of the silk films prepared at different temperatures were investigated and compared by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), uniaxial tensile studies, and enzymatic degradation studies. The results revealed that this new physical processing method accurately controls structure, in turn providing control of mechanical properties, thermal stability, enzyme degradation rate, and human mesenchymal stem cell interactions. The mechanistic basis for the control is through the temperature controlled regulation of water vapor, to control crystallization. Control of silk structure via TCWVA represents a significant improvement in the fabrication of silk-based biomaterials, where control of structure-property relationships is key to regulating material properties. This new approach to control crystallization also provides an entirely new green approach, avoiding common methods which use organic solvents (methanol, ethanol) or organic acids. The method described here for silk proteins would also be universal for many other structural proteins (and likely other biopolymers), where water controls chain interactions related to material properties.
Hu, Xiao; Shmelev, Karen; Sun, Lin; Gil, Eun-Seok; Park, Sang-Hyug; Cebe, Peggy; Kaplan, David L.
Release and cellular acceptance of multiple drugs loaded silk fibroin particles.
In this article, silk fibroin particles with average diameter of 980 nm were fabricated via self assembly. Exceptional loading efficiency and release patterns of hydrophobic and protein drugs were observed. Furthermore, smoother release patterns were observed with increase loading of the hydrophobic and protein model drugs, only about 23% FITC-BSA and 34% RhB were released from the silk particles at their highest corresponding loading in 50 days. Most importantly, osteoblasts' viability was augmented during co-culture with silk fibroin particles, as shown by Alamar Blue assay. Attachment of the particles and delivery of model drugs to cells were confirmed by fluorescence images and flow cytometry. Hence, the silk fibroin particles could be potential biomaterial for application in controlled release and pharmaceutics. PMID:21920418
Shi, Pujiang; Goh, James C H
Structure and Properties of Nephila Clavipes Dragline Silk Polymer
NASA Astrophysics Data System (ADS)
Silk, spun from an aqueous state at room temperature by a variety of organisms, is the most commonly spun extracellular fibrous protein. It comprises polypeptide chains with regions which can crystallize and regions which are predominantly amorphous. The polymer chains in the crystalline regions form anti-parallel pleated sheet structures with an orthorhombic unit cell. Dragline silk is a structural material produced by a variety of spiders. It has been genetically tailored to meet a specific purpose. Dragline silk exhibits high extensibility and tensile strength approaching that of high-strength synthetic fibers. The specific energy to break it can exceed some steels and synthetic fibers. Samples of Nephila clavipes (golden orb-weaver) dragline silk were extracted from live specimens and examined with a series of experimental techniques including optical, scanning electron, and atomic force microscopy, wide and small angle X-ray diffraction and birefringence compensation. Computer modeling of the mechanical properties of the crystallite was also performed. An assortment of features at a variety of length scales was observed by microscopy. These occur on both the as-spun and abraded silk surfaces. The silk was observed to undergo large deformations without evidence of failure, suggesting the absence of a microfibrillar structure. There was no conclusive evidence for either a microfibrillar or a skin core structure. Meridional and equatorial SAXD peaks were observed at Bragg spacings of 79 AA and 250 AA, respectively. Analysis of the WAXD patterns indicated that the silk belongs in Warwicker's category 3b and that the minimum dimensions of the crystals are approximately 38 AA in the molecular direction and 16 x 23 AA in the transverse directions. The crystal modulus was determined with WAXD to be 16.7 GPa, applying the assumption of uniform stress. This is lower than the 200 GPa modulus calculated with molecular modeling. These results and other factors indicate the inapplicability of the widely used assumption of uniform stress. The X-ray data show increased alignment of the crystals along the fiber axis with stress. The birefringence of the silk fibers increases monotonically to failure which indicates increasing molecular alignment. The change in birefringence appears to be governed by the amorphous contribution.
Mahoney, David Vincent
Rate-dependent behavior of the amorphous phase of spider dragline silk.
The time-dependent stress-strain behavior of spider dragline silk was already observed decades ago, and has been attributed to the disordered sequences in silk proteins, which compose the soft amorphous matrix. However, the actual molecular origin and magnitude of internal friction within the amorphous matrix has remained inaccessible, because experimentally decomposing the mechanical response of the amorphous matrix from the embedded crystalline units is challenging. Here, we used atomistic molecular dynamics simulations to obtain friction forces for the relative sliding of peptide chains of Araneus diadematus spider silk within bundles of these chains as a representative unit of the amorphous matrix in silk fibers. We computed the friction coefficient and coefficient of viscosity of the amorphous phase to be in the order of 10(-6) Ns/m and 10(4) Ns/m(2), respectively, by extrapolating our simulation data to the viscous limit. Finally, we used a finite element method for the amorphous phase, solely based on parameters derived from molecular dynamics simulations including the newly determined coefficient of viscosity. With this model the time scales of stress relaxation, creep, and hysteresis were assessed, and found to be in line with the macroscopic time-dependent response of silk fibers. Our results suggest the amorphous phase to be the primary source of viscosity in silk and open up the avenue for finite element method studies of silk fiber mechanics including viscous effects. PMID:24896131
Patil, Sandeep P; Markert, Bernd; Gr�ter, Frauke
Impact of processing parameters on the haemocompatibility of Bombyx mori silk films
Silk has traditionally been used for surgical sutures due to its lasting strength and durability; however, the use of purified silk proteins as a scaffold material for vascular tissue engineering goes beyond traditional use and requires application-orientated biocompatibility testing. For this study, a library of Bombyx mori silk films was generated and exposed to various solvents and treatment conditions to reflect current silk processing techniques. The films, along with clinically relevant reference materials, were exposed to human whole blood to determine silk blood compatibility. All substrates showed an initial inflammatory response comparable to polylactide-co-glycolide (PLGA), and a low to moderate haemostasis response similar to polytetrafluoroethylene (PTFE) substrates. In particular, samples that were water annealed at 25 �C for 6 h demonstrated the best blood compatibility based on haemostasis parameters (e.g. platelet decay, thrombin-antithrombin complex, platelet factor 4, granulocytes-platelet conjugates) and inflammatory parameters (e.g. C3b, C5a, CD11b, surface-associated leukocytes). Multiple factors such as treatment temperature and solvent influenced the biological response, though no single physical parameter such as ?-sheet content, isoelectric point or contact angle accurately predicted blood compatibility. These findings, when combined with prior in vivo data on silk, support a viable future for silk-based vascular grafts.
Seib, F. Philipp; Maitz, Manfred F.; Hu, Xiao; Werner, Carsten; Kaplan, David L.
Structure modifications induced in silk fibroin by enzymatic treatments. A Raman study
NASA Astrophysics Data System (ADS)
Raman spectroscopy was used to investigate various enzyme-catalyzed reactions onto silk fibroin, i.e. the biodegradation of Tussah ( Antheraea pernyi) silk fibroin films by a proteolytic enzyme, the oxidation of domestic ( Bombyx mori) silk fibroin by mushroom tyrosinase and the subsequent grafting of chitosan onto oxidized silk. The spectra of Tussah silk fibroin films exposed to a bacterial protease for different times demonstrated that the cleavage of sensitive peptide bonds in the amorphous glycine-rich domains resulted in the loss of various amino acid residues (Tyr, Trp, Asp, etc.). The bands attributed to the crystalline alanine-rich sequences increased in intensity, and the ?-sheet molecular conformation was not affected by biodegradation. Following oxidation with mushroom tyrosinase, the tyrosine bands of Bombyx mori fibroin decreased in intensity but did not disappear. The increase of the I853/ I829 intensity ratio indicated that the Tyr residues not accessible to the enzyme were located in a strongly hydrophobic environment. Raman spectroscopy provided evidence that chitosan was effectively grafted onto oxidized silk, probably via the Schiff-base mechanism, as shown by the behavior of the imine band at about 1646 cm -1. Grafting chitosan onto silk fibroin resulted in a ?-sheet?random coil conformational transition of the protein component in the bioconjugated product.
Monti, Patrizia; Freddi, Giuliano; Sampaio, Sandra; Tsukada, Masuhiro; Taddei, Paola
Enhanced Cellular Adhesion on Titanium by Silk Functionalized with titanium binding and RGD peptides
Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. Quartz Crystal Microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived RGD peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by Scanning Electron Microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk-peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required.
Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J.; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L.; Egles, Christophe
Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides.
Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. A quartz crystal microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived arginine-glycine-aspartic acid (RGD) peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by scanning electron microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required. PMID:22975628
Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L; Egles, Christophe
Identification and synthesis of novel biomaterials based on spider structural silk fibers
NASA Astrophysics Data System (ADS)
The diversity in function and mechanical behavior of spider silks, and the ability to produce these silks recombinantly, have tremendous potential in creating a new class of biomimetic materials. Here we investigate the structural and mechanical properties of pyriform silks from the golden orb-weaver, Nephila clavipes. Nanoscale indentation measurements using atomic force microscopy on natural pyriform silk suggests that this biomaterial has high toughness that may be suitable for dissipating high amounts of mechanical energy. We also observed the occurrence of highly organized nanocrystals within the pyriform silk fibers that may contribute to the remarkable energy dissipation capability of these silks. It has been demonstrated that poly-(Gly-Ala) and poly-Ala stretches within the internal block repeat modules of dragline silk fibroins form nanocrystals, and these nanocrystalline structures may be responsible for the high extensibility of the dragline silks. In contrast, amino acid sequence analysis shows that PySp2 does not contain the same motifs. In the absence of poly-(Gly-Ala) and poly-Ala repeats, we hypothesized that PySp2 contains new protein motifs sufficient to polymerize into functional structures. To investigate the functional contributions of these novel motifs during pyriform fiber formation, we expressed different recombinant PySp2 fibroins with various segments spanning its block repeat units. We demonstrate that PySp2 recombinant proteins with the Pro-rich sub-block domain (PXP motifs, where X= sub-set of the amino acids A, L, or R) and/or the Ser + Gln + Ala-rich sub-block domain (QQSSVAQS motifs) are sufficient for artificial fiber formation. Moreover, we show that recombinant PySp2 proteins that contain a single block repeat unit can self-assemble into foam-like nanostructures. Collectively, our findings support the use of PySp2 recombinant proteins for a wide range of biomimetic materials with morphologies ranging from fibers to porous structures.
Hsia, Yang; Gnesa, Eric; Tang, Simon; Jeffery, Felicia; Geurts, Paul; Zhao, Liang; Franz, Andreas; Vierra, Craig
Macroporous silk fibroin cryogels.
Silk fibroin cryogels with remarkable properties were obtained from frozen fibroin solutions (4.2-12.6%) at subzero temperatures between -5 and -22 �C. This was achieved by the addition of ethylene glycol diglycidyl ether (EGDE) into the cryogelation system. EGDE triggers the conformational transition of fibroin from random coil to ?-sheet structure and hence fibroin gelation. One of the unique features of fibroin cryogels is their elasticity that allows them to resist complete compression without any crack development, during which water inside the cryogel is removed. The compressed cryogel immediately swells during unloading to recover its original shape. The scaffolds obtained by freeze-drying of the cryogels consist of regular, interconnected pores of diameters ranging from 50 to 10 ?m that could be regulated by the synthesis parameters. The mechanical compressive strength and the modulus of the scaffolds increase with decreasing pore diameter, that is, with decreasing gelation temperature or, with increasing fibroin or EGDE concentrations in the feed. The scaffolds produced at 12.6% fibroin exhibit a very high compressive modulus (50 MPa) making them good candidates as bone scaffold materials. PMID:23360211
Ak, Fatih; Oztoprak, Zeynep; Karakutuk, Ilknur; Okay, Oguz
Differential Binding of theBombyxSilk Gland-Specific Factor SGFB to Its Target DNA Sequence Drives Posterior-Cell-Restricted Expression
The gene encoding the silk protein P25 inBombyx moriis expressed in the posterior silk gland (PSG) cells and repressed in the middle silk gland (MSG) cells. To identify the factors involved in this transcription- dependent spatial restriction, we examined the P25 chromatin in PSG and MSG nuclei by DNase I-aided ligation-mediated PCR and analyzed the expression of variousP25-lacZconstructs in biolistically
A quicker degradation rate is yielded by a novel kind of transgenic silk fibroin consisting of shortened silk fibroin heavy chains fused with matrix metalloproteinase cleavage sites.
Degradation performance of silk fibroin is an important property for its medical applications. Herein we constructed a shortened silk fibroin heavy chain protein fused with a matrix metalloproteinase cleavage site (SSFH-MMP) along with a glutathione S-transferase tag ahead. The digestion assay shows it can be cut by matrix metalloproteinase-2 (MMP-2) at its MMP cleavage site. Furthermore, we introduced the SSFH-MMP into silk fibroin by genetic modification of silkworms in order to increase the degradation rate of the silk fibroin. After acquisition of a race of transgenic silkworms with the coding sequence of the MMP cleavage site in their genomic DNA, we tested some properties of their silk fibroin designated TSF-MMP. The results show that the TSF-MMP has MMP cleavage sites and yields a quicker degradation rate during dilution in MMP-2 enzyme buffer or implantation into tumor tissues compared with that of normal silk fibroin. Moreover, the TSF-MMP is in vitro non-toxic to human�bone marrow mesenchymal stem cells (hBM-MSCs) indicating that the TSF-MMP may become a biomaterial with a quicker degradation rate for its medical applications. PMID:24801061
Huang, Guoping; Yang, Danfeng; Sun, Chunfeng; Huang, Jianping; Chen, Keping; Zhang, Chunxia; Chen, Huiqing; Yao, Qin
Crystal growth of calcium carbonate in silk fibroin/sodium alginate hydrogel
NASA Astrophysics Data System (ADS)
As known, silk fibroin-like protein plays a pivotal role during the formation of calcium carbonate (CaCO3) crystals in the nacre sheets. Here, we have prepared silk fibroin/sodium alginate nanofiber hydrogels to serve as templates for calcium carbonate mineralization. In this experiment, we report an interesting finding of calcium carbonate crystal growth in the silk fibroin/sodium alginate nanofiber hydrogels by the vapor diffusion method. The experimental results indicate calcium carbonate crystals obtained from nanofiber hydrogels with different proportions of silk fibroin/sodium alginate are mixture of calcite and vaterite with unusual morphologies. Time-dependent growth study was carried out to investigate the crystallization process. It is believed that nanofiber hydrogels play an important role in the process of crystallization. This study would help in understanding the function of organic polymers in natural mineralization, and provide a novel pathway in the design and synthesis of new materials related unique morphology and structure.
Ming, Jinfa; Zuo, Baoqi
Study on preirradiation grafting of methacrylamide onto natural silk fabrics.
National Technical Information Service (NTIS)
In order to improve the properties of natural silk fabrics, the mechanism and method of the radiation induced graft copolymerization of monomers onto natural silk fabrics were more studied. Grafting of methacrylamide onto natural silk fabrics by the preir...
Xiang Zhengyu Wan Dairong
ERIC Educational Resources Information Center
Demonstrates that the Judeans traveled along the Ancient Silk Route. Discusses the Iranian influence on the formation of Jewish religious ideas. Considers the development of Jewish trade networks, focusing on the Radanites (Jewish traders), the Jewish presence in the Far East, and the survival of Judaism in central Asia. (CMK)
Foltz, Richard
Thromboelastometric and platelet responses to silk biomaterials.
Silkworm's silk is natural biopolymer with unique properties including mechanical robustness, all aqueous base processing and ease in fabrication into different multifunctional templates. Additionally, the nonmulberry silks have cell adhesion promoting tri-peptide (RGD) sequences, which make it an immensely potential platform for regenerative medicine. The compatibility of nonmulberry silk with human blood is still elusive; thereby, restricts its further application as implants. The present study, therefore, evaluate the haematocompatibility of silk biomaterials in terms of platelet interaction after exposure to nonmulberry silk of Antheraea mylitta using thromboelastometry (ROTEM). The mulberry silk of Bombyx mori and clinically used Uni-Graft W biomaterial serve as references. Shortened clotting time, clot formation times as well as enhanced clot strength indicate the platelet mediated activation of blood coagulation cascade by tested biomaterials; which is comparable to controls. PMID:24824624
Kundu, Banani; Schlimp, Christoph J; N�rnberger, Sylvia; Redl, Heinz; Kundu, S C
Thromboelastometric and platelet responses to silk biomaterials
Silkworm's silk is natural biopolymer with unique properties including mechanical robustness, all aqueous base processing and ease in fabrication into different multifunctional templates. Additionally, the nonmulberry silks have cell adhesion promoting tri-peptide (RGD) sequences, which make it an immensely potential platform for regenerative medicine. The compatibility of nonmulberry silk with human blood is still elusive; thereby, restricts its further application as implants. The present study, therefore, evaluate the haematocompatibility of silk biomaterials in terms of platelet interaction after exposure to nonmulberry silk of Antheraea mylitta using thromboelastometry (ROTEM). The mulberry silk of Bombyx mori and clinically used Uni-Graft W biomaterial serve as references. Shortened clotting time, clot formation times as well as enhanced clot strength indicate the platelet mediated activation of blood coagulation cascade by tested biomaterials; which is comparable to controls.
Kundu, Banani; Schlimp, Christoph J.; Nurnberger, Sylvia; Redl, Heinz; Kundu, S. C.
Fabrication and Biocompatibility of Electrospun Silk Biocomposites
Silk fibroin has attracted great interest in tissue engineering because of its outstanding biocompatibility, biodegradability and minimal inflammatory reaction. In this study, two kinds of biocomposites based on regenerated silk fibroin are fabricated by electrospinning and post-treatment processes, respectively. Firstly, regenerated silk fibroin/tetramethoxysilane (TMOS) hybrid nanofibers with high hydrophilicity are prepared, which is superior for fibroblast attachment. The electrospinning process causes adjacent fibers to �weld� at contact points, which can be proved by scanning electron microscope (SEM). The water contact angle of silk/tetramethoxysilane (TMOS) composites shows a sharper decrease than pure regenerated silk fibroin nanofiber, which has a great effect on the early stage of cell attachment behavior. Secondly, a novel tissue engineering scaffold material based on electrospun silk fibroin/nano-hydroxyapatite (nHA) biocomposites is prepared by means of an effective calcium and phosphate (Ca�P) alternate soaking method. nHA is successfully produced on regenerated silk fibroin nanofiber within several min without any pre-treatments. The osteoblastic activities of this novel nanofibrous biocomposites are also investigated by employing osteoblastic-like MC3T3-E1 cell line. The cell functionality such as alkaline phosphatase (ALP) activity is ameliorated on mineralized silk nanofibers. All these results indicate that this silk/nHA biocomposite scaffold material may be a promising biomaterial for bone tissue engineering.
Wei, Kai; Kim, Byoung-Suhk; Kim, Ick-Soo
Modification of silk fibroin using diazonium coupling chemistry and the effects on hMSC proliferation and differentiation.
A simple chemical modification method using diazonium coupling chemistry was developed to tailor the structure and hydrophilicity of silk fibroin protein. The extent of modification using several aniline derivatives was characterized using UV-vis and 1H NMR spectroscopies, and the resulting protein structure was analyzed with ATR-FTIR spectroscopy. Introduction of hydrophobic functional groups facilitated rapid conversion of the protein from a random coil to a beta-sheet structure, while addition of hydrophilic groups inhibited this process. hMSCs were grown on these modified silks to assess the biocompatibility of these materials. The hydrophilicity of the silk derivatives was found to affect the growth rate and morphology, but hMSCs were able to attach, proliferate and differentiate into an osteogenic lineage on all of the silk derivatives. PMID:18417206
Murphy, Amanda R; St John, Peter; Kaplan, David L
Dissecting the structural determinants for the difference in mechanical stability of silk and amyloid beta-sheet stacks.
Stacking of ?-sheets results in a protein super secondary structure with remarkable mechanical properties. ?-Stacks are the determinants of a silk fiber's resilience and are also the building blocks of amyloid fibrils. While both silk and amyloid-type crystals are known to feature a high resistance against rupture, their structural and mechanical similarities and particularities are yet to be fully understood. Here, we systematically compare the rupture force and stiffness of amyloid and spider silk poly-alanine ?-stacks of comparable sizes using Molecular Dynamics simulations. We identify the direction of force application as the primary determinant of the rupture strength; ?-sheets in silk are orientated along the fiber axis, i.e. the pulling direction, and consequently require high forces in the several nanoNewton range for shearing ?-strands apart, while ?-sheets in amyloid are oriented vertically to the fiber, allowing a zipper-like rupture at sub-nanoNewton forces. A secondary factor rendering amyloid ?-stacks softer and weaker than their spider silk counterparts is the sub-optimal side-chain packing between ?-sheets due to the sequence variations of amyloid-forming proteins as opposed to the perfectly packed poly-alanine ?-sheets of silk. Taken together, amyloid fibers can reach the stiffness of silk fibers in spite of their softer and weaker ?-sheet arrangement as they are missing a softening amorphous matrix. PMID:23633029
Xiao, Senbo; Xiao, Shijun; Gr�ter, Frauke
The natural silk spinning process. A nucleation-dependent aggregation mechanism?
The spinning mechanism of natural silk has been an open issue. In this study, both the conformation transition from random coil to beta sheet and the beta sheet aggregation growth of silk fibroin are identified in the B. mori regenerated silk fibroin aqueous solution by circular dichroism (CD) spectroscopy. A nucleation-dependent aggregation mechanism, similar to that found in prion protein, amyloid beta (Abeta) protein, and alpha-synuclein protein with the conformation transition from a soluble protein to a neurotoxic, insoluble beta sheet containing aggregate, is a novel suggestion for the silk spinning process. We present evidence that two steps are involved in this mechanism: (a) nucleation, a rate-limiting step involving the conversion of the soluble random coil to insoluble beta sheet and subsequently a series of thermodynamically unfavorable association of beta sheet unit, i.e. the formation of a nucleus or seed; (b) once the nucleus forms, further growth of the beta sheet unit becomes thermodynamically favorable, resulting a rapid extension of beta sheet aggregation. The aggregation growth follows a first order kinetic process with respect to the random coil fibroin concentration. The increase of temperature accelerates the beta sheet aggregation growth if the beta sheet seed is introduced into the random coil fibroin solution. This work enhances our understanding of the natural silk spinning process in vivo. PMID:11737214
Li, G; Zhou, P; Shao, Z; Xie, X; Chen, X; Wang, H; Chunyu, L; Yu, T
Pyriform Spidroin 1, a Novel Member of the Silk Gene Family That Anchors Dragline Silk Fibers in Attachment Discs of the Black Widow Spider, Latrodectus hesperus*
Spiders spin high performance threads that have diverse mechanical properties for specific biological applications. To better understand the molecular mechanism by which spiders anchor their threads to a solid support, we solubilized the attachment discs from black widow spiders and performed in-solution tryptic digests followed by MS/MS analysis to identify novel peptides derived from glue silks. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and cDNA library screening, we isolated a novel member of the silk gene family called pysp1 and demonstrate that its protein product is assembled into the attachment disc silks. Alignment of the PySp1 amino acid sequence to other fibroins revealed conservation in the non-repetitive C-terminal region of the silk family. MS/MS analysis also confirmed the presence of MaSp1 and MaSp2, two important components of dragline silks, anchored within the attachment disc materials. Characterization of the ultrastructure of attachment discs using scanning electron microscopy studies support the localization of PySp1 to small diameter fibers embedded in a glue-like cement, which network with large diameter dragline silk threads, producing a strong, adhesive material. Consistent with elevated PySp1 mRNA levels detected in the pyriform gland, MS analysis of the luminal contents extracted from the pyriform gland after tryptic digestion support the assertion that PySp1 represents one of the major constituents manufactured in the pyriform gland. Taken together, our data demonstrate that PySp1 is spun into attachment disc silks to help affix dragline fibers to substrates, a critical function during spider web construction for prey capture and locomotion.
Blasingame, Eric; Tuton-Blasingame, Tiffany; Larkin, Leah; Falick, Arnold M.; Zhao, Liang; Fong, Justine; Vaidyanathan, Veena; Visperas, Anabelle; Geurts, Paul; Hu, Xiaoyi; La Mattina, Coby; Vierra, Craig
Complex gene expression in the dragline silk producing glands of the Western black widow (Latrodectus hesperus)
Background Orb-web and cob-web weaving spiders spin dragline silk fibers that are among the strongest materials known. Draglines are primarily composed of MaSp1 and MaSp2, two spidroins (spider fibrous proteins) expressed in the major ampullate (MA) silk glands. Prior genetic studies of dragline silk have focused mostly on determining the sequence of these spidroins, leaving other genetic aspects of silk synthesis largely uncharacterized. Results Here, we used deep sequencing to profile gene expression patterns in the Western black widow, Latrodectus hesperus. We sequenced millions of 3?-anchored �tags� of cDNAs derived either from MA glands or control tissue (cephalothorax) mRNAs, then associated the tags with genes by compiling a reference database from our newly constructed normalized L. hesperus cDNA library and published L. hesperus sequences. We were able to determine transcript abundance and alternative polyadenylation of each of three loci encoding MaSp1. The ratio of MaSp1:MaSp2 transcripts varied between individuals, but on average was similar to the estimated ratio of MaSp1:MaSp2 in dragline fibers. We also identified transcription of TuSp1 in MA glands, another spidroin family member that encodes the primary component of egg-sac silk, synthesized in tubuliform glands. In addition to the spidroin paralogs, we identified 30 genes that are more abundantly represented in MA glands than cephalothoraxes and represent new candidates for involvement in spider silk synthesis. Conclusions Modulating expression rates of MaSp1 variants as well as MaSp2 and TuSp1 could lead to differences in mechanical properties of dragline fibers. Many of the newly identified candidate genes likely encode secreted proteins, suggesting they could be incorporated into dragline fibers or assist in protein processing and fiber assembly. Our results demonstrate previously unrecognized transcript complexity in spider silk glands.
NSDL National Science Digital Library
Secrets of the Silk Road is another worthy addition to the growing body of Web-based information about the Silk Road (see, for example, The International Dunhuang Project, founded in 1998 and mentioned in several Scout Reports), launched by the Bowers Museum of Santa Ana, California and presented by University of Pennsylvania Museum of Archaeology and Anthropology. This site includes artifacts documenting the history of the Silk Road in the vast Tarim Basin in the Far Western Xinjiang Uyghur Autonomous Region of China. Three well-preserved mummies from the Tarim Basin have been brought to the United States for the exhibition, along with lavish grave goods. Visitors to the website can view images of "The Beauty of Xiaohe," a female mummy from 1800-1500 BCE and other treasures ranging from a Chrysanthemum Shaped Dessert from the 5th-3rd century BCE, to gold objects such as mask from 5th-6th century CE or the Gold Plaque with Lion Design, 5th-3rd Century BCE.
Nanocomposite gold-silk nanofibers
Cell-biomaterial interactions can be controlled by modifying the surface chemistry or nanotopography of the material, to induce cell proliferation and differentiation if desired. Here we combine both approaches in forming silk nanofibers (SNFs) containing gold nanoparticles (AuNPs) and subsequently chemically modifying the fibers. Silk fibroin mixed with gold seed nanoparticles was electrospun to form SNFs doped with gold seed nanoparticles (SNFseed). Following gold reduction, there was a two-fold increase in particle diameter confirmed by the appearance of a strong absorption peak at 525 nm. AuNPs were dispersed throughout the AuNP-doped silk nanofibers (SNFsAu). The Young�s modulus of the SNFsAu was almost 70% higher than that of SNFs. SNFsAu were modified with the arginine-glycine-aspartic acid (RGD) peptide. Human mesenchymal stem cells that were cultured on RGD-modified SNFAu had a more than two-fold larger cell area compared to the cells cultured on bare SNFs; SNFAu also increase cell size. We suggest that this approach can be used to alter the cell-material interface in tissue engineering and other applications.
Cohen-Karni, Tzahi; Jeong, Kyung Jae; Tsui, Jonathan H.; Reznor, Gally; Mustata, Mirela; Wanunu, Meni; Graham, Adam; Marks, Carolyn; Bell, David C.; Langer, Robert S.; Kohane, Daniel S.
Ordering recombinant silk-elastin-like nanofibers on the microscale
NASA Astrophysics Data System (ADS)
Self-assembled peptide/polypeptide nanofibers are appealing building blocks for creating complex three-dimensional structures. However, ordering assembled peptide/polypeptide nanofibers into three-dimensional structures on the microscale remains challenging and often requires the employment of top-down approaches. We report that silk-elastin-like protein polymers self-assemble into nanofibers in physiologically relevant conditions, the assembled nanofibers further form fiber clusters on the microscale, and the nanofiber clusters eventually coalesce into three-dimensional structures with distinct nanoscale and microscale features. It is believed that the interplay between fiber growth and molecular diffusion leads to the ordering of the assembled silk-elastin-like nanofibers at the microscale.
Zeng, Like; Teng, Weibing; Jiang, Linan; Cappello, Joseph; Wu, Xiaoyi
On the strength of ?-sheet crystallites of Bombyx mori silk fibroin.
Silk fibroin, a natural multi-domain protein, has attracted great attention due to its superior mechanical properties such as ultra-high strength and stretchability, biocompatibility, as well as its versatile biodegradability and processability. It is mainly composed of ?-sheet crystallites and amorphous domains. Although its strength is well known to be controlled by the dissociation of protein chains from ?-sheet crystallites, the way that water as the solvent affects its strength and the reason that its theoretically predicted strength is several times higher than experimental measurement remain unclear. We perform all-atom molecular dynamics simulations on a ?-sheet crystallite of Bombyx mori silk. We find that water solvent reduces the number and strength of hydrogen bonds between ?-chains, and thus greatly weakens the strength of silk fibroin. By dissociating protein chains at different locations from the crystallite, we also find that the pulling strength for the interior chains is several times higher than that for the surface/corner chains, with the former being consistent with the theoretically predicted value, while the latter on par with the experimental value. It is shown that the weakest rupture strength controls the failure strength of silk fibre. Hence, this work sheds light on the role of water in the strength of silk fibroin and also provides clues on the origin of the strength difference between theory and experiment. PMID:24789564
Cheng, Yuan; Koh, Leng-Duei; Li, Dechang; Ji, Baohua; Han, Ming-Yong; Zhang, Yong-Wei
Seljuk Muqarnas along the Silk Road
The film Seljuk Muqarnas along the Silk Road gives an overview of muqarnas, stalactite vaults, in Seljuk style architecture (1038-1194). The muqarnas are located in portals and niches of car- avansaraies, madrassas and mosques. Starting with the Sultan Han near Kayseri we follow the Silk Road westward till Konya and finally show the Arslanhane Camii in Ankara. Video recordings alternate
Silvia Harmsen; Daniel Jungblut; Susanne Kromker
Controllable transition of silk fibroin nanostructures: an insight into in vitro silk self-assembly process.
Silk fiber is one of the strongest and toughest biological materials with hierarchical structures, where nanofibril with size <20nm is a critical factor in determining its excellent mechanical properties. Although silk nanofibrils have been found in natural and regenerated silk solutions, there is no way to actively control nanofibril formation in aqueous solution. This study shows a simple but effective method of preparing silk nanofibrils by regulating the silk self-assembly process. Through a repeated drying-dissolving process, a silk fibroin solution composed of metastable nanoparticles was first prepared and then used to reassemble nanofibrils with different sizes and secondary conformations under various temperatures and concentrations. These nanofibrils have a similar size to that of natural fibers, providing a suitable unit to further assemble the hierarchical structure in vitro. Several important issues, such as the relationships between silk nanofibrils, secondary conformations and viscosity, are also investigated, giving a new insight into the self-assembly process. In summary, besides rebuilding silk nanofibrils in aqueous solution, this study provides an important model for furthering the understanding of silk structures, properties and forming mechanisms, making it possible to regenerate silk materials with exceptional properties in the future. PMID:23628774
Bai, S; Liu, S; Zhang, C; Xu, W; Lu, Q; Han, H; Kaplan, D L; Zhu, H
Cell proliferation and migration in silk fibroin 3D scaffolds.
Pore architecture in 3D polymeric scaffolds is known to play a critical role in tissue engineering as it provides the vital framework for the seeded cells to organize into a functioning tissue. In this report, we investigated the effects of different freezing temperature regimes on silk fibroin protein 3D scaffold pore microstructure. The fabricated scaffolds using freeze-dry technique were used as a 3D model to monitor cell proliferation and migration. Pores of 200-250microm diameter were formed by slow cooling at temperatures of -20 and -80 degrees C but were found to be limited in porosity and pore interconnectivity as observed through scanning electron microscopic images. In contrast, highly interconnected pores with 96% porosity were observed when silk solutions were rapidly frozen at -196 degrees C. A detailed study was conducted to assess the affect of pore size, porosity and interconnectivity on human dermal fibroblast cell proliferation and migration on these 3D scaffolds using confocal microscopy. The cells were observed to migrate within the scaffold interconnectivities and were found to reach scaffold periphery within 28 days of culture. Confocal images further confirmed normal cell attachment and alignment of actin filaments within the porous scaffold matrix with well-developed nuclei. This study indicates rapid freeze-drying technique as an alternative method to fabricate highly interconnected porous scaffolds for developing functional 3D silk fibroin matrices for potential tissue engineering, biomedical and biotechnological applications. PMID:19249094
Mandal, Biman B; Kundu, Subhas C
In vivo degradation of three-dimensional silk fibroin scaffolds.
Three-dimensional porous scaffolds prepared from regenerated silk fibroin using either an all-aqueous process or a process involving an organic solvent, hexafluoroisopropanol (HFIP), have shown promise in cell culture and tissue engineering applications. However, their biocompatibility and in vivo degradation have not been fully established. The present study was conducted to systematically investigate how processing method (aqueous vs. organic solvent) and processing variables (silk fibroin concentration and pore size) affect the short-term (up to 2 months) and long-term (up to 1 year) in vivo behavior of the protein scaffolds in both nude and Lewis rats. The samples were analyzed by histology for scaffold morphological changes and tissue ingrowth, and by real-time RT-PCR and immunohistochemistry for immune responses. Throughout the period of implantation, all scaffolds were well tolerated by the host animals and immune responses to the implants were mild. Most scaffolds prepared from the all-aqueous process degraded to completion between 2 and 6 months, while those prepared from organic solvent (hexafluoroisopropanol (HFIP)) process persisted beyond 1 year. Due to widespread cellular invasion throughout the scaffold, the degradation of aqueous-derived scaffolds appears to be more homogeneous than that of HFIP-derived scaffolds. In general and especially for the HFIP-derived scaffolds, a higher original silk fibroin concentration (e.g. 17%) and smaller pore size (e.g. 100-200microm) resulted in lower levels of tissue ingrowth and slower degradation. These results demonstrate that the in vivo behavior of the three-dimensional silk fibroin scaffolds is related to the morphological and structural features that resulted from different scaffold preparation processes. The insights gained in this study can serve as a guide for processing scenarios to match desired morphological and structural features and degradation time with tissue-specific applications. PMID:18502501
Wang, Yongzhong; Rudym, Darya D; Walsh, Ashley; Abrahamsen, Lauren; Kim, Hyeon-Joo; Kim, Hyun S; Kirker-Head, Carl; Kaplan, David L
Evaluation of Gel Spun Silk-Based Biomaterials in a Murine Model of Bladder Augmentation
Currently, gastrointestinal segments are considered the gold standard for bladder reconstructive procedures. However, significant complications including chronic urinary tract infection, metabolic abnormalities, urinary stone formation, bowel dysfunction, and secondary malignancies are associated with this approach. Biomaterials derived from silk fibroin may represent a superior alternative due their robust mechanical properties, biodegradable features, and processing plasticity. In the present study, we evaluated the efficacy of a gel spun silk-based matrix for bladder augmentation in a murine model. Over the course of 70 d implantation period, H&E and Masson�s trichrome (MTS) analysis revealed that silk matrices were capable of supporting both urothelial and smooth muscle regeneration at the defect site. Prominent uroplakin and contractile protein expression (?-actin, calponin, and SM22?) was evident by immunohistochemical analysis demonstrating maturation of the reconstituted bladder wall compartments. Gel spun silk matrices also elicited a minimal acute inflammatory reaction following 70 d of bladder integration, in contrast to parallel assessments of small intestinal submucosa (SIS) and polyglycolic acid (PGA) matrices which routinely promoted evidence of fibrosis and chronic inflammatory responses. Voided stain on paper analysis revealed that silk augmented animals displayed similar voiding patterns in comparison to non surgical controls by 42 d of implantation. In addition, cystometric evaluations of augmented bladders at 70 d post-op demonstrated that silk scaffolds supported significant increases in bladder capacity, voided volume, and flow rate while maintaining similar degrees of compliance relative to the control group. These results provide evidence for the utility of gel spun silk-based matrices for functional bladder tissue engineering applications.
Mauney, Joshua R.; Cannon, Glenn M.; Lovett, Michael L.; Gong, Edward M.; DiVizio, Dolores; Kaplan, David L.; Adam, Rosalyn M.; Estrada, Carlos R.
In vitro chondrogenic differentiation of human adipose-derived stem cells with silk scaffolds
Human adipose-derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with natural and synthetic biomaterials. In the present study, we hypothesized that porous aqueous-derived silk protein scaffolds would be suitable for chondrogenic differentiation of human adipose-derived stem cells. Human adipose-derived stem cells were cultured up to 6 weeks, and cell proliferation and chondrogenic differentiation were investigated and compared with those in conventional micromass culture. Cell proliferation, glycosaminoglycan, and collagen levels in aqueous-derived silk scaffolds were significantly higher than in micromass culture. Transcript levels of SOX9 and type II collagen were also upregulated in the cell�silk constructs at 6 weeks. Histological examination revealed that the pores of the silk scaffolds were filled with cells uniformly distributed. In addition, chondrocyte-specific lacunae formation was evident and distributed in the both groups. The results suggest the biodegradable and biocompatible three-dimensional aqueous-derived silk scaffolds provided an improved environment for chondrogenic differentiation compared to micromass culture.
Kim, Hyeon Joo; Park, Sang-Hyug; Durham, Jennah; Gimble, Jeffrey M; Kaplan, David L
The expression analysis of silk gland-enriched intermediate-size non-coding RNAs in silkworm Bombyx mori.
Small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. Silkworm is an important model for studies on insect genetics and control of Lepidopterous pests. We have previously identified 189 novel intermediate-size ncRNAs in silkworm Bombyx mori, including 40 ncRNAs that showed altered expression in different developmental stages. Here we characterized the functions of these 40 ncRNAs by measuring their expressions in six tissues of the fifth instar larvae using Northern blot and real-time polymerase chain reaction assays. We identified nine ncRNAs (four small nucleolar RNAs and five unclassified ncRNAs) that were enriched in silk gland, including four ncRNAs that showed silk gland-specific expression. We further showed that three of nine silk gland-enriched ncRNAs were predominantly expressed in the anterior silk gland, whereas another three ncRNAs were highly accumulated in the posterior silk gland, suggesting that they may play different roles in fibroin synthesis. Furthermore, an unclassified ncRNA, Bm-152, exhibited converse expression pattern with its antisense host gene gartenzwerg in diverse tissues, and might regulate the expression of gartenzwerg through RNA-protein complex. In addition, two silk gland-enriched ncRNAs Bm-102 and Bm-159 can be found in histone modification complex, which indicated that they might play roles through epigenetic modifications. Taken together, we provided the first expression and preliminary functional analysis of silk gland-enriched ncRNAs, which will help understand the molecular mechanism of silk gland-development and fibroin synthesis. PMID:24124013
Li, Dan-Dan; Liu, Zong-Cai; Huang, Lei; Jiang, Qian-Ling; Zhang, Kun; Qiao, Hui-Li; Jiao, Zhu-Jin; Yao, Lun-Guang; Liu, Ren-Yi; Kan, Yun-Chao
New internal structure of spider dragline silk revealed by atomic force microscopy.
Atomic force microscopy was used to study the three-dimensional nanometer-scale structure of the dragline silk of Nephila clavipes from microtomed sections of the silk. Contrary to a previously proposed model of randomly distributed protein crystallites interspersed in amorphous regions, a highly organized skin-core structure of the fiber was observed. The skin appeared to be thin with no discernible distinct features. The core consists of pleated fibril-like structures, which are arranged in two concentric cylinders. Upon stretching, the pleats were smoothed out substantially. The mechanical properties of spider silk can quite straightforwardly be related to the newly observed structures. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4
Li, S F; McGhie, A J; Tang, S L
Determination of molecular weight of silk fibroin by non-gel sieving capillary electrophoresis.
A simple non-gel sieving capillary electrophoresis (NGSCE) method was established to determine the MW of silk fibroin using CE. The background electrolyte with a pH of 8.8 was based on three components: polyethylene glycol, tris(hydroxymethyl)aminomethane, and sodium dodecyl sulfate (SDS). NGSCE showed a good linear relationship with satisfactory reproducibility between the migration time and the MW of standard proteins. It was found that the regenerated silk fibroin had an MW around 83 kDa with a wide MW distribution (MWD). This absolute value is lower than the result obtained from SDS-polyacrylamide gel electrophoresis due to the different principles of the methods, but their similar MWD shapes indicated that NGSCE could be a feasible, highly sensitive, rapid method for determination of the MW of silk fibroin. PMID:20922945
Wei, Wei; Zhang, Yaopeng; Shao, Huili; Hu, Xuechao
Non-periodic lattice crystals in the hierarchical microstructure of spider (major ampullate) silk.
A commonly adopted model for the microstructure of Nephila clavipes major ampullate silk (MAS) is similar to that used for Bombyx mori (silkworm) silk: a simple composite wherein discrete, essentially perfect crystals are dispersed throughout an amorphous protein matrix. However, inconsistencies arise when researchers using complementary microstructural characterisation techniques attempt to explain their results within that framework. We present here the findings of our parallel studies in x-ray diffraction, electron microscopy, and molecular modeling. These results, combined with other data gleaned from the literature, lead us to propose a revised description of the spider silk microstructure. The new model recognizes that the 70-500 nm sized ordered regions in MAS cannot be constructed from a simple motif of repeating monomers, and develops the concept of non-periodic lattice (NPL) crystals to characterize these structures. The local composition, symmetry, and perfection of order vary over distances that are small compared to the size of an NPL crystal. PMID:9128438
Thiel, B L; Guess, K B; Viney, C
Dating silk by capillary electrophoresis mass spectrometry.
A new capillary electrophoresis mass spectrometry (CE-MS) technique is introduced for age estimation of silk textiles based on amino acid racemization rates. With an L to D conversion half-life of ~2500 years for silk (B. mori) aspartic acid, the technique is capable of dating silk textiles ranging in age from several decades to a few-thousand-years-old. Analysis required only ~100 ?g or less of silk fiber. Except for a 2 h acid hydrolysis at 110 �C, no other sample preparation is required. The CE-MS analysis takes ~20 min, consumes only nanoliters of the amino acid mixture, and provides both amino acid composition profiles and D/L ratios for ~11 amino acids. PMID:21913691
Moini, Mehdi; Klauenberg, Kathryn; Ballard, Mary
Beating the Heat - Fast Scanning Melts Silk Beta Sheet Crystals
Beta-pleated-sheet crystals are among the most stable of protein secondary structures, and are responsible for the remarkable physical properties of many fibrous proteins, such as silk, or proteins forming plaques as in Alzheimer's disease. Previous thinking, and the accepted paradigm, was that beta-pleated-sheet crystals in the dry solid state were so stable they would not melt upon input of heat energy alone. Here we overturn that assumption and demonstrate that beta-pleated-sheet crystals melt directly from the solid state to become random coils, helices, and turns. We use fast scanning chip calorimetry at 2,000?K/s and report the first reversible thermal melting of protein beta-pleated-sheet crystals, exemplified by silk fibroin. The similarity between thermal melting behavior of lamellar crystals of synthetic polymers and beta-pleated-sheet crystals is confirmed. Significance for controlling beta-pleated-sheet content during thermal processing of biomaterials, as well as towards disease therapies, is envisioned based on these new findings.
Cebe, Peggy; Hu, Xiao; Kaplan, David L.; Zhuravlev, Evgeny; Wurm, Andreas; Arbeiter, Daniela; Schick, Christoph
Beating the Heat - Fast Scanning Melts Silk Beta Sheet Crystals
NASA Astrophysics Data System (ADS)
Beta-pleated-sheet crystals are among the most stable of protein secondary structures, and are responsible for the remarkable physical properties of many fibrous proteins, such as silk, or proteins forming plaques as in Alzheimer's disease. Previous thinking, and the accepted paradigm, was that beta-pleated-sheet crystals in the dry solid state were so stable they would not melt upon input of heat energy alone. Here we overturn that assumption and demonstrate that beta-pleated-sheet crystals melt directly from the solid state to become random coils, helices, and turns. We use fast scanning chip calorimetry at 2,000 K/s and report the first reversible thermal melting of protein beta-pleated-sheet crystals, exemplified by silk fibroin. The similarity between thermal melting behavior of lamellar crystals of synthetic polymers and beta-pleated-sheet crystals is confirmed. Significance for controlling beta-pleated-sheet content during thermal processing of biomaterials, as well as towards disease therapies, is envisioned based on these new findings.
Cebe, Peggy; Hu, Xiao; Kaplan, David L.; Zhuravlev, Evgeny; Wurm, Andreas; Arbeiter, Daniela; Schick, Christoph
Silk Roads or Steppe Roads? The Silk Roads in World History.
ERIC Educational Resources Information Center
Explores the prehistory of the Silk Roads, reexamines their structure and history in the classical era, and explores shifts in their geography in the last one thousand years. Explains that a revised understanding of the Silk Roads demonstrates how the Afro-Eurasian land mass has been linked by networks of exchange since the Bronze Age. (CMK)
Christian, David
Acellular bi-layer silk fibroin scaffolds support tissue regeneration in a rabbit model of onlay urethroplasty.
Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a rabbit model of urethra repair. A bi-layer silk fibroin matrix was fabricated by a solvent-casting/salt leaching process in combination with silk fibroin film casting to generate porous foams buttressed by homogeneous silk fibroin films. Ventral onlay urethroplasty was performed with silk fibroin grafts (Group 1, N = 4) (Width � Length, 1 � 2 cm(2)) in adult male rabbits for 3 m of implantation. Parallel control groups consisted of animals receiving small intestinal submucosa (SIS) implants (Group 2, N = 4) or urethrotomy alone (Group 3, N = 3). Animals in all groups exhibited 100% survival prior to scheduled euthanasia and achieved voluntary voiding following 7 d of initial catheterization. Retrograde urethrography of each implant group at 3 m post-op revealed wide urethral calibers and preservation of organ continuity similar to pre-operative and urethrotomy controls with no evidence of contrast extravasation, strictures, fistulas, or stone formation. Histological (hematoxylin and eosin and Masson's trichrome), immunohistochemical, and histomorphometric analyses demonstrated that both silk fibroin and SIS scaffolds promoted similar extents of smooth muscle and epithelial tissue regeneration throughout the original defect sites with prominent contractile protein (?-smooth muscle actin and SM22?) and cytokeratin expression, respectively. De novo innervation and vascularization were also evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Following 3 m post-op, minimal acute inflammatory reactions were elicited by silk fibroin scaffolds characterized by the presence of eosinophil granulocytes while SIS matrices promoted chronic inflammatory responses indicated by mobilization of mononuclear cell infiltrates. The results of this study demonstrate that bi-layer silk fibroin scaffolds represent promising biomaterials for onlay urethroplasty, capable of promoting similar degrees of tissue regeneration in comparison to conventional SIS scaffolds, but with reduced immunogenicity. PMID:24632740
Chung, Yeun Goo; Tu, Duong; Franck, Debra; Gil, Eun Seok; Algarrahi, Khalid; Adam, Rosalyn M; Kaplan, David L; Estrada, Carlos R; Mauney, Joshua R
Acellular Bi-Layer Silk Fibroin Scaffolds Support Tissue Regeneration in a Rabbit Model of Onlay Urethroplasty
Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a rabbit model of urethra repair. A bi-layer silk fibroin matrix was fabricated by a solvent-casting/salt leaching process in combination with silk fibroin film casting to generate porous foams buttressed by homogeneous silk fibroin films. Ventral onlay urethroplasty was performed with silk fibroin grafts (Group 1, N?=?4) (Width�Length, 1�2 cm2) in adult male rabbits for 3 m of implantation. Parallel control groups consisted of animals receiving small intestinal submucosa (SIS) implants (Group 2, N?=?4) or urethrotomy alone (Group 3, N?=?3). Animals in all groups exhibited 100% survival prior to scheduled euthanasia and achieved voluntary voiding following 7 d of initial catheterization. Retrograde urethrography of each implant group at 3 m post-op revealed wide urethral calibers and preservation of organ continuity similar to pre-operative and urethrotomy controls with no evidence of contrast extravasation, strictures, fistulas, or stone formation. Histological (hematoxylin and eosin and Masson's trichrome), immunohistochemical, and histomorphometric analyses demonstrated that both silk fibroin and SIS scaffolds promoted similar extents of smooth muscle and epithelial tissue regeneration throughout the original defect sites with prominent contractile protein (?-smooth muscle actin and SM22?) and cytokeratin expression, respectively. De novo innervation and vascularization were also evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Following 3 m post-op, minimal acute inflammatory reactions were elicited by silk fibroin scaffolds characterized by the presence of eosinophil granulocytes while SIS matrices promoted chronic inflammatory responses indicated by mobilization of mononuclear cell infiltrates. The results of this study demonstrate that bi-layer silk fibroin scaffolds represent promising biomaterials for onlay urethroplasty, capable of promoting similar degrees of tissue regeneration in comparison to conventional SIS scaffolds, but with reduced immunogenicity.
Franck, Debra; Gil, Eun Seok; Algarrahi, Khalid; Adam, Rosalyn M.; Kaplan, David L.; Estrada Jr., Carlos R.; Mauney, Joshua R.
Studies on Silk Proteins. I. The Properties and Constitution of Fibroin. The Conversion of Fibroin into a Water-Soluble Form and Its Bearing on the Phenomenon of Denaturation
Some of the constituent amino-acids of fibroin (degummed silk) are determined. Special attention is directed to histidine, owing to its use in the calculation of the molecular weight of fibroin. A value of 0\\\\cdot 45% has been found by methods in which the histidine is isolated as nitranilate or di-(3:4-dichlorobenzenesulphonate). Other values obtained are serine 12\\\\cdot 6%, threonine 1\\\\cdot 5%,
D. Coleman; F. O. Howitt
The performance of silk scaffolds in a rat model of augmentation cystoplasty.
The diverse processing plasticity of silk-based biomaterials offers a versatile platform for understanding the impact of structural and mechanical matrix properties on bladder regenerative processes. Three distinct groups of 3-D matrices were fabricated from aqueous solutions of Bombyx mori silk fibroin either by a gel spinning technique (GS1 and GS2 groups) or a solvent-casting/salt-leaching method in combination with silk film casting (FF group). SEM analyses revealed that GS1 matrices consisted of smooth, compact multi-laminates of parallel-oriented silk fibers while GS2 scaffolds were composed of porous (pore size range, 5-50 ?m) lamellar-like sheets buttressed by a dense outer layer. Bi-layer FF scaffolds were comprised of porous foams (pore size, ~400 ?m) fused on their external face with a homogenous, nonporous silk film. Silk groups and small intestinal submucosa (SIS) matrices were evaluated in a rat model of augmentation cystoplasty for 10 weeks of implantation and compared to cystotomy controls. Gross tissue evaluations revealed the presence of intra-luminal stones in all experimental groups. The incidence and size of urinary calculi was the highest in animals implanted with gel spun silk matrices and SIS with frequencies ?57% and stone diameters of 3-4 mm. In contrast, rats augmented with FF scaffolds displayed substantially lower rates (20%) and stone size (2 mm), similar to the levels observed in controls (13%, 2 mm). Histological (hematoxylin and eosin, Masson's trichrome) and immunohistochemical (IHC) analyses showed comparable extents of smooth muscle regeneration and contractile protein (?-smooth muscle actin and SM22?) expression within defect sites supported by all matrix groups similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent uroplakin and p63 protein expression in all experimental groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by Fox3-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. In comparison to other biomaterial groups, cystometric analyses at 10 weeks post-op revealed that animals implanted with the FF matrix configuration displayed superior urodynamic characteristics including compliance, functional capacity, as well as spontaneous non voiding contractions consistent with control levels. Our data demonstrate that variations in scaffold processing techniques can influence the in vivo functional performance of silk matrices in bladder reconstructive procedures. PMID:23545287
Seth, Abhishek; Chung, Yeun Goo; Gil, Eun Seok; Tu, Duong; Franck, Debra; Di Vizio, Dolores; Adam, Rosalyn M; Kaplan, David L; Estrada, Carlos R; Mauney, Joshua R
The Performance of Silk Scaffolds in a Rat Model of Augmentation Cystoplasty
The diverse processing plasticity of silk-based biomaterials offers a versatile platform for understanding the impact of structural and mechanical matrix properties on bladder regenerative processes. Three distinct groups of 3-D matrices were fabricated from aqueous solutions of Bombyx mori silk fibroin either by a gel spinning technique (GS1 and GS2 groups) or a solvent-casting/salt-leaching method in combination with silk film casting (FF group). SEM analyses revealed that GS1 matrices consisted of smooth, compact multi-laminates of parallel-oriented silk fibers while GS2 scaffolds were composed of porous (pore size range, 5�50�m) lamellar-like sheets buttressed by a dense outer layer. Bi-layer FF scaffolds were comprised of porous foams (pore size, ~400 �m) fused on their external face with a homogenous, nonporous silk film. Silk groups and small intestinal submucosa (SIS) matrices were evaluated in a rat model of augmentation cystoplasty for 10 weeks of implantation and compared to cystotomy controls. Gross tissue evaluations revealed the presence of intra-luminal stones in all experimental groups. The incidence and size of urinary calculi was the highest in animals implanted with gel spun silk matrices and SIS with frequencies ?57% and stone diameters of 3�4mm. In contrast, rats augmented with FF scaffolds displayed substantially lower rates (20%) and stone size (2mm), similar to the levels observed in controls (13%, 2mm). Histological (hematoxylin and eosin, Masson's trichrome) and immunohistochemical (IHC) analyses showed comparable extents of smooth muscle regeneration and contractile protein (?-smooth muscle actin and SM22?) expression within defect sites supported by all matrix groups similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent uroplakin and p63 protein expression in all experimental groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by Fox3-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. In comparison to other biomaterial groups, cystometric analyses at 10 weeks post-op revealed that animals implanted with the FF matrix configuration displayed superior urodynamic characteristics including compliance, functional capacity, as well as spontaneous non voiding contractions consistent with control levels. Our data demonstrate that variations in scaffold processing techniques can influence the in vivo functional performance of silk matrices in bladder reconstructive procedures.
Seth, Abhishek; Chung, Yeun Goo; Gil, Eun Seok; Tu, Duong; Franck, Debra; Di Vizio, Dolores; Adam, Rosalyn M.; Kaplan, David L.; Estrada, Carlos R.; Mauney, Joshua R.
Silk / absorbable polyester hybrid medical devices and applications thereof
US Patent & Trademark Office Database
Silk/absorbable polyester hybrid medical devices for tissue repair and regeneration are formed of degummed, naturally produced, multifilament silk yarn in combination with at least one absorbable polyester in the form of a surface coating, with or without an antimicrobial agent, for producing value-added braided silk sutures and multifilament yarn, as well as silk/absorbable polyester fiber composites, for producing tailored hybrid sutures, meshes, and scaffolds for tissue regeneration.
Atomic force microscopy of orb-spider-web-silks to measure surface nanostructuring and evaluate silk fibers per strand
NASA Astrophysics Data System (ADS)
Atomic force microscopy (AFM) study is used to measure the surface topology and roughness of radial and capture spider silks on the micro- and nanoscale. This is done for silks of the orb weaver spider Argiope keyserlingi. Capture silk has a surface roughness that is five times less than that for radial silk. The capture silk has an equivalent flatness of ?/100 (5-6 nm deep surface features) as an optical surface. This is equivalent to a very highly polished optical surface. AFM does show the number of silk fibers that make up a silk thread but geometric distortion occurs during sample preparation. This prevented AFM from accurately measuring the silk topology on the microscale in this study.
Kane, D. M.; Naidoo, N.; Staib, G. R.
Silk Film Culture System for in vitro Analysis and Biomaterial Design
Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.
Lawrence, Brian D.; Pan, Zhi; Weber, Michael D.; Kaplan, David L.; Rosenblatt, Mark I.
Amphiphilic Spider Silk-Like Block Copolymers with Tunable Physical Properties and Morphology for Biomedical Applications
NASA Astrophysics Data System (ADS)
Silk-based materials are important candidates for biomedical applications because of their excellent biocompatibility and biodegradability. To generate silk amphiphilic biopolymers with potential use in guided tissue repair and drug delivery, a novel family of spider silk-like block copolymers was synthesized by recombinant DNA technology. Block copolymer thermal properties, structural conformations, protein-water interactions, and self-assembly morphologies were studied with respect to well controlled protein amino acid sequences. A theoretical model was used to predict the heat capacity of the protein and protein-water complex. Using thermal analysis, two glass transitions were observed: Tg1 is related to conformational changes caused by bound water removal, while Tg2 (>Tg1) is the glass transition of dry protein. Real-time infrared spectroscopy and X-ray diffraction confirmed that different secondary structural changes occur during the two Tg relaxations. Using scanning electron microscopy, fibrillar networks and hollow vesicles are observed, depending on protein block copolymer sequence. This study provides a deeper understanding of the relationship between protein physical properties and amino acid sequence, with implications for design of other protein-based materials.
Huang, Wenwen; Krishnaji, Sreevidhya; Kaplan, David; Cebe, Peggy
Microfabrication of a spider-silk analogue through the liquid rope coiling instability
NASA Astrophysics Data System (ADS)
Spider capture silk outperforms most synthetic materials in terms of specific toughness. We developed a technique to fabricate tough microstructured fibers inspired by the molecular structure of the spider silk protein. To fabricate microfibers (with diameter �30?m) with various mechanical properties, we yield the control of their exact geometry to the liquid rope coiling instability. This instability causes a thread of honey to wiggle as it buckles when hitting a surface. Similarly, we flow a filament of viscous polymer solution towards a substrate moving perpendicularly at a slower velocity than the filament flows. The filament buckles repetitively giving rise to periodic meanders and stitch patterns. As the solvent evaporates, the filament solidifies into a fiber with a geometry bestowed by the instability. Microtraction tests performed on fibers show interesting links between the mechanical properties and the instability patterns. Some coiling patterns give rise to high toughness due to the sacrificial bonds created when the viscous filament loops over itself and fuse. The sacrificial bonds in the microstructured fiber play an analogous role to that of the hydrogen bonds present in the molecular structure of the silk protein which give its toughness to spider silk.
Gosselin, Frederick P.; Therriault, Daniel; Levesque, Martin
Non-equilibrium silk fibroin adhesives.
Regenerated silkworm silk solutions formed metastable, soft-solid-like materials (e-gels) under weak electric fields, displaying interesting mechanical characteristics such as dynamic adhesion and strain stiffening. Raman spectroscopy, in situ electric field dynamic oscillatory rheology and polarized optical microscopy indicated that silk fibroin electrogelation involved intermolecular self-assembly of silk molecules into amorphous, micron-scale, micellar structures and the formation of relatively long lifetime, intermicellar entanglement crosslinks. Overall, the electrogelation process did not require significant intramolecular beta-strand or intermolecular beta-sheet formation, unlike silk hydrogels. The kinetics of e-gel formation could be tuned by changing the field strength and assembly conditions, such as silk concentration and solution pH, while e-gel stiffness was partially reversible by removal of the applied field. Transient adhesion testing indicated that the adhesive characteristics of e-gels could at least partially be attributed to a local increase in proton concentration around the positive electrode due to the applied field and surface effects. A working model of electrogelation was described en route to understanding the origins of the adhesive characteristics. PMID:20026216
Yucel, Tuna; Kojic, Nikola; Leisk, Gary G; Lo, Tim J; Kaplan, David L
Biocompatibility Assessment of Novel Collagen-Sericin Scaffolds Improved with Hyaluronic Acid and Chondroitin Sulfate for Cartilage Regeneration
Cartilage tissue engineering (CTE) applications are focused towards the use of implantable biohybrids consisting of biodegradable scaffolds combined with in vitro cultured cells. Hyaluronic acid (HA) and chondroitin sulfate (CS) were identified as the most potent prochondrogenic factors used to design new biomaterials for CTE, while human adipose-derived stem cells (ASCs) were proved to display high chondrogenic potential. In this context, our aim was not only to build novel 3D porous scaffolds based on natural compounds but also to evaluate their in vitro biological performances. Therefore, for prospective CTE, collagen-sericin (Coll-SS) scaffolds improved with HA (5% or 10%) and CS (5% or 10%) were used as temporary physical supports for ASCs and were analyzed in terms of structural, thermal, morphological, and swelling properties and cytotoxic potential. To complete biocompatibility data, ASCs viability and proliferation potential were also assessed. Our studies revealed that Coll-SS hydrogels improved with 10% HA and 5% CS displayed the best biological performances in terms of cell viability, proliferation, morphology, and distribution. Thus, further work will address a novel 3D system including both HA 10% and CS 5% glycoproteins, which will probably be exposed to prochondrogenic conditions in order to assess its potential use in CTE applications.
Galateanu, Bianca; Albu, Madalina
A new class of animal collagen masquerading as an insect silk.
Collagen is ubiquitous throughout the animal kingdom, where it comprises some 28 diverse molecules that form the extracellular matrix within organisms. In the 1960s, an extracorporeal animal collagen that forms the cocoon of a small group of hymenopteran insects was postulated. Here we categorically demonstrate that the larvae of a sawfly species produce silk from three small collagen proteins. The native proteins do not contain hydroxyproline, a post translational modification normally considered characteristic of animal collagens. The function of the proteins as silks explains their unusual collagen features. Recombinant proteins could be produced in standard bacterial expression systems and assembled into stable collagen molecules, opening the door to manufacture a new class of artificial collagen materials. PMID:24091725
Sutherland, Tara D; Peng, Yong Y; Trueman, Holly E; Weisman, Sarah; Okada, Shoko; Walker, Andrew A; Sriskantha, Alagacone; White, Jacinta F; Huson, Mickey G; Werkmeister, Jerome A; Glattauer, Veronica; Stoichevska, Violet; Mudie, Stephen T; Haritos, Victoria S; Ramshaw, John A M
Synthesis and characterization of biocompatible nanodiamond-silk hybrid material.
A new hybrid material consisting of nanodiamonds (NDs) and silk has been synthesized and investigated. NDs can contain bright fluorescence centers, important for bioprobes to image biological structures at the nanoscale and silk provides a transparent, robust matrix for these nanoparticles in-vivo or in-vitro. The ND-silk hybrid films were determined to be highly transparent in the visible to near infrared wavelength range. The NDs embedded in silk exhibited significant enhancement of emission relative to air, correlating with theoretical predictions. Furthermore, animal toxicity tests confirmed ND-silk films to be non-toxic in an in-vivo mice model. PMID:24575352
Khalid, Asma; Lodin, Rebecca; Domachuk, Peter; Tao, Hu; Moreau, Jodie E; Kaplan, David L; Omenetto, Fiorenzo G; Gibson, Brant C; Tomljenovic-Hanic, Snjezana
Salt-leached silk scaffolds with tunable mechanical properties
Substrate mechanical properties have remarkable influences on cell behavior and tissue regeneration. Although salt-leached silk scaffolds have been used in tissue engineering, applications in softer tissue regeneration can be encumbered with excessive stiffness. In the present study, silk-bound water interactions were regulated by controlling processing to allow the preparation of salt-leached porous scaffolds with tunable mechanical properties. Increasing silk-bound water interactions resulted in reduced silk II (beta-sheet crystal) formation during salt-leaching, which resulted in a modulus decrease in the scaffolds. The microstructures as well as degradation behavior were also changed, implying that this water control and salt-leaching approach can be used to achieve tunable mechanical properties. Considering the utility of silk in various fields of biomedicine, the results point to a new approach to generate silk scaffolds with controllable properties to better mimic soft tissues, by combining scaffold preparation methods and silk self-assembly in aqueous solutions.
Yao, Danyu; Dong, Sen; Lu, Qiang; Hu, Xiao; Kaplan, David L; Zhang, Bingbo; Zhu, Hesun
Development of silk-based scaffolds for tissue engineering of bone from human adipose derived stem cells
Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and demonstrated ability to support bone formation, in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASC), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol - HFIP), pore sizes (250�500?m vs. 500�1000?m) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes with decellularized trabecular bone (DCB) included as a �gold standard�. The scaffolds were seeded with hASC and cultured for 7 weeks in osteogenic media. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400�600?m porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2, and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation.
Correia, Cristina; Bhumiratana, Sarindr; Yan, Le-Ping; Oliveira, Ana L.; Gimble, Jeffrey M.; Rockwood, Danielle; Kaplan, David L.; Sousa, Rui A.; Reis, Rui L.; Vunjak-Novakovic, Gordana
High-resolution NMR characterization of a spider-silk mimetic composed of 15 tandem repeats and a CRGD motif.
Multidimensional solution NMR spectroscopic techniques have been used to obtain atomic level information about a recombinant spider silk construct in hexafluoro-isopropanol (HFIP). The synthetic 49 kDa silk-like protein mimics authentic silk from Nephila clavipes, with the inclusion of an extracellular matrix recognition motif. 2D (1)H-(15)N HSQC NMR spectroscopy reveals 33 cross peaks, which were assigned to amino acid residues in the semicrystalline repeat units. Signals from the amorphous segments in the primary sequence were weak and broad, suggesting that this region is highly dynamic and undergoing conformational exchange. An analysis of the deviations of the (13)C(alpha), (13)C(beta), and (13)CO chemical shifts relative to the expected random coil values reveals two highly alpha-helical regions from amino acid 12-19 and 26-32, which comprise the polyalanine track and a GGLGSQ sequence. This finding is further supported by phi-value analysis and sequential and medium-range NOE interactions. Pulsed field gradient NMR measurements indicate that the topology of the silk mimetic in HFIP is nonglobular. Moreover, the 3D (15)N-NOESY HSQC spectrum exhibits few long-range NOEs. Similar spectral features have been observed for repeat modules in other polypeptides and are characteristic of an elongated conformation. The results provide a residue-specific description of a silk sequence in nonaqueous solution and may be insightful for understanding the fold and topology of highly concentrated, stable silk before spinning. Additionally, the insights obtained may find application in future design and large-scale production and storage of synthetic silks in organic solvents. PMID:19177364
McLachlan, Glendon D; Slocik, Joseph; Mantz, Robert; Kaplan, David; Cahill, Sean; Girvin, Mark; Greenbaum, Steve
High-resolution NMR characterization of a spider-silk mimetic composed of 15 tandem repeats and a CRGD motif
Multidimensional solution NMR spectroscopic techniques have been used to obtain atomic level information about a recombinant spider silk construct in hexafluoro-isopropanol (HFIP). The synthetic 49 kDa silk-like protein mimics authentic silk from Nephila clavipes, with the inclusion of an extracellular matrix recognition motif. 2D 1H-15N HSQC NMR spectroscopy reveals 33 cross peaks, which were assigned to amino acid residues in the semicrystalline repeat units. Signals from the amorphous segments in the primary sequence were weak and broad, suggesting that this region is highly dynamic and undergoing conformational exchange. An analysis of the deviations of the 13C?, 13C?, and 13CO chemical shifts relative to the expected random coil values reveals two highly ?-helical regions from amino acid 12�19 and 26�32, which comprise the polyalanine track and a GGLGSQ sequence. This finding is further supported by ?-value analysis and sequential and medium-range NOE interactions. Pulsed field gradient NMR measurements indicate that the topology of the silk mimetic in HFIP is nonglobular. Moreover, the 3D 15N-NOESY HSQC spectrum exhibits few long-range NOEs. Similar spectral features have been observed for repeat modules in other polypeptides and are characteristic of an elongated conformation. The results provide a residue-specific description of a silk sequence in nonaqueous solution and may be insightful for understanding the fold and topology of highly concentrated, stable silk before spinning. Additionally, the insights obtained may find application in future design and large-scale production and storage of synthetic silks in organic solvents.
McLachlan, Glendon D; Slocik, Joseph; Mantz, Robert; Kaplan, David; Cahill, Sean; Girvin, Mark; Greenbaum, Steve
Modular evolution of egg case silk genes across orb-weaving spider superfamilies
Spider silk proteins (fibroins) are renowned for their extraordinary mechanical properties and biomimetic potential. Despite extensive evolutionary, ecological, and industrial interest in these fibroins, only a fraction of the known silk types have been characterized at the molecular level. Here we report cDNA and genomic sequences of the fibroin TuSp1, which appears to be the major component of tubuliform gland silk, a fiber exclusively synthesized by female spiders for egg case construction. We obtained TuSp1 sequences from 12 spider species that represent the extremes of phylogenetic diversity within the Orbicularia (orb-weaver superfamilies, Araneoidea and Deinopoidea) and finer scale sampling within genera. TuSp1 encodes tandem arrays of an ?200-aa-long repeat unit and individual repeats are readily aligned, even among species that diverged >125 million years ago. Analyses of these repeats across species reveal the strong influence of concerted evolution, resulting in intragenic homogenization. However, deinopoid TuSp1 repeats also contain insertions of coding, minisatellite-like sequences, an apparent result of replication slippage and nonreciprocal recombination. Phylogenetic analyses of 37 spider fibroin sequences support the monophyly of TuSp1 within the spider fibroin gene family, consistent with a single origin of this ortholog group. The diversity of taxa and silks examined here confirms that repetitive architecture is a general feature of this gene family. Moreover, we show that TuSp1 provides a clear example of modular evolution across a range of phylogenetic levels.
Garb, Jessica E.; Hayashi, Cheryl Y.
Molecular characterization and silk gland expression of Bombyx engrailed and invected genes.
Genetic analysis in Drosophila has shown that engrailed (en) plays an important role in segmentation and neurogenesis. A closely related gene, invected (in), is coexpressed with en in the posterior developmental compartments where en is known to specify cell state. We report here the isolation of two en-like cDNAs from the middle silk glands of Bombyx mori larvae. Sequence analysis revealed that they are the counterparts of Drosophila en and in. Four highly conserved domains, including the homeodomain, were identified in these En and In proteins from Bombyx and Drosophila. In addition, two en-specific and one in-specific domains could also be found. These structurally homologous genes might share a similar role in Bombyx development. They were found to be coexpressed in the middle silk gland but not in the posterior silk gland during the fourth molt/fifth intermolt period. We speculate that these Bombyx en-like genes might be involved in the compartmentalization of the silk gland. Images
Hui, C C; Matsuno, K; Ueno, K; Suzuki, Y
Porous Silk Scaffolds for Delivery of Growth Factors and Stem Cells to Enhance Bone Regeneration
Stem cell-based tissue engineering shows promise for bone regeneration and requires artificial microenvironments to enhance the survival, proliferation and differentiation of the seeded cells. Silk fibroin, as a natural protein polymer, has unique properties for tissue regeneration. The present study aimed to evaluate the influence of porous silk scaffolds on rat bone marrow stem cells (BMSCs) by lenti-GFP tracking both in vitro and in vivo in cranial bone defects. The number of cells seeded within silk scaffolds in rat cranial bone defects increased from 2 days to 2 weeks after implantation, followed by a decrease at eight weeks. Importantly, the implanted cells survived for 8 weeks in vivo and some of the cells might differentiate into endothelial cells and osteoblasts induced by the presence of VEGF and BMP-2 in the scaffolds to promote angiogenesis and osteogenesis. The results demonstrate that porous silk scaffolds provide a suitable niche to maintain long survival and function of the implanted cells for bone regeneration.
Xu, Ling; Zhang, Xiaochen; Wu, Qianju; Zhang, Xiuli; Kaplan, David L.; Jiang, Xinquan
Phononic band gap and mechanical anisotropy in spider silk
NASA Astrophysics Data System (ADS)
Spider dragline silk is a semi-crystalline biopolymer exhibiting superior properties compared to synthetic polymers with similar chemical structure, such as polyamides. This is ascribed to the hierarchical nanostructure that is created in the spinning duct. During this process the aqueous solution of the two protein constituents of dragline silk is crystallized, while the macromolecules maintain their high orientation, leading to a high value of the Young's modulus (in the order of 10 GPa) along the fiber. We employed spontaneous Brillouin light scattering to measure the longitudinal modulus (M//,,M) along the two symmetry directions of the native fiber with increased (decreased) pre-strain created by stretching (supercontracting after hydration). A strong mechanical anisotropy is found; at about 18% strain M///M�5. Most important, an unexpected finding is the first observation of a unidirectional hypersonic phononic band gap in biological structures. This relates to the existence of a strain-dependent correlation length of the mechanical modulus in the submicron range along the fiber axis.
Papadopoulos, Periklis; Gomopoulos, Nikos; Kremer, Friedrich; Fytas, George
Stimuli-responsive capsules prepared from regenerated silk fibroin microspheres.
Microcapsules are synthesized via the self-assembly of silk-fibroin microspheres with polycaprolactone in a colliodosome preparation procedure. The microsphere building blocks with controlled diameter are prepared from the naturally occurring biopolymer, silk fibroin, and then act as stabilizers for oil-in-water (O/W) emulsion and organized themselves on the surface of chloroform droplets to form the capsules. The concentration of the protein-based microspheres and the binding polycacrolactone is used to tailor the size, as well as the permeability of the resultant capsules. Furthermore, microencapsulation of fluorescently labeled macromolecules (20-2000 KDa) is demonstrated. The permeability of the capsules is observed to be stimuli responsive, making it possible to incorporate a pH-triggered payload release from the capsules. The relatively simple preparation of capsules with controlled dimensions and tunable permeability, alongside the biocompatibility and biodegradability of both polymer components makes these promising materials for potential use in smart drug-delivery systems. PMID:24532252
Cheng, Cheng; Teasdale, Ian; Br�ggemann, Oliver
The use of injectable sonication-induced silk hydrogel for VEGF165 and BMP-2 delivery for elevation of the maxillary sinus floor
Sonication-induced silk hydrogels were previously prepared as an injectable bone replacement biomaterial, with a need to improve osteogenic features. Vascular endothelial growth factor (VEGF165) and bone morphogenic protein-2 (BMP-2) are key regulators of angiogenesis and osteogenesis, respectively, during bone regeneration. Therefore, the present study aimed at evaluating in situ forming silk hydrogels as a vehicle to encapsulate dual factors for rabbit maxillary sinus floor augmentation. Sonication-induced silk hydrogels were prepared in vitro and the slow release of VEGF165 and BMP-2 from these silk gels was evaluated by ELISA. For in vivo studies for each time point (4 and 12 weeks), 24 sinus floors elevation surgeries were made bilaterally in 12 rabbits for the following four treatment groups: silk gel (group Silk gel), silk gel/VEGF165 (group VEGF), silk gel/BMP-2 (group BMP-2), silk gel/VEGF165/BMP-2 (group V+B) (n=6 per group). Sequential florescent labeling and radiographic observations were used to record new bone formation and mineralization, along with histological and histomorphometric analysis. At week 4, VEGF165 promoted more tissue infiltration into the gel and accelerated the degradation of the gel material. At this time point, the bone area in group V+B was significantly larger than those in the other three groups. At week 12, elevated sinus floor heights of groups BMP-2 and V+B were larger than those of the Silk gel and VEGF groups, and the V+B group had the largest new bone area among all groups. In addition, a larger blood vessel area formed in the remaining gel areas in groups VEGF and V+B. In conclusion, VEGF165 and BMP-2 released from injectable and biodegradable silk gels promoted angiogenesis and new bone formation, with the two factors demonstrating an additive effect on bone regeneration. These results indicate that silk hydrogels can be used as an injectable vehicle to deliver multiple growth factors in a minimally invasive approach to regenerate irregular bony cavities.
Zhang, Wenjie; Wang, Xiuli; Wang, Shaoyi; Zhao, Jun; Xu, Lianyi; Zhu, Chao; Zeng, Deliang; Chen, Jake; Zhang, Zhiyuan; Kaplan, David L.; Jiang, Xinquan
Silk ionomers for encapsulation and differentiation of human MSCs
The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-L-lysine or silk-poly-L-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-L-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-L-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages.
Calabrese, Rossella; Kaplan, David L.
The Ancient Art of Silk Painting
ERIC Educational Resources Information Center
In this article, the author describes a silk-painting project with a sea-creature theme for eighth-grade students. Other themes can be used such as geometric quilt designs, tropical rain forest, large flowers, Art Nouveau motifs, portraits and more. (Contains 2 resources.)
Yonker, Kim
Constructing Knowledge with Silk Road Visuals
ERIC Educational Resources Information Center
In this study a group of elementary teachers use illustrations, rather than written text, to introduce their students to the peoples and places of the ancient silk routes. The illustrations are from two picture books; "Marco Polo," written by Gian Paolo Cesaerani and illustrated by Piero Ventura (1977), and "We're Riding on a Caravan: An Adventure�
Bisland, Beverly Milner
Solid lipid nanoparticles coated with silk fibroin
Solid lipid nanoparticles (SLNs) composed of stearic acid (SA) and ceramide (Cer) were prepared by an emulsification and solidification method using sodium lauryl sulfate (an anionic surfactant) as a stabilizer. And then, the SLN was coated with silk fibroin (SF) under an acidic condition by an electrostatic interaction. The size was tens of nanometers to hundreds of nanometers, and the
Teak Kwan Kwon; Kun Bin Lim; Jin-Chul Kim
Opportunities for Intervention in Thailand's Silk Subsector.
National Technical Information Service (NTIS)
The silk subsector holds enormous promise as a potential engine of income growth for poor village households. But to take advantage of the growth, most producers will have to shift from the low-productivity, low-growth traditional markets to the rapidly g...
S. Haggblade N. Ritchie
Green process to prepare silk fibroin/gelatin biomaterial scaffolds.
A new all-aqueous and green process is described to form three-dimensional porous silk fibroin matrices with control of structural and morphological features. Silk-based scaffolds are prepared using lyophilization. Gelatin is added to the aqueous silk fibroin solution to change the silk fibroin conformation and silk fibroin-water interactions through adjusting the hydrophilic interactions in silk fibroin-gelatin-water systems to restrain the formation of separate sheet like structures in the material, resulting in a more homogenous structure. Water annealing is used to generate insolubility in the silk fibroin-gelatin scaffold system, thereby avoiding the use of organic solvents such as methanol to lock in the beta-sheet structure. The adjusting of the concentration of gelatin, as well as the concentration of silk fibroin, leads to control of morphological and functional properties of the scaffolds. The scaffolds were homogeneous in terms of interconnected pores, with pore sizes ranging from 100 to 600 microm, depending on the concentration of silk fibroin used in the process. At the same time, the morphology of the scaffolds changed from lamellar sheets to porous structures based on the increase in gelatin content. Compared with salt-leaching aqueous-derived scaffolds and hexafluoroisopropanol (HFIP)-derived scaffolds, these freeze-dried scaffolds had a lower content of beta-sheet, resulting in more hydrophilic features. Most of gelatin was entrapped in the silk fibroin-gelatin scaffolds, without the burst release in PBS solution. During in vitro cell culture, these silk fibroin-gelatin scaffolds had improved cell-compatibility than salt-leaching silk fibroin scaffolds. This new process provides useful silk fibroin-based scaffold systems for use in tissue engineering. Furthermore, the whole process is green, including all-aqueous, room temperature and pressure, and without the use of toxic chemicals or solvents, offering new ways to load bioactive drugs or growth factors into the process. PMID:19924684
Lu, Qiang; Zhang, Xiaohui; Hu, Xiao; Kaplan, David L
The Influence of Elasticity and Surface Roughness on Myogenic and Osteogenic-Differentiation of Cells on Silk-Elastin Biomaterials
The interactions of C2C12 myoblasts and human bone marrow stem cells (hMSCs) with silk-tropoelastin biomaterials, and the capacity of each to promote attachment, proliferation, and either myogenic- or osteogenic-differentiation were investigated. Temperature-controlled water vapor annealing was used to control beta-sheet crystal formation to generate insoluble silk-tropoelastin biomaterial matrices at defined ratios of the two proteins. These ratios controlled surface roughness and micro/nano-scale topological patterns, and elastic modulus, stiffness, yield stress, and tensile strength. A combination of low surface roughness and high stiffness in the silk-tropoelastin materials promoted proliferation and myogenic-differentiation of C2C12 cells. In contrast, high surface roughness with micro/nano-scale surface patterns was favored by hMSCs. Increasing the content of human tropoelastin in the silk-tropoelastin materials enhanced the proliferation and osteogenic-differentiation of hMSCs. We conclude that the silk-tropoelastin composition facilitates fine tuning of the growth and differentiation of these cells.
Hu, Xiao; Park, Sang-Hyug; Gil, Eun Seok; Xia, Xiao-Xia; Weiss, Anthony S.; Kaplan, David L.
Synthesis, characterization and immunogenicity of silk fibroin-L-asparaginase bioconjugates.
L-asparaginase (ASNase) is one basic drug in the treatment of acute lymphoblastic leukemia (ALL). Because its half-life time is too short and it is easy to arouse allergic reaction, use in practical clinic is considerably limited. Silk fibroin (SF) with different molecular mass from 40 to 120 kDa is a natural biocompatible protein and could be used as a novel bioconjugate for enzyme modification to overcome its usual shortcomings mentioned above. When the enzyme was bioconjugated covalently with the water-soluble fibroin by glutaraldehyde, the enzyme kinetic properties and immune characteristics in vivo of the resulting silk fibroin-L-asparaginase (SF-ASNase) bioconjugates were investigated in detail. The results show that the modified ASNase was characterized by its higher residual activity (nearly 80%), increased heat and storage stability and resistance to trypsin digestion, and its longer half-life (63 h) than that of intact ASNase (33 h). The abilities of intact and modified ASNases to arouse allergic reaction are 2(4) and 2(1) antibody titers, respectively. Bioconjugation of silk fibroin significantly helps to reduce the immunogenicity and antigenicity of the enzyme. The apparent Michaelis constants of the modified ASNase (K(m(app))=0.844 x 10(-3)mol L(-1)) was approximately six times lower than that of enzyme alone, which suggests that the affinity of the enzyme to substrate l-asparagine elevated when bioconjugated covalently with silk fibroin. SF-ASNase bioconjugates could overcome the common shortcomings of the native form. Therefore, the modified ASNase coupled with silk fibroin has the potential values of being studied and developed as a new bioconjugate drug. PMID:16102867
Zhang, Yu-Qing; Zhou, Wei-Lin; Shen, Wei-De; Chen, Yu-Hua; Zha, Xin-Min; Shirai, Koji; Kiguchi, Kenji
Functionalization of silk fibroin with NeutrAvidin and biotin.
New methods are needed to modify silk biomaterials with bioactive molecules for tissue engineering and drug delivery. In the present study, silk fibroin in solution or in microsphere format was coupled with NeutrAvidin via carbodiimide chemistry. Silk fibroin retained its self-assembly features after reaction. It was found that more than four NeutrAvidin molecules bound to one silk molecule. Non-specific binding of biotin or NeutrAvidin to silk microspheres could be reduced by pre-treatment of the microspheres with BSA or post-treatment with detergent. The NeutrAvidin-coupled silk microspheres were coupled with biotinylated anti-CD3 antibody and the functionalized microspheres were able to specifically bind to the CD3 positive T-lymphocytic cell line Jurkat. PMID:20824692
Wang, Xiaoqin; Kaplan, David L
Inkjet printing of silk nest arrays for cell hosting.
An inkjet printing approach is presented for the facile fabrication of microscopic arrays of biocompatible silk "nests" capable of hosting live cells for prospective biosensors. The patterning of silk fibroin nests were constructed by the layer-by-layer (LbL) assembly of silk polyelectrolytes chemically modified with poly-(l-lysine) and poly-(l-glutamic acid) side chains. The inkjet-printed silk circular regions with a characteristic "nest" shape had diameters of 70-100 ?m and a thickness several hundred nanometers were stabilized by ionic pairing and by the formation of the silk II crystalline secondary structure. These "locked-in" silk nests remained anchored to the substrate during incubation in cell growth media to provide a biotemplated platform for printing-in, immobilization, encapsulation and growth of cells. The process of inkjet-assisted printing is versatile and can be applied on any type of substrate, including rigid and flexible, with scalability and facile formation. PMID:24605757
Suntivich, Rattanon; Drachuk, Irina; Calabrese, Rossella; Kaplan, David L; Tsukruk, Vladimir V
Controlling the Molecular Structure and Physical Properties of Artificial Honeybee Silk by Heating or by Immersion in Solvents
Honeybee larvae produce silken cocoons that provide mechanical stability to the hive. The silk proteins are small and non-repetitive and therefore can be produced at large scale by fermentation in E. coli. The recombinant proteins can be fabricated into a range of forms; however the resultant material is soluble in water and requires a post production stabilizing treatment. In this study, we describe the structural and mechanical properties of sponges fabricated from artificial honeybee silk proteins that have been stabilized in aqueous methanol baths or by dry heating. Aqueous methanol treatment induces formation of �-sheets, with the amount of �-sheet dictated by methanol concentration. Formation of �-sheets renders sponges insoluble in water and generates a reversibly compressible material. Dry heat treatments at 190�C produce a water insoluble material, that is stiffer than the methanol treated equivalent but without significant secondary structural changes. Honeybee silk proteins are particularly high in Lys, Ser, Thr, Glu and Asp. The properties of the heat treated material are attributed to generation of lysinoalanine, amide (isopeptide) and/or ester covalent cross-links. The unique ability to stabilize material by controlling secondary structure rearrangement and covalent cross-linking allows us to design recombinant silk materials with a wide range of properties.
Huson, Mickey G.; Church, Jeffrey S.; Poole, Jacinta M.; Weisman, Sarah; Sriskantha, Alagacone; Warden, Andrew C.; Campbell, Peter M.; Ramshaw, John A. M.; Sutherland, Tara D.
Enhanced chondrogenic responses of articular chondrocytes onto porous silk fibroin scaffolds treated with microwave-induced argon plasma
Silk fibroin (SF) as a naturally occurring degradable fibrous protein with unique mechanical properties, excellent biocompatibility and processability has demonstrated strong potential for skeletal tissue engineering. Recent studies has mostly focused on nanofibrous SF (NSF) as a novel chondrogenic scaffold since its structure is very similar to collagen fibrous structure of natural extracellular matrix (ECM). However, less attention has been
Hyun Sook Baek; Young Hwan Park; Chang Seok Ki; Jong-Chul Park; Dong Kyun Rah
Controlling the molecular structure and physical properties of artificial honeybee silk by heating or by immersion in solvents.
Honeybee larvae produce silken cocoons that provide mechanical stability to the hive. The silk proteins are small and non-repetitive and therefore can be produced at large scale by fermentation in E. coli. The recombinant proteins can be fabricated into a range of forms; however the resultant material is soluble in water and requires a post production stabilizing treatment. In this study, we describe the structural and mechanical properties of sponges fabricated from artificial honeybee silk proteins that have been stabilized in aqueous methanol baths or by dry heating. Aqueous methanol treatment induces formation of �-sheets, with the amount of �-sheet dictated by methanol concentration. Formation of �-sheets renders sponges insoluble in water and generates a reversibly compressible material. Dry heat treatments at 190�C produce a water insoluble material, that is stiffer than the methanol treated equivalent but without significant secondary structural changes. Honeybee silk proteins are particularly high in Lys, Ser, Thr, Glu and Asp. The properties of the heat treated material are attributed to generation of lysinoalanine, amide (isopeptide) and/or ester covalent cross-links. The unique ability to stabilize material by controlling secondary structure rearrangement and covalent cross-linking allows us to design recombinant silk materials with a wide range of properties. PMID:23300639
Huson, Mickey G; Church, Jeffrey S; Poole, Jacinta M; Weisman, Sarah; Sriskantha, Alagacone; Warden, Andrew C; Campbell, Peter M; Ramshaw, John A M; Sutherland, Tara D
Mussel collagen molecules with silk-like domains as load-bearing elements in distal byssal threads
Mechanically stressed biological materials like tendon, spider silk or mussel byssal threads are typically composite materials comprising multi-domain proteins, in which molecular building blocks contribute to overall material function.Mussel byssal threads are the anchorage of sessile mytilid mussels, which withstand recurring external loads from waves and tides. A single thread is elastic and ductile proximally, while the distal portion exhibits
Anja Hagenau; Periklis Papadopoulos; Friedrich Kremer; Thomas Scheibel
Osteoinductive silk�silica composite biomaterials for bone regeneration
Osteoinductive and biodegradable composite biomaterials for bone regeneration were prepared by combining silk fibroin with silica particles. The influence of these composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on silk\\/silica films. The addition of the silica to the silk films influenced gene expression
Aneta J. Mieszawska; Nikolaos Fourligas; Irene Georgakoudi; Nadia M. Ouhib; David J. Belton; Carole C. Perry; David L. Kaplan
Rheology of Self-Assembling Silk Fibroin Solutions
NASA Astrophysics Data System (ADS)
A robust procedure for preparation of aqueous silk fibroin solutions with a range of concentration up to 25 wt% from domestic Bombyx mori cocoon shells has been established. We have carried out molecular and rheometric characterizations of silk fibroin solutions, and constructed an equilibrium phase diagram. The sharp sol-gel transition can be exploited for rapid solidification of micro-morphological structure. We will discuss the correlations between fluid formulation, rheological properties and processibility of silk fibroin in the talk.
Zhou, Rui; Chen, Song-Bi; Yuan, Xue-Feng
Modulating Materials by Orthogonally Oriented ?-Strands: Composites of Amyloid and Silk Fibroin Fibrils.
Amyloid fibrils and silk fibroin (SF) fibrils are proteinaceous aggregates occurring either naturally or as artificially reconstituted fibrous systems, in which the constituent ?-strands are aligned either orthogonally or parallel to the fibril main axis, conferring complementary physical properties. Here, it is shown how the combination of these two classes of protein fibrils with orthogonally oriented ?-strands results in composite materials with controllable physical properties at the molecular, mesoscopic, and continuum length scales. PMID:24845975
Ling, Shengjie; Li, Chaoxu; Adamcik, Jozef; Shao, Zhengzhong; Chen, Xin; Mezzenga, Raffaele
Spider Silk as Guiding Biomaterial for Human Model Neurons
Over the last years, a number of therapeutic strategies have emerged to promote axonal regeneration. An attractive strategy is the implantation of biodegradable and nonimmunogenic artificial scaffolds into injured peripheral nerves. In previous studies, transplantation of decellularized veins filled with spider silk for bridging critical size nerve defects resulted in axonal regeneration and remyelination by invading endogenous Schwann cells. Detailed interaction of elongating neurons and the spider silk as guidance material is unknown. To visualize direct cellular interactions between spider silk and neurons in vitro, we developed an in vitro crossed silk fiber array. Here, we describe in detail for the first time that human (NT2) model neurons attach to silk scaffolds. Extending neurites can bridge gaps between single silk fibers and elongate afterwards on the neighboring fiber. Culturing human neurons on the silk arrays led to an increasing migration and adhesion of neuronal cell bodies to the spider silk fibers. Within three to four weeks, clustered somata and extending neurites formed ganglion-like cell structures. Microscopic imaging of human neurons on the crossed fiber arrays in vitro will allow for a more efficient development of methods to maximize cell adhesion and neurite growth on spider silk prior to transplantation studies.
Roloff, Frank; Strauss, Sarah; Vogt, Peter M.; Bicker, Gerd; Radtke, Christine
Spider silk as guiding biomaterial for human model neurons.
Over the last years, a number of therapeutic strategies have emerged to promote axonal regeneration. An attractive strategy is the implantation of biodegradable and nonimmunogenic artificial scaffolds into injured peripheral nerves. In previous studies, transplantation of decellularized veins filled with spider silk for bridging critical size nerve defects resulted in axonal regeneration and remyelination by invading endogenous Schwann cells. Detailed interaction of elongating neurons and the spider silk as guidance material is unknown. To visualize direct cellular interactions between spider silk and neurons in vitro, we developed an in vitro crossed silk fiber array. Here, we describe in detail for the first time that human (NT2) model neurons attach to silk scaffolds. Extending neurites can bridge gaps between single silk fibers and elongate afterwards on the neighboring fiber. Culturing human neurons on the silk arrays led to an increasing migration and adhesion of neuronal cell bodies to the spider silk fibers. Within three to four weeks, clustered somata and extending neurites formed ganglion-like cell structures. Microscopic imaging of human neurons on the crossed fiber arrays in vitro will allow for a more efficient development of methods to maximize cell adhesion and neurite growth on spider silk prior to transplantation studies. PMID:24949480
Roloff, Frank; Strau�, Sarah; Vogt, Peter M; Bicker, Gerd; Radtke, Christine
Differential binding of the Bombyx silk gland-specific factor SGFB to its target DNA sequence drives posterior-cell-restricted expression.
The gene encoding the silk protein P25 in Bombyx mori is expressed in the posterior silk gland (PSG) cells and repressed in the middle silk gland (MSG) cells. To identify the factors involved in this transcription-dependent spatial restriction, we examined the P25 chromatin in PSG and MSG nuclei by DNase I-aided ligation-mediated PCR and analyzed the expression of various P25-lacZ constructs in biolistically treated silk glands. P25 promoter activation depends on two cis-acting elements. One coincides with the target sequence of SGFB, a silk gland-specific factor present in all silk gland nuclei, but bound to its target DNA sequence in only PSG cells. The interaction of the other element with a factor that we named PSGF is also exclusive to PSG cells. Placed ahead of a non-P25-related basal promoter, the SGFB and PSGF elements are sufficient to drive posterior-cell transcription. Collectively, our data support the hypothesis that the spatial restriction of P25 expression is driven by the stabilization of SGFB onto its target sequence by the action of PSGF.
Horard, B; Julien, E; Nony, P; Garel, A; Couble, P
Volume constancy during stretching of spider silk.
The characterization of silk properties requires a reliable measurement of stress-strain curves from tensile tests, which calls for a detailed analysis of what is considered the cross section of the sample and how it varies during the experiments. Here, spider silk fibers from the major ampullate gland (MAS) of Argiope trifasciata spiders are tensile tested, and the cross-sectional area is measured under different strained configurations. It has been found that the fiber volume remains practically constant during stretching, and deformation proceeds homogeneously in all the fibers. The conservation of volume is validated independently of the type of fiber and the strain level. This result, applied to compute true stress-strain curves for different MAS fibers, shows that the description of their properties depends noticeably on which set of tensile parameters is chosen (true or engineering), and that engineering values could lead to misinterpretation of experiments that combine results from different strain ranges. PMID:16827584
Guinea, G V; P�rez-Rigueiro, J; Plaza, G R; Elices, M
Pollination Timing Effects on Kernel Set and Silk Receptivity in Four Maize Hybrids
have drastic impacts on grain or seed production profit- ability, and may be influenced by silk characteristics for Silk receptivity and kernel set vary with time of pollination and a given hybrid or inbred. environment. Our objectives were to evaluate differences in kernel set, silk elongation patterns, and duration of silk receptivity for four Cell elongation requires maintenance of sufficient
Steven R. Anderson; Michael J. Lauer; John B. Schoper; Richard M. Shibles
Project for a Comprehensive Study of the Silk Roads
From time immemorial the Silk Road, with its hundreds of byways, was considered the highway that linked the worlds of East and West. In the mid dle of the twentieth century, scholarly research revealed the crucial role played by oases in linking the North to the South; these oases, even more than the transversal lines of the Silk Roads, played
Vadim Eliseyev
Geographic Perspectives with Elementary Students: The Silk Road
ERIC Educational Resources Information Center
The purpose of this study is to investigate elementary students' explanations of how physical features of the land influence the location of humanly defined structures including trade routes, such as the silk routes. The silk routes were a series of caravan trade routes that extended from Turkey to China and were located as far south as India and�
Bisland, Beverly Milner
Visual Literacy with Picture Books: The Silk Road
ERIC Educational Resources Information Center
The ancient Silk Routes connecting China to Europe across the rugged mountains and deserts of central Asia are one of the primary examples of transculturation in world history. Traders on these routes dealt not only in goods such as silk and horses but also made possible the spread of art forms as well as two major religions, Buddhism and Islam. �
Bisland, Beverly Milner Lee
The failure mode of natural silk epoxy triggered composite tubes
NASA Astrophysics Data System (ADS)
In this study the quasi static compression test over natural silk epoxy triggered composite tubes has been carried out, the natural silk epoxy composite tubes consist of 24 layer of woven natural silk as reinforcement and thermoset epoxy resin as matrix which both of them i e natural silk and epoxy have excellent mechanical properties More over the natural silk have better moisture resistance in comparison with other natural reinforcements, the length of tubes are 50, 80 and 120 mm The natural silk epoxy composite tubes are associated with an external trigger which includes 4 steel pieces welded on downside flat plate fixture The hand lay up fabrication method has been used to make the natural silk epoxy composite tubes Instron universal testing machine with 250 KN load capacity has been employed to accomplish this investigation The failure modes of natural silk epoxy triggered composite tubes has been investigated by representative photographs which has been taken by a high resolution camera(12 2 Mp) during the quasi static compression test, from the photographs is observed the failure modes is progressive local buckling
Eshkour, R. A.; Ariffin, A. K.; Zulkifli, R.; Sulong, A. B.; Azhari, C. H.
Innovative and Multidirectional Applications of Natural Fibre, Silk - A Review
Realization of current trends and innovative uses of insect fibre, silk is required to exploit its compatibility, eco friendly and value addition potential. The nutritive value as human diet for cardiac and diabetic patients, component for cosmetic preparations indicate silk application rate. The bio-compatibility made it a base material for tissue wall, membrane, muscle ligament, blood vessel, nerve gadget, cartilage
R. Manohar Reddy
An Experimental study on Electrospinning of silk fibroin
Silk fibroin is one of candidate materials for biomedical application, because it has good biocompatibility and minimal inflammatory reaction. Electrospinning is a simple method capable of producing nano scale fibers from both synthetic and natural polymers for biomedical applications. In this study nonwoven matrices of silk fibroin (SF) nanofibers were prepared by electrospinning of regenerated SF solution. Effects of electric
Nasim Amiralian; Mahdi Nouri; Mohammad Haghighat
China-Indian Silk Trade: Current Production and Future Prospects
As an important traditional labor-intensive industry of both India and China, the cocoon silk industry has long made great contributions to the ecological environment protection, rural economic development and the increase in export income of both countries. India is not only a very important cocoon silk trading partner, but an important production competitor of China. In recent years, there has
Liu Feng; Sun Shimin; Qiao Xianjuan
Intervertebral Disk Tissue Engineering Using Biphasic Silk Composite Scaffolds
Scaffolds composed of synthetic, natural, and hybrid materials have been investigated as options to restore intervertebral disk (IVD) tissue function. These systems fall short of the lamellar features of the native annulus fibrosus (AF) tissue or focus only on the nucleus pulposus (NP) tissue. However, successful regeneration of the entire IVD requires a combination approach to restore functions of both the AF and NP. To address this need, a biphasic biomaterial structure was generated by using silk protein for the AF and fibrin/hyaluronic acid (HA) gels for the NP. Two cell types, porcine AF cells and chondrocytes, were utilized. For the AF tissue, two types of scaffold morphologies, lamellar and porous, were studied with the porous system serving as a control. Toroidal scaffolds formed out of the lamellar, and porous silk materials were used to generate structures with an outer diameter of 8?mm, inner diameter of 3.5?mm, and a height of 3?mm (the interlamellar distance in the lamellar scaffold was 150�250??m, and the average pore sizes in the porous scaffolds were 100�250??m). The scaffolds were seeded with porcine AF cells to form AF tissue, whereas porcine chondrocytes were encapsulated in fibrin/HA hydrogels for the NP tissue and embedded in the center of the toroidal disk. Histology, biochemical assays, and gene expression indicated that the lamellar scaffolds supported AF-like tissue over 2 weeks. Porcine chondrocytes formed the NP phenotype within the hydrogel after 4 weeks of culture with the AF tissue that had been previously cultured for 2 weeks, for a total of 6 weeks of cultivation. This biphasic scaffold simulating in combination of both AF and NP tissues was effective in the formation of the total IVD in vitro.
Park, Sang-Hyug; Gil, Eun Seok; Cho, Hongsik; Mandal, Biman B.; Tien, Lee W.; Min, Byoung-Hyun
Apatite-coated Silk Fibroin Scaffolds to Healing Mandibular Border Defects in Canines
Tissue engineering has become a new approach for repairing bony defects. Highly porous osteoconductive scaffolds perform the important role for the success of bone regeneration. By biomimetic strategy, apatite-coated porous biomaterial based on silk fibroin scaffolds (SS) might provide an enhanced osteogenic environment for bone-related outcomes. To assess the effects of apatite-coated silk fibroin (mSS) biomaterials for bone healing as a tissue engineered bony scaffold, we explored a tissue engineered bony graft using mSS seeded with osteogenically induced autologous bone marrow stromal cells (bMSCs) to repair inferior mandibular border defects in a canine model. The results were compared with those treated with bMSCs/SS constructs, mSS alone, SS alone, autologous mandibular grafts and untreated blank defects. According to radiographic and histological examination, new bone formation was observed from 4 weeks post-operation, and the defect site was completely repaired after 12 months for the bMSCs/mSS group. In the bMSCs/SS group, new bone formation was observed with more residual silk scaffold remaining at the center of the defect compared with the bMSCs/mSS group. The engineered bone with bMSCs/mSS achieved satisfactory bone mineral densities (BMD) at 12 months post-operation close to those of normal mandible (p>0.05). The quantities of newly formed bone area for the bMSCs/mSS group was higher than the bMSCs/SS group (p<0.01), but no significant differences were found when compared with the autograft group (p>0.05). In contrast, bony defects remained in the center with undegraded silk fibroin scaffold and fibrous connective tissue, and new bone only formed at the periphery in the groups treated with mSS or SS alone. The results suggested apatite-coated silk fibroin scaffolds combined with bMSCs could be successfully used to repair mandibular critical size border defects and the premineralization of these porous silk fibroin protein scaffolds provided an increased osteoconductive environment for bMSCs to regenerate sufficient new bone tissue.
Zhao, Jun; Zhang, Zhiyuan; Wang, Shaoyi; Sun, Xiaojuan; Zhang, Xiuli; Chen, Jake; Kaplan, David L.; Jiang, Xinquan
?-Sheet nanocrystalline domains formed from phosphorylated serine-rich motifs in caddisfly larval silk: a solid state NMR and XRD study.
Adhesive silks spun by aquatic caddisfly (order Trichoptera) larvae are used to build both intricate protective shelters and food harvesting nets underwater. In this study, we use (13)C and (31)P solid-state NMR and wide angle X-ray diffraction (WAXD) as tools to elucidate molecular protein structure of caddisfly larval silk from the species Hesperophylax consimilis . Caddisfly larval silk is a fibroin protein based biopolymer containing mostly repetitive amino acid motifs. NMR and X-ray results provide strong supporting evidence for a structural model in which phosphorylated serine repeats (pSX)4 complex with divalent cations Ca(2+) and Mg(2+) to form rigid nanocrystalline ?-sheet structures in caddisfly silk. (13)C NMR data suggests that both phosphorylated serine and neighboring valine residues exist in a ?-sheet conformation while glycine and leucine residues common in GGX repeats likely reside in random coil conformations. Additionally, (31)P chemical shift anisotropy (CSA) analysis indicates that the phosphates on phosphoserine residues are doubly ionized, and are charge-stabilized by divalent cations. Positively charged arginine side chains also likely play a role in charge stabilization. Finally, WAXD results finds that the silk is at least 7-8% crystalline, with ?-sheet interplane spacings of 3.7 and 4.5 �. PMID:23452243
Addison, J Bennett; Ashton, Nicholas N; Weber, Warner S; Stewart, Russell J; Holland, Gregory P; Yarger, Jeffery L
?-Sheet Nanocrystalline Domains Formed from Phosphorylated Serine-Rich Motifs in Caddisfly Larval Silk: A Solid State NMR and XRD Study
Adhesive silks spun by aquatic caddisfly (order Trichoptera) larvae are used to build both intricate protective shelters and food harvesting nets underwater. In this study, we use 13C and 31P solid-state Nuclear Magnetic Resonance (NMR) and Wide Angle X-ray Diffraction (WAXD) as tools to elucidate molecular protein structure of caddisfly larval silk from the species Hesperophylax consimilis. Caddisfly larval silk is a fibroin protein based biopolymer containing mostly repetitive amino acid motifs. NMR and X-ray results provide strong supporting evidence for a structural model in which phosphorylated serine repeats (pSX)4 complex with divalent cations Ca2+ and Mg2+ to form rigid nanocrystalline ?-sheet structures in caddisfly silk. 13C NMR data suggests that both phosphorylated serine and neighboring valine residues exist in a ?-sheet secondary structure conformation while glycine and leucine residues common in GGX repeats likely reside in random coil conformations. Additionally, 31P chemical shift anisotropy (CSA) analysis indicates that the phosphates on phosphoserine residues are doubly ionized, and are charge-stabilized by divalent cations. Positively charged arginine side chains also likely play a role in charge stabilization. Finally, WAXD results finds that the silk is at least 7�8% crystalline, with ?-sheet inter-plane spacings of 3.7 and 4.5 �.
Addison, J. Bennett; Ashton, Nicholas N.; Weber, Warner S.; Stewart, Russell J.; Holland, Gregory P.; Yarger, Jeffery L.
Templated native silk smectic gels
NASA Technical Reports Server (NTRS)
One aspect of the present invention relates to a method of preparing a fibrous protein smectic hydrogel by way of a solvent templating process, comprising the steps of pouring an aqueous fibrous protein solution into a container comprising a solvent that is not miscible with water; sealing the container and allowing it to age at about room temperature; and collecting the resulting fibrous protein smectic hydrogel and allowing it to dry. Another aspect of the present invention relates to a method of obtaining predominantly one enantiomer from a racemic mixture, comprising the steps of pouring an aqueous fibrous protein solution into a container comprising a solvent that is not miscible with water; sealing the container and allowing it to age at about room temperature; allowing the enantiomers of racemic mixture to diffuse selectively into the smectic hydrogel in solution; removing the smectic hydrogel from the solution; rinsing predominantly one enantiomer from the surface of the smectic hydrogel; and extracting predominantly one enantiomer from the interior of the smectic hydrogel. The present invention also relates to a smectic hydrogel prepared according to an aforementioned method.
Jin, Hyoung-Joon (Inventor); Park, Jae-Hyung (Inventor); Valluzzi, Regina (Inventor)
Helicoidal multi-lamellar features of RGD-functionalized silk biomaterials for corneal tissue engineering
RGD-coupled silk protein-biomaterial lamellar systems were prepared and studied with human cornea fibroblasts (hCFs) to match functional requirements. A strategy for corneal tissue engineering was pursued to replicate the structural hierarchy of human corneal stroma within thin stacks of lamellae-like tissues, in this case constructed from scaffolds constructed with RGD-coupled, patterned, porous, mechanically robust and transparent silk films. The influence of RGD-coupling on the orientation, proliferation, ECM organization, and gene expression of hCFs was assessed. RGD surface modification enhanced cell attachment, proliferation, alignment and expression of both collagens (type I and V) and proteoglycans (decorin and biglycan). Confocal and histological images of the lamellar systems revealed that the bio-functionalized silk human cornea 3D constructs exhibited integrated corneal stroma tissue with helicoidal multi-lamellar alignment of collagen-rich and proteoglycan-rich extracellular matrix, with transparency of the construct. This biomimetic approach to replicate corneal stromal tissue structural hierarchy and architecture demonstrates a useful strategy for engineering human cornea. Further, this approach can be exploited for other tissue systems due to the pervasive nature of such helicoids in most human tissues.
Gil, Eun Seok; Mandal, Biman B.; Park, Sang-Hyug; Marchant, Jeffrey K.; Omenetto, Fiorenzo G.; Kaplan, David L.
Multifunctionalized electrospun silk fibers promote axon regeneration in central nervous system
The repair of central nerves remains a major challenge in regenerative neurobiology. Regenerative guides possessing critical features such as cell adhesion, physical guiding and topical stimulation are needed. To generate such a guide, silk protein materials are prepared using electrospinning. The silk is selected for this study due to its biocompatibility and ability to be electrospun for the formation of aligned biofunctional nanofibers. The addition of Brain Derived Neurotrophic Factor (BDNF), Ciliary Neurotrophic Factor (CNTF) or both to the electrospun fibers enable enhanced function without impact to the structure or the surface morphology. Only a small fraction of the loaded growth factors is released over time allowing the fibers to continue to provide these factors to the cells for extended periods of time. The entrapped factors remain active and available to the cells as rat retinal ganglion cells (RGCs) exhibit longer axonal growth when in contact with the biofunctionalized fibers. Compare to non-functionalized fibers, the growth of neurites increased 2 fold on fibers containing BDNF, 2.5 fold with fibers containing CNTF and by almost 3-fold on fibers containing both factors. The results demonstrate the potential of aligned and functionalized electrospun silk fibers to promote nerve growth in the central nervous system, underlying the great potential of complex biomaterials in neuroregenerative strategies following axotomy and nerve crush traumas.
Wittmer, Corinne R.; Claudepierre, Thomas; Reber, Michael; Wiedemann, Peter; Garlick, Jonathan A.; Kaplan, David
Evaluation of Silk Biomaterials in Combination with Extracellular Matrix Coatings for Bladder Tissue Engineering with Primary and Pluripotent Cells
Silk-based biomaterials in combination with extracellular matrix (ECM) coatings were assessed as templates for cell-seeded bladder tissue engineering approaches. Two structurally diverse groups of silk scaffolds were produced by a gel spinning process and consisted of either smooth, compact multi-laminates (Group 1) or rough, porous lamellar-like sheets (Group 2). Scaffolds alone or coated with collagen types I or IV or fibronectin were assessed independently for their ability to support attachment, proliferation, and differentiation of primary cell lines including human bladder smooth muscle cells (SMC) and urothelial cells as well as pluripotent cell populations, such as murine embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells. AlamarBlue evaluations revealed that fibronectin-coated Group 2 scaffolds promoted the highest degree of primary SMC and urothelial cell attachment in comparison to uncoated Group 2 controls and all Group 1 scaffold variants. Real time RT-PCR and immunohistochemical (IHC) analyses demonstrated that both fibronectin-coated silk groups were permissive for SMC contractile differentiation as determined by significant upregulation of ?-actin and SM22? mRNA and protein expression levels following TGF?1 stimulation. Prominent expression of epithelial differentiation markers, cytokeratins, was observed in urothelial cells cultured on both control and fibronectin-coated groups following IHC analysis. Evaluation of silk matrices for ESC and iPS cell attachment by alamarBlue showed that fibronectin-coated Group 2 scaffolds promoted the highest levels in comparison to all other scaffold formulations. In addition, real time RT-PCR and IHC analyses showed that fibronectin-coated Group 2 scaffolds facilitated ESC and iPS cell differentiation toward both urothelial and smooth muscle lineages in response to all trans retinoic acid as assessed by induction of uroplakin and contractile gene and protein expression. These results demonstrate that silk scaffolds support primary and pluripotent cell responses pertinent to bladder tissue engineering and that scaffold morphology and fibronectin coatings influence these processes.
Franck, Debra; Gil, Eun Seok; Adam, Rosalyn M.; Kaplan, David L.; Chung, Yeun Goo; Estrada, Carlos R.; Mauney, Joshua R.
Analysis of Transcripts Expressed in One-Day-Old Larvae and Fifth Instar Silk Glands of Tasar Silkworm, Antheraea mylitta
Antheraea mylitta is one of the wild nonmulberry silkworms, which produces tasar silk. An EST project has been undertaken to understand the gene expression profile of A. mylitta silk gland. Two cDNA libraries, one from the whole bodies of one-day-old larvae and the other from the silkglands of fifth instar larvae, were constructed and sequenced. A total of 2476 good-quality ESTs (1239 clones) were obtained and grouped into 648 clusters containing 390 contigs and 258 singletons to represent 467 potential unigenes. Forty-five sequences contained putative coding region, and represented potentially novel genes. Among the 648 clusters, 241 were categorized according to Gene Ontology hierarchy and showed presence of several silk and immune-related genes. The A. mylitta ESTs have been organized into a freely available online database �AmyBASE�. These data provide an initial insight into the A. mylitta transcriptome and help to understand the molecular mechanism of silk protein production in a Lepidopteran species.
Maity, Samita; Goel, Sagar I.; Roy, Sobhan; Ghorai, Suvankar; Bhattacharyya, Swati; Venugopalan, Aravind; Ghosh, Ananta K.
Synthesis and properties of multiwalled carbon nanotubes incorporated organic silk fibroin cryogels.
A new monolithic organic cryogel was synthesized from a silk fibroin/multiwalled carbon nanotube (MWCNT) hydrogel by the freeze-drying method in order to maintain the silk fibroin network. The silk fibroin/MWCNT hydrogel was prepared by the regeneration of silk fibroin using an aqueous silk fibroin solution in which MWCNTs were dispersed by in-situ methods. It was observed that the formation of the silk fibroin/MWCNT hydrogel was accelerated by increasing the MWCNT concentration. Fourier transform infrared spectrum analysis, X-ray diffraction, nitrogen adsorption-desorption, and electron microscopy analysis techniques were used to characterize the silk fibroin/MWCNT cryogel. The MWCNTs were embedded in the silk fibroin based cryogel with disordered porous structures. In addition, the MWCNTs induced a change in the crystal structure of the silk fibroin and also increased the proportions of micropores and mesopores in the supermacroporous cryogels when they were introduced in the appropriate amounts. PMID:19908758
Kwon, Soon-Min; Kim, Hun-Sik; Jin, Hyoung-Joon
Silk-pectin hydrogel with superior mechanical properties, biodegradability, and biocompatibility.
A new method is developed to prepare silk hydrogels and silk-pectin hydrogels via dialysis against methanol to obtain hydrogels with high concentrations of silk fibroin. The relationship between the mechanical and biological properties and the structure of the silk-pectin hydrogels is subsequently evaluated. The present results suggest that pectin associates with silk molecules when the silk concentration exceeds 15?wt%, suggesting that a silk concentration of over 15?wt% is critical to construct interacting silk-pectin networks. The silk-pectin hydrogel reported here is composed of a heterogeneous network, which is different from fiber-reinforced, interpenetrated networks and double-network hydrogels, as well as high-stiffness hydrogels (elastic modulus of 4.7?�?0.9?MPa, elastic stress limit of 3.9?�?0.1?MPa, and elastic strain limit of 48.4?�?0.5%) with regard to biocompatibility and biodegradability. PMID:24610718
Numata, Keiji; Yamazaki, Shoya; Katashima, Takuya; Chuah, Jo-Ann; Naga, Naofumi; Sakai, Takamasa
Doxorubicin-loaded silk films: drug-silk interactions and in vivo performance in human orthotopic breast cancer
Breast cancer is the most common of all malignant diseases in women. Systemic chemotherapy provides low clinical benefit for locoregional control of the disease, while localised chemotherapy may provide a therapeutic advantage. In this study, doxorubicin-loaded silk films were directly applied to tumours. Affinity binding studies demonstrated that the adsorption of doxorubicin onto silk was partially dependent on crystallinity. By manipulating silk crystallinity, or ?-sheet content, the doxorubicin release rate could be controlled ranging from immediate release to prolonged release over >4 weeks. Following successful in vitro studies, the therapeutic impact of doxorubicin-loaded silk films on primary tumour growth and metastasis was assessed in mice using a humanised orthotopic breast cancer model (adenocarcinoma). Both soluble and stabilised silk films loaded with doxorubicin had a significantly greater primary tumour response than the equivalent dose of doxorubicin administered intravenously in the absence of the silk film carrier. In addition to reducing primary tumour growth, stabilised silk films loaded with doxorubicin also reduced metastatic spread and autopsy indicated that these films were not associated with any local or systemic toxicities. Collectively, these results suggest that the future use of this approach for localised chemotherapy is promising.
Seib, F. Philipp; Kaplan, David L.
Plasticity in Major Ampullate Silk Production in Relation to Spider Phylogeny and Ecology
Spider major ampullate silk is a high-performance biomaterial that has received much attention. However, most studies ignore plasticity in silk properties. A better understanding of silk plasticity could clarify the relative importance of chemical composition versus processing of silk dope for silk properties. It could also provide insight into how control of silk properties relates to spider ecology and silk uses. We compared silk plasticity (defined as variation in the properties of silk spun by a spider under different conditions) between three spider clades in relation to their anatomy and silk biochemistry. We found that silk plasticity exists in RTA clade and orbicularian spiders, two clades that differ in their silk biochemistry. Orbiculariae seem less dependent on external spinning conditions. They probably use a valve in their spinning duct to control friction forces and speed during spinning. Our results suggest that plasticity results from different processing of the silk dope in the spinning duct. Orbicularian spiders seem to display better control of silk properties, perhaps in relation to their more complex spinning duct valve.
Boutry, Cecilia; Rezac, Milan; Blackledge, Todd Alan
Effects of Microwave Radiation on Selected Mechanical Properties of Silk
NASA Astrophysics Data System (ADS)
Impressive mechanical properties have served to peak interest in silk as an engineering material. In addition, the ease with which silk can be altered through processing has led to its use in various biomaterial applications. As the uses of silk branch into new territory, it is imperative (and inevitable) to discover the boundary conditions beyond which silk no longer performs as expected. These boundary conditions include factors as familiar as temperature and humidity, but may also include other less familiar contributions, such as exposure to different types of radiation. The inherent variations in mechanical properties of silk, as well as its sensitivity to moisture, suggest that in an engineering context silk is best suited for use in composite materials; that way, silk can be shielded from ambient moisture fluctuations, and the surrounding matrix allows efficient load transfer from weaker fibers to stronger ones. One such application is to use silk as a reinforcing fiber in epoxy composites. When used in this way, there are several instances in which exposure to microwave radiation is likely (for example, as a means of speeding epoxy cure rates), the effects of which remain mostly unstudied. It will be the purpose of this dissertation to determine whether selected mechanical properties of B. mori cocoon silk are affected by exposure to microwave radiation, under specified temperature and humidity conditions. Results of our analyses are directly applicable wherever exposure of silk to microwave radiation is possible, including in fiber reinforced epoxy composites (the entire composite may be microwaved to speed epoxy cure time), or when silk is used as a component in the material used to construct the radome of an aircraft (RADAR units use frequencies in the microwave range of the electromagnetic spectrum), or when microwave energy is used to sterilize biomaterials (such as cell scaffolds) made of silk. In general, we find that microwave exposure does not detract from the average mechanical properties of silk, but that it may increase the spread of data points around that average. Along the way, we come to a number of useful conclusions, summarized here: Regarding silk in general: � Storage conditions can have a significant and enduring effect on tensile properties of degummed B. mori silk. Samples stored in a sealed container with desiccant (silica gel) have a lower yield stress and yield strain than samples stored without desiccant and they also relax more rapidly in stress relaxation tests. The ability of this silk to resist plastic deformation is optimized at intermediate hydration levels. Sensitivity to the humidity levels encountered by samples prior to testing complicates the interpretation of results, and makes inter-laboratory comparisons challenging. Silk storage conditions should therefore be reported---and, ideally, standardized---to enable useful comparison between studies. (Abstract shortened by UMI.)
Reed, Emily Jane
Immobilized Lentivirus Vector on Chondroitin Sulfate-Hyaluronate Acid-Silk Fibroin Hybrid Scaffold for Tissue-Engineered Ligament-Bone Junction
The lack of a fibrocartilage layer between graft and bone remains the leading cause of graft failure after anterior cruciate ligament (ACL) reconstruction. The objective of this study was to develop a gene-modified silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS) hybrid scaffold for reconstructing the fibrocartilage layer. The scaffold was fabricated by lyophilizing the CHS mixture with braided silk cables. The scanning electronic microscopy (SEM) showed that microporous CHS sponges were formed around silk cables. Each end of scaffold was modified with lentiviral-mediated transforming growth factor-?3 (TGF-?3) gene. The cells on scaffold were transfected by bonded lentivirus. In vitro culture demonstrated that mesenchymal stem cells (MSCs) on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of cartilage-specific genes also increased with culture time. After 2 weeks, the MSCs were distributed uniformly throughout scaffold. Deposited collagen was also found to increase. The chondral differentiation of MSCs was verified by expressions of collagen II and TGF-?3 genes in mRNA and protein level. Histology also confirmed the production of cartilage extracellular matrix (ECM) components. The results demonstrated that gene-modified silk cable-reinforced CHS scaffold was capable of supporting cell proliferation and differentiation to reconstruct the cartilage layer of interface.
Sun, Liguo; Li, Hongguo; Qu, Ling; Zhu, Rui; Fan, Xiangli; Xue, Yingsen; Xie, Zhenghong; Fan, Hongbin
Conductive Au nanowires regulated by silk fibroin nanofibers
NASA Astrophysics Data System (ADS)
Conductive Au-biopolymer composites have promising applications in tissue engineering such as nerve tissue regeneration. In this study, silk fibroin nanofibers were formed in aqueous solution by regulating silk self-assembly process and then used as template for Au nanowire fabrication. We performed the synthesis of Au seeds by repeating the seeding cycles for several times in order to increase the density of Au seeds on the nanofibers. After electroless plating, densely decorated Au seeds grew into irregularly shaped particles following silk nanofiber to fill the gaps between particles and finally form uniform continuous nanowires. The conductive property of the Au-silk fibroin nanowires was studied with current-voltage ( I-V) measurement. A typical ohmic behavior was observed, which highlighted their potential applications in nerve tissue regeneration.
Dong, Bo-Ju; Lu, Qiang
21 CFR 878.5030 - Natural nonabsorbable silk surgical suture.
Code of Federal Regulations, 2013 CFR
... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES Surgical Devices § 878.5030 Natural nonabsorbable silk surgical suture. (a) Identification....
Silk fibroin and polyethylene glycol-based biocompatible tissue adhesives
Tissue sealants have emerged in recent years as strong candidates for hemostasis. A variety of formulations are currently commercially available and though they satisfy many of the markets� needs there are still key aspects of each that need improvement. Here we present a new class of blends, based on silk fibroin and chemically active polyethylene glycols (PEGs) with strong adhesive properties. These materials are cytocompatible, crosslink within seconds via chemical reaction between thiols and maleimides present on the constituent PEGs and have the potential to further stabilize through ?-sheet formation by silk. Based on the silk concentration in the final formulation, the adhesive properties of these materials are comparable or better than the current leading PEG-based sealant. In addition, the silk-PEG based materials show decreased swelling and longer degradation times. Such properties would make them suitable for applications for which the current sealants are contraindicated.
Serban, Monica A.; Panilaitis, Bruce; Kaplan, David L.
Study on Bombyx mori silk treated by oxygen plasma.
Study of the morphology, aggregation structure and properties of Bombyx mori silk treated by low temperature oxygen plasma showed that slight flutes appeared on the surface of Bombyx mori silk fiber and that its surface structure changed after plasma treatment. The conformation also changed and crystalline degree decreased. The stannic filling rate of treated fiber was improved. Because of etching, the weight of the fiber decreased but the breaking strength changed little after short-time treatment. PMID:15236475
Chen, Yu-Yue; Lin, Hong; Ren, Yu; Wang, Hong-Wei; Zhu, Liang-Jun
Study on Bombyx mori silk treated by oxygen plasma
Study of the morphology, aggregation structure and properties ofBombyx mori silk treated by low temperature oxygen plasma showed that slight flutes appeared on the surface ofBombyx mori silk fiber and that its surface structure changed after plasma treatment. The conformation also changed and crystalline degree\\u000a decreased. The stannic filling rate of treated fiber was improved. Because of etching, the weight
Chen Yu-yue; Lin Hong; Ren Yu; Wang Hong-wei; Zhu Liang-jun
SilkDB: a knowledgebase for silkworm biology and genomics
The Silkworm Knowledgebase (SilkDB) is a web- based repository for the curation, integration and study of silkworm genetic and genomic data. With the recent accomplishment of a 6X draft genome sequence of the domestic silkworm (Bombyx mori), SilkDB provides an integrated representation of the large-scale, genome-wide sequence assembly, cDNAs, clusters of expressed sequence tags (ESTs), transposable elements (TEs), mutants, single
Jing Wang; Qingyou Xia; Ximiao He; Mingtao Dai; Jue Ruan; Jie Chen; Guo Yu; Haifeng Yuan; Yafeng Hu; Ruiqiang Li; Tao Feng; Chen Ye; Cheng Lu; Jun Wang; Songgang Li; Gane Ka-shu Wong; Huanming Yang; Jian Wang; Zhonghuai Xiang; Zeyang Zhou; Jun Yu
Beating the heat--fast scanning melts silk beta sheet crystals.
Beta-pleated-sheet crystals are among the most stable of protein secondary structures, and are responsible for the remarkable physical properties of many fibrous proteins, such as silk, or proteins forming plaques as in Alzheimer's disease. Previous thinking, and the accepted paradigm, was that beta-pleated-sheet crystals in the dry solid state were so stable they wo