Abstract
Functionalized biomaterial scaffolds targeted at improving axonal regeneration by enhancing guided axonal growth provide a promising approach for the repair of spinal cord injury. Collagen neural conduits provide structural guidance for neural tissue regeneration, and in this study it is shown that these conduits can also act as a reservoir for sustained gene delivery. Either a G-luciferase marker gene or a neurotrophin-3-encoding gene, complexed to a non-viral, cyclized, PEGylated transfection vector, was loaded within a multichannel collagen conduit. The complexed genes were then released in a controlled fashion using a dual release system both in vitro and in vivo. For evaluation of their biological performance, the loaded conduits were implanted into the completely transected rat thoracic spinal cord (T8-T10). Aligned axon regeneration through the channels of conduits was observed one month post-surgery. The conduits delivering neurotrophin-3 polyplexes resulted in significantly increased neurotrophin-3 levels in the surrounding tissue and a statistically higher number of regenerated axons versus the control conduits (Po0.05). This study suggests that collagen neural conduits delivering a highly effective non-viral therapeutic gene may hold promise for repair of the injured spinal cord. 1 The application of cell therapy, molecular therapy and biomaterial scaffolds in SCI has increased the understanding of the factors contributing to, and the requirements for, spinal cord repair.
Abstract
2-12 However, significant improvements are still required for the advancement of therapeutic strategy to clinical practice.
Abstract
A number of natural and synthetic biomaterial scaffolds have been implanted in models of SCI. These include compression, hemisection and complete transection SCI models.
INTRODUCTION
Spinal cord injury (SCI) is a devastating condition, which often results in life-long disability and a broad range of secondary complications. 1 The application of cell therapy, molecular therapy and biomaterial scaffolds in SCI has increased the understanding of the factors contributing to, and the requirements for, spinal cord repair. [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] However, significant improvements are still required for the advancement of therapeutic strategy to clinical practice.
INTRODUCTION
A number of natural and synthetic biomaterial scaffolds have been implanted in models of SCI. These include compression, hemisection and complete transection SCI models. [11] [12] [13] [14] Compression allows study in a more clinically relevant model for spinal cord repair. However, analysis of regeneration in this setting can be confusing, as origins of regenerating axons or processes make it difficult to isolate (for example, regenerating axons may be sprouting from remaining uninjured axons). 13 Complete transection allows analysis of repair in a more isolated environment, and regenerative processes can be more readily traced to their site of origin. For regeneration of the injured cord, biomaterial scaffolds can provide the structural support necessary for repair and provide appropriate architecture for topographical guidance to the regenerating axons. Biomaterials-based scaffolds have been applied to the injured spinal cord in a number of configurations and designs (for example, single-and multichannel conduits and hydrogels) and have shown the ability to promote regeneration within the injured spinal cord. 11, 13, 15, 16 These scaffolds serve to bypass or prevent the formation of the glial scar and allow axons to regenerate in a more growth-permissive environment. However, when the regenerated axon number reaches the limitation of their re-growth capacity in such scaffolds, functional restoration becomes limited. This limited regenerative capacity may be primarily due to a poor regenerative environment with an insubstantial supply of growth-promoting factors.
INTRODUCTION
One method of extending the regenerative capacity is the use of cell or molecular therapies. Cell therapies such as the use of peripheral Schwann cells or stem cells enclosed within the biomaterial scaffold have proven efficacy in enhancing spinal cord repair. 13, [17] [18] [19] [20] However, a lot remains to be understood about the interaction and behavior of the transplanted cells in such a hostile microenvironment. In particular, studies have shown that implanted neural stem cells result in increased allodynia over the course of the treatment. 13, 21 Alternatively, molecular therapies that enhance axonal regeneration by delivering growthpromoting and survival factors (for example, nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 (NT-3) and/or reduce inhibitory components of the glial scar (chondroitinase ABC) have shown promise for repair. [17] [18] [19] [22] [23] [24] [25] [26] However, one major limitation restricts their applicability for repair. The use of such therapies shows limited improvement when used in single dose administration, as they cannot maintain a constant biological effect in vivo and multiple doses are required for maintenance. 27, 28 Ideally, this biological effect would be 1 maintained throughout the regenerative process, without the need for multiple injections or further invasive therapy. Gene therapy offers an alternative strategy, which would allow the maintenance of such biological effects. NT-3, an attractive candidate growth factor for neuronal repair, is involved in axon path-finding by mediating its chemoattractive property and promoting growth of the spinal cord axons. 29, 30 Viral vectors are a well-established system for gene delivery to the central nervous system because of their long-term, stable transduction. 31, 32 A previous study demonstrated that neural conduits delivering viral vectors encoding NT-3 can improve axonal growth within the injured spinal cord. 33 Although recombinant viral vectors efficiently transduce cells, their clinical therapeutic efficacy may be compromised by the potential risk of insertional mutagenesis, immunogenicity and difficulty of scaling-up. [34] [35] [36] Alternatively, non-viral gene vectors avoid these perceived safety concerns by generating functional proteins while remaining episomal in the host cell. To employ a non-viral strategy a highly efficient gene vector with a low-toxicity profile and a vector that protects and delivers the plasmid of interest is required. The high transfection capability of 2-(dimethylamino) ethyl methacrylate-based polymers has recently been shown through in vitro studies. 37, 38 In this study a cyclized 2-(dimethylamino) ethyl methacrylatebased polymer that consists of single polymer chain looped with itself in a knot structure (distinctly different from hyperbranched or dendrimer polymers) was used to deliver a NT-3-encoding gene in vivo.
INTRODUCTION
In this investigation, upregulation of growth-promoting cues through sustained delivery of a non-viral gene vector, was used in combination with a multichannel collagen conduit to promote repair of the injured spinal cord. Previous studies in our group have demonstrated that multichannel collagen conduits that resemble the structure of the multiple nerve basal lamina tubes can control axonal dispersion in peripheral nerve regeneration. 39 Similar devices using synthetic 7-channel conduits made from a variety of polymers have been successfully used for spinal cord repair. 2, 13, 16, 40 However, the use of a more natural material, such as collagen, has a number of major advantages over synthetic polymers and may hold additional benefits for repair. It possesses multiple cell-adhesive and signaling domains that have been shown to improve axonal growth and repair. 41 The conduit material properties such as the rate of biodegradation and its mechanical properties can be directly controlled by crosslinking of the core solution. 42 The flexibility of the fabrication process and properties of the collagen molecule allow the incorporation/ loading of factors within or on the surface of conduit material; such that they are released in a controlled and regulated manner. In this instance non-viral polyplexes are incorporated for gene therapy. It was hypothesized that controlled delivery of a NT-3 growth-promoting gene from the multichannel collagen conduit would result in greater axonal regeneration than simply relying on structural guidance alone.
RESULTS
Release profile of polyplexes from multichannel collagen conduits and in vitro cell transfection Multichannel collagen conduits were fabricated with the tube outer diameter, length and channel diameter of 2.6, 2.0, and 0.66 mm respectively (Figure 1 ). The fabrication method of the multichannel collagen conduits was modified from the previously reported method. 42, 43 To create additional guidance cues for spinal axon growth, a layer of aligned collagen fibers was fabricated into the inner wall of the channels (Figures 1d-f) .
RESULTS
The knot-complexed pGluc showed a rapid release from the collagen film and from the 7-channel neural conduits during the first 24 h in phosphate-buffered saline (PBS) solution at 37 1C. About 50% of the loaded polyplexes were released within 24 h and an additional 30% of the loaded polyplexes were released in the following 13 days (Figure 2a) . The conduits surface-loaded with polyplexes also showed significantly higher transfection capability than did the collagenase-digested conduits for both 3T3 fibroblast cells and adipose-derived stem cells (Figures 2b and c) . By combining the two methods, the biomaterial conduit ideally obviated the trade-off between fast release (high transfection) for the initial transection and sustained delivery (lower transfection capability) for maintenance of NT-3 expression. In addition, polyplexes released from collagenase-digested conduits showed significantly higher transfection than that seen from a comparison group comprised of polyplexes formed with a commercially available polyethylenimine (PEI). To assess the ability of the delivered gene vector to promote repair, the number of regenerated axons from cross-sections at both one-quarter and three-quarter positions of the isolated conduit was counted. At both one-quarter and three-quarter positions, the NT-3 group showed a significantly greater number of axons for the entire scaffold than did the control group ( Figure 5 ). The axon number of the NT-3 group at one-quarter and three-quarter positions of the isolated conduit (1711.3 ± 687.1 and 1662.4 ± 558.5, respectively) was significantly higher than that seen in the control group (Po0.05) (Figure 6b ).
RESULTS
To confirm enhanced expression of human NT-3 within the injured spinal cord, NT-3 protein level was quantified using an enzyme-linked immunosorbent assay (ELISA) assay (Figure 6a ). The NT-3 protein content (expressed as weight ratio of NT-3 (pg) to tissue (mg)) in the NT-3 group (122.5 pg mg À 1 ± 24.7 pg mg À 1 )
RESULTS
was significantly higher than that of the control group (63.3 pg mg
Measurement of microglia in the regenerated neural tissue
The cross-sections of the tissue samples were labeled with anti-ED-1 antibody to identify and quantify the presence of Conduits carrying NT-3 for spinal cord repair L Yao et al phagocytic cells (that is, microglia). The average number of microglia in the observed fields for the control group and NT-3 group was 4.6 ± 1.6 and 3.8 ± 1.5 respectively (Figure 6c ). No significant difference was found between groups.
Measurement of microglia in the regenerated neural tissue
Assessment of functional recovery The Basso, Beattie and Bresnahan (BBB) locomoter rating scale was assessed to evaluate the functional recovery of the animals at 2 weeks and 4 weeks post-surgery. At 4 weeks, the BBB score of the (a-g) shows axons in the channels of the seven-channel control conduit taken at the one-quarter position of the conduit; (a 0 -g 0 ) shows axons in the matching channels of control conduit at three-quarter position of the conduit; (h-n) shows axons in the seven-channels at one-quarter position of the treated conduit carrying NT-3 polyplexes; (h 0 -n 0 ) shows axons in the matching channels of NT-3 polyplex conduit at the three-quarter position of the conduit. Scale bar, 200 mm. 
DISCUSSION
Primary injury of spinal cord causes direct neurological damage to the spinal cord leading to the necrosis of tissue at the site of injury due to trauma and local ischemia from damage to the surrounding vasculature. The injury provokes a cascade-like secondary reaction and further injury to the neural tissue. Tissue destruction causes the death of neurons and glial cells, demyelination of intact axons, increased ischemia, inflammation and results in swelling in the segments immediately above and below the site of injury. 15 A number of cells migrate to the site of injury (for example, macrophages, microglia, neuroglia such as astrocytes and eventually Schwann cells from the periphery) and participate in both the inflammatory response and scar tissue formation. 44, 45 The cyst/glial scar formed from reactive astrocytes and the infiltrated cells forms both a chemical and physical barrier to axonal repair. The implantation of biomaterial nerve conduits have been previously shown to prevent/reduce scar formation and simultaneously creating a suitable microenvironment for repair. 11, 13, 14, 16, 46 The use of multichannel neural conduits has demonstrated the ability to provide structural support and guidance to regenerating nerve tissue while retaining their structural integrity. Seven-channel conduits have been made from a number of synthetic materials including poly(lactide-coglycolide), poly(e-caprolactone fumarate), oligo(polyethyelene glycol) fumarate and have been used alone or in combination with either cells or exogenous growth factors, and been implanted within the injured spinal cord. 13, 16 Oriented neural tissue was observed to grow from both rostral and caudal sides within the channels of the biomaterial scaffolds and, in some cases, regenerated axons extended up to 14 mm distal from the conduit into the cord evident from fast blue tracing of the regenerated tracts. 9, 11, 14, 16, 47 Based on these studies a natural seven-channel conduit was fabricated from collagen type I with similar dimensional properties to that of the previous studies (that is, an internal channel diameter of 660 mm).
DISCUSSION
It was shown that aligned collagen fibers within a biomaterial conduit resulted in significantly increased axonal regeneration and more accurate nerve regeneration. 43, [48] [49] [50] [51] In this study, we fabricated aligned collagen fibers into the inner wall of the conduit channels. It was assumed that the addition of structure to the inner wall of the conduit would provide additional topographical guidance to the regenerating axons. Resulting from this combination, randomly oriented axons were observed at the boundaries of the conduit entry zone (Figures 4a and f) . Directly at the conduit entry zone, axons were seen to become oriented by the narrow conduit channels both rostrally and caudally (Figures 4d and c, respectively) . Moving further into the conduit, clear, aligned (Figures 4b and e) and parallel axonal growth was seen to grow within the channels of the conduits. This data suggests that the combination of collagen fibers and channels can effectively direct axonal growth.
DISCUSSION
Although many of the previously used multichannel conduits were fabricated using synthetic polymers, we have used bovinederived collagen type I as a biomaterial to fabricate these conduits for the intrinsic presence of cell-adhesive and signaling domains, which offer advantages over synthetic polymers for axonal regeneration. 9, 11, 13, 14, 16 Furthermore, the mechanical and degradable properties of collagen conduits have been thoroughly characterized and can be readily tailored using a suitable crosslinking regime. 42, 52 In particular, the degradation rate of such collagen neural conduits can be modified by adjusting the crosslinking strength according to the duration of the in vivo experiments offering advantages over synthetic polymers, which degrade at a slower rate.
DISCUSSION
To promote axonal regeneration, neurotrophins are major therapeutic factors that have been delivered to the injured central nervous system. However, because of their relatively short half-life, neurotrophins can function only transiently. 27, 28 Non-viral gene delivery into the spinal cord offers a promising approach for the treatment of spinal cord traumatic injury. In a study by Takahashi et al. 53 , adult rats with spinal cord hemisection were injected with lipoplexed DNA plasmid encoding human Bcl-2 gene into the normal side the spinal cord (or caudal to the hemisection). Administration of the Bcl-2 gene in the spinal cord prevented retrograde cell death and also minimized atrophy. In another study, a lipoplexed plasmid encoding glial cell-derived neurotrophic factor was injected after SCI. Real-time polymerase chain reaction showed the increased expression of glial cellderived neurotrophic factor mRNA in the injected areas at 7 days after injection. Four weeks after glial cell-derived neurotrophic factor gene transfer, regeneration down the corticospinal tracts was confirmed using anterograde tract tracing. This study demonstrated that in vivo transfer of glial cell-derived neurotrophic factor cDNA can promote axonal regeneration and demonstrate some locomotion functional recovery. 54 The use of polymeric vectors has also been reported recently, where plasmid DNA complexed with polyethylene glycol-grafted PEI showed enhanced gene expression after repeated intrathecal injections. 
Conduits carrying NT-3 for spinal cord repair L Yao et al
DNA delivered in a controlled manner by a combinatorial biomaterials approach, may overcome these problems.
Conduits carrying NT-3 for spinal cord repair L Yao et al
In addition to the structural guidance for axonal re-growth, the constant release of the plasmid DNA, from the scaffolds, could continuously transfect cells and maintain expression of therapeutic molecules. An example of such a study used surfacemediated DNA delivery from multichannel poly(lactide-co-glycolide) bridges coated with fibronectin to deliver lipoplexes to the spinal cord hemisection model. 11 The lipoplex-mediated expression in the neural tissue persisted for at least 3 weeks.
Conduits carrying NT-3 for spinal cord repair L Yao et al
In this study, a highly effective transfection agent was required, which transfects neuronal cell types such as astrocytes. 56 To this end the recently developed cyclic knot polymer was used to complex and protect the plasmid encoding NT-3. By subsequent loading to collagen conduits, a combined approach was utilized coupling conduit implantation with sustained gene delivery in a single surgical procedure. The higher number of regenerated axons in the NT-3 group ,therefore, corresponds to the increased NT-3 level (as confirmed by ELISA) in the neural tissue implanted with conduits. This study suggests that the collagen neural conduit delivering a functional gene is an effective approach to improve axonal regeneration. The functional recovery of the rats after injury was studied using BBB scoring. The BBB score of NT-3 group is higher than control group at both 2 weeks and 4 weeks after the surgery. The result suggested that pNT-3 in the conduits improved axonal regeneration, which may result in better functional recovery. Although the difference was not statistically significant, this study indicated that an increased animal number in the functional study may yield significance as there was a high variation within the experimental groups. For future studies it will be interesting to see if these regenerated axons extend distally into the conduit, and how that regeneration distance would compare to previous studies. This may be achieved through the use of appropriate anterograde and retrograde tracers. 57 Spinal cord injury results in the influx of a large number of different cell types to the site of injury (for example, macrophages, microglia, oligdendrocyte precursors, meningeal cells and astrocytes). Although these cells can be potentially transfected, it was critical to analyze whether the amount of cyclic knot polymer used for complexing the NT-3 plasmid resulted in a greater infiltration of a phagocytic cells, which may be inhibitory for repair. 58 As previously reported, seven-channel conduits have been shown to create an isolated microenvironment for repair. 13 It was critical to analyze whether the introduction of such polyplexes into the channels of the conduit resulted in an increased influx of phagocytic cells, that is, microglia. However, no significant differences in ED-1 positive cells was found between groups indicating that the 4 mg of in situ-loaded complexed plasmid, plus the 3 mg loaded on the surface, cause no rise in the number of microglia recruited to the site of implantation. Further study of the interaction of the surrounding tissue may be warranted to ensure there are no adverse effects in that area, future studies will address this.
Conduits carrying NT-3 for spinal cord repair L Yao et al
In conclusion, multichannel collagen conduits can be functionalized with polyplexes by two methods: in situ loading or surface loading, each displaying a different transfection profile in vitro. By combining the two loading methods, NT-3 levels were shown to be statistically increased during nerve regeneration and maintained up to 4 weeks in vivo. This cyclic knot polymer was used for enhanced and sustained NT-3 delivery within the injured spinal cord and shown to maintain expression levels up to one month in vivo. Sustained expression resulted in significantly increased aligned axonal regeneration within the channels of the multichannel collagen conduit compared with the control group. This study suggests that the combination of biomaterial scaffolds and sustained non-viral gene delivery provides synergistic effects of structural guidance and effective molecular therapy for spinal cord regeneration.
MATERIALS AND METHODS

Fabrication of multichannel collagen conduit with inner layer of parallel collagen fibers
Collagen fibers were fabricated by extruding an atellocollagen solution (5 mg ml
Fabrication of multichannel collagen conduit with inner layer of parallel collagen fibers
) at a rate of (0.3 ml min À 1 ) using a syringe pump (KD-Scientific 200, KD-Scientific, Holliston, MA, USA) into a fiber formation buffer (118 mM phosphate buffer and 20% polyethylene glycol (molecular weight 8000), pH 7.55, 37 1C) for 5 min and then transferring the fibers into an incubation buffer (6.0 mM phosphate buffer and 75 mM sodium chloride, pH 7.1, 37 1C) for another 5 min. The collagen fibers were crosslinked with 0.5 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.5 mM N-hydroxysuccinimide in 50 mM 2-(N-morpholino)ethansulfonate, pH 5.5 for 30 min, as per previous work. 43 Thereafter, the collagen fibers were thoroughly washed with distilled water for 1 h to remove excess crosslinker and subsequently air-dried (Figure 1d) .
Fabrication of multichannel collagen conduit with inner layer of parallel collagen fibers
The aligned, extruded collagen fibers were attached onto the surface of seven stainless steel wires to be incorporated into the multichannel conduit fabrication process. The conduit was fabricated by sequentially inserting stainless steel wires into the molds and air-drying the collagen on stainless steel wires. The air-dried collagen conduit on the wires was then treated with a crosslinking solution (30 mM carbodiimide and 10 mM N-hydroxysuccinimide in 50 mM 2-(N-morpholino)ethansulfonate solution), pH 5.5 for 5 h. After washing with 0.1 M NaH 2 PO 4 and distilled water, the multichannel collagen conduit was freeze-dried on the wire. Molds and wires were removed from the collagen conduits after freeze-drying.
Cyclized knot polymer synthesis
The transfection agent was synthesized as previously reported. 38 Briefly, a highly controlled synthesis method, known as deactivation-enhanced atom transfer radical polymerization, was employed. 59 This allows the polymerization of single or multiple di-vinyl monomers without the formation of an insoluble gel. 59, 60 The three monomers ethylene glycol dimethacrylate, used as the knotting agent; 2-(dimethylamino) ethyl methacrylate, the complexing agent; and poly(ethylene glycol) methyl ether methacrylate, for PEGylation were polymerized in a 'one-pot' reaction at 50 1C in tetrahydrofluran at a ratio of 10:82:8, respectively using a copper/ligand complex activated by the addition of ascorbic acid. By the suppression of gelation, several grams of single copolymer polymer chains cyclized upon themselves at a molecular weight of 32 kDa were obtained and were then purified by hexane precipitation. This was followed by extensive dialysis in distilled water and subsequent freeze-drying leaving the polymer ready for use as a dry powder.
Cyclized knot polymer synthesis
In vitro polyplex release profile from the collagen conduits and subsequent functionality analysis To study the release profile of polyplexes from the conduit, 1 mg of Gaussia princeps luciferase plasmid (pGluc) labeled with Cy5 using a Label ITCy5 Labeling Kit (Mirus Bio, Madison, WI, USA) was complexed to the knot polymer by addition at a 4:1 polymer-to-plasmid w/w ratio in dH 2 O for 15 min. 61 Collagen films were fabricated by spreading the collagen solution on a flat Teflon surface (weigh boat) and air-dried. The collagen films were then crosslinked with 1 mM carbodiimide and 1 mM N-hydroxysuccinimide for 1 h. The labeled polyplexes were loaded onto the crosslinked collagen film or multichannel collagen conduits post-assembly, as outlined in Figure 7b . The polyplexes-loaded scaffolds were placed into a 96-well plate with 200 ml PBS solution and incubated at 37 1C. At 24-hour, 48-hour, 1-week and 2-week time points, the conduits were transferred to another row of wells containing fresh PBS solution. At the end of the 2-week time period, the conduits were removed from the final set of wells and the PBS solution from each time point was analyzed using a Varioskan Flash Multimode Reader (Thermo Scientific, Essex, UK).
Cyclized knot polymer synthesis
To study the functionality of polyplexes delivered by means of the collagen conduits, two methods of loading and subsequent release were performed, with subsequent transfection analysis as outlined in Figures 7a and b. For this study pGluc complexed either with the knot polymer or PEI (at a 2:1 polymer to plasmid w/w ratio) (Sigma, St Louis, MO, USA) were used. The scaffolds were then loaded with these polyplexes in one of two ways: (1) the polyplexes were mixed with the initial atellocollagen solution used to fabricate the conduit followed by crosslinking with 1 mM carbodiimide and 1 mM N-hydroxysuccinimide for 1 h to form an in situloaded conduit directly ( Figure 7a) ; (2) the polyplexes were added to the surface/inner channels (that is, surface loaded) of the conduit fabricated using collagen solution alone without polyplexes (Figure 7b ). To study Conduits carrying NT-3 for spinal cord repair L Yao et al functionality of the in situ-loaded polyplexes, conduits were digested with bacterial collagenase type II (Sigma C6885), which was dissolved in 35 ml PBS (3 U of collagenase per mg of collagen sample). The solution was then collected for transfection analysis. To study the functionality of the surface-loaded polyplexes, the treated conduits were placed directly into 48-well plate wells with cultured cells. To establish the in vitro efficacy of the released polyplexes, both the standard cell line 3T3 fibroblasts and adipose-derived stem cells were used in the study. The cells were seeded at a density of 8000 cells per well in 48-well plates for the gene transfection study. Twenty-four hours after cell seeding, the conduits loaded with polyplexes or the solution containing the digested conduits was added into the cell culture wells. After another 72 h, the cell culture medium was collected for analysis using BioLux Gaussia Luciferase Assay Kit (E3300) (New England Biolabs, Ipswich, MA, USA).
Cyclized knot polymer synthesis
Fabrication of multichannel collagen conduits functionalized with NT-3 polyplexes pNT-3 (pcDNA3.3-TOPO (Invitrogen, Grand Island, NY, USA) human NT-3) was complexed with the knot polymer at a polymer-to-plasmid weight ratio of 4:1. The polyplexes were incorporated into the conduits in a twostep procedure outlined in Figure 7c . First, pNT-3 (4 mg) polyplexes were added into the collagen solution (12 mg ml À 1 ) and mixed gently to ensure uniform distribution of the polyplexes within the solution (in situ loading). Then the solution was used to fabricate the multichannel conduits with inner layer aligned collagen fibers. Second, after the conduits were fabricated, pNT-3 (3 mg) polyplexes were loaded onto the surface and within the channels of the multichannel collagen conduits before being air-dried (surface loaded). This created a conduit that combined surface loading and in situ loading based on the results of previously described release studies.
Surgical procedure
In this study a total of 32 adult female Sprague Dawley rats weighing between 220 and 280 g were used. The multichannel conduits with or without polyplexes were implanted into the completely transected rat spinal cord. All experimental procedures were conducted in accordance with national and institutional guidelines, and all procedures were approved by the institutional animal ethics committee and by the National Board under the Cruelty to Animals Act. The rats were randomly assigned to two subgroups: control collagen conduits (without polyplexes) and conduits containing pNT-3 polyplexes (NT-3 group). Animals were anesthetized with intraperitoneal injection of ketamine and xylazine. The back was shaved and aseptically prepared using povidone-iodine. Vet ointment was applied to protect the eyes from dehydration during the relatively long procedure. To maintain body temperature during surgery, animals were kept on a heating pad at 37 1C. A microsurgical microscope was used throughout the surgical procedure. After skin incision and laminectomy at the T8-T10 vertebral levels, the spinal cord was completely transected with a no. 11 blade. After transection, the spinal cord was retracted from the lesion site. A cotton sponge was inserted into the gap of the transected spinal cord to staunch excess bleeding and create a 2 mm gap. A 2-mm-long multichannel collagen conduit was implanted in the gap and aligned with the rostral and caudal spinal cord stumps. Muscles and skin were then sutured separately. Post-operative care was included twice daily monitoring for wound sepsis and bladder expression. Antibiotics (Baytril, Bayer HealthCare, LLC, KS, USA) and analgesics (Buprenorphine, Fort Dodge Animal Health, Southampton, UK) were administered for the first week post-surgery, and saline was administered 3 days post-surgery.
Tissue processing
One month post-surgery, the animals were killed by an overdose of sodium pentobarbitone (intraperitoneally) (for immunohistochemical studies) or CO 2 asphyxiation (for protein analysis). For the immunohistochemical study, 11 rats in each group were transcardially perfused with 4% paraformaldehyde in PBS. The spinal cord was then removed and post fixed in 4% paraformaldehyde. The samples were cryoprotected in 30% sucrose and the region containing the scaffold was embedded in Cryogel (Instrumedics, Richmond, IL, USA) and frozen for sectioning. The samples were then continuously sectioned transversely or longitudinally. For protein analysis, the spinal cords of five rats in each group were isolated without performing transcardial perfusion. The isolated tissue was frozen using liquid nitrogen and stored in a À 80 1C freezer for further analysis.
Immunohistochemistry
The sample sections for immunohistochemical analysis were permeabilized with 0.5% Triton X-100. Then the sections were exposed to blocking solution (2% goat serum, 2% bovine serum albumin). All antibodies were diluted in PBS with 1% bovine serum albumin. The sections were incubated with primary antibody for two hours at room temperature. Figure 7 . Two methods used for loading the multichannel neural with polyplexes with subsequent analyses. Conduits for in vitro analysis were loaded by mixing polyplexes and the collagen solution before fabrication (a) or loaded to the surface post-fabrication (b). A dual loading method of in situ loading followed by surface loading was used for the preparation of scaffolds for in vivo analysis (c).
Immunohistochemistry
The cross-sections (taken at one-quarter length and three-quarter length intervals) and the longitudinal sections of the isolated neural conduit samples were labeled with anti-neurofilament monoclonal antibody (1:200, N2912, Sigma) to identify and quantify regenerating axons. Regenerated axons from cross-sections at both one-quarter and three-quarter positions of the isolated conduit (rostral to the caudal) were labeled with antineurofilament antibodies and the axon number was counted.
Immunohistochemistry
The cross-sections at the mid-point of the scaffold-cord interface were labeled with an anti-ED-1 antibody (1:250, MAB1435, Millipore, Billerica, MA, USA). Following exposure to primary antibodies, the sections were incubated with DyLight 488-conjugated donkey anti-mouse (1:250, Jackson ImmunoResearch Europe, Suffolk, UK) for 1 h. Nuclei were stained with 4,6-diamidino-2-phenylindole. Fluorescent images of the sections were viewed under a microscope (Olympus dp70, Olympus, Tokyo, Japan). Six fields of view were randomly selected and captured at a magnification of 400 Â for each section using image analysis software (Image Pro, Media Cybernetics, Buckinghamshire, UK). The number of ED-1-labeled cells was measured using a 192-point grid. A 192-point grid was overlaid on each field of view, and the number of ED-1 positive cells that intersected with a grid point was counted.
ELISA assay
The frozen tissue samples containing the scaffolds were suspended in tissue extraction reagent (Sigma) and then homogenized using a TissueRuptor (Qiagen, Valencia, CA, USA). Homogenate was centrifuged at 10 000 g for 10 min to remove particulates. The amount of NT-3 in the tissue homogenate was quantified using an ELISA kit (DY267, R&D Systems, Minneapolis, MN, USA). The absorbance of the samples was read at 450 nm using a Varioskan Flash Multimode Reader (Thermo Scientific, Essex, UK). The NT-3 content in the homogenate was then analyzed and normalized to the total protein content, as analyzed using the Bio-Rad Protein assay (Bio-Rad, Hercules, CA, USA).
ELISA assay
Assessment of locomotor recovery BBB locomotor score was used to evaluate motor functional recovery of experimental and control animals 2 and 4 weeks after surgery. Each rat was observed in a clean and open plastic box for 5 min to record hind-limb joint movements, weight support, toe clearance, tail position and coordination of gait and paw position.
Statistical analysis
The data were expressed as means ± s.d. and analyzed by using a one-way ANOVA (post-hoc Bonferroni) with SPSS version 17.0 software package (SPSS, Chicago, IL, USA). P o0.05 was considered statistically significant.

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