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Regenerative Medicine
Engineering Vascularized Bone: Osteogenic and Proangiogenic Potential of Murine Periosteal Cells†‡§
Version of Record online: 22 OCT 2012
DOI: 10.1002/stem.1210
Copyright © 2012 AlphaMed Press
Volume 30, Issue 11, pages 2460–2471, November 2012
Additional Information
How to Cite
van Gastel, N., Torrekens, S., Roberts, S. J., Moermans, K., Schrooten, J., Carmeliet, P., Luttun, A., Luyten, F. P. and Carmeliet, G. (2012), Engineering Vascularized Bone: Osteogenic and Proangiogenic Potential of Murine Periosteal Cells. STEM CELLS, 30: 2460–2471. doi: 10.1002/stem.1210
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Author contributions: N.v.G.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; S.T., S.J.R., and K.M.: collection and assembly of data; J.S.: conception and design; P.C. and A.L.: provision of study material; F.P.L.: conception and design, data analysis and interpretation, and provision of study material; G.C.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript.
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Disclosure of potential conflicts of interest is found at the end of this article.
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First published online in STEM CELLSEXPRESS August 22, 2012.
Publication History
- Issue online: 22 OCT 2012
- Version of Record online: 22 OCT 2012
- Accepted manuscript online: 21 AUG 2012 08:32AM EST
- Manuscript Accepted: 25 JUL 2012
- Manuscript Received: 4 JUN 2012
Funded by
- Fund for Scientific Research Flanders. Grant Numbers: FWO; G.0500.08, G.0982.11
- Flemish Government
- Long-term Structural Funding—Methusalem
- European Commission. Grant Number: FP7-StG-IMAGINED 203291
- KU Leuven (IOF Knowledge Platform "Prometheus". Grant Number: IOFKP/07/004
- Stem Cell Institute of Leuven
- Agency for Innovation by Science and Technology in Flanders (IWT)
- Prometheus, the Leuven Research & Development Division of Skeletal Tissue Engineering of the KU Leuven
- Periosteum;
- Stem cells;
- Osteogenesis;
- Angiogenesis;
- Tissue engineering;
- Hematopoiesis
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
One of the key challenges in bone tissue engineering is the timely formation of blood vessels that promote the survival of the implanted cells in the construct. Fracture healing largely depends on the presence of an intact periosteum but it is still unknown whether periosteum-derived cells (PDC) are critical for bone repair only by promoting bone formation or also by inducing neovascularization. We first established a protocol to specifically isolate murine PDC (mPDC) from long bones of adult mice. Mesenchymal stem cells were abundantly present in this cell population as more than 50% of the mPDC expressed mesenchymal markers (CD73, CD90, CD105, and stem cell antigen-1) and the cells exhibited trilineage differentiation potential (chondrogenic, osteogenic, and adipogenic). When transplanted on a collagen-calcium phosphate scaffold in vivo, mPDC attracted numerous blood vessels and formed mature bone which comprises a hematopoiesis-supportive stroma. We explored the proangiogenic properties of mPDC using in vitro culture systems and showed that mPDC promote the survival and proliferation of endothelial cells through the production of vascular endothelial growth factor. Coimplantation with endothelial cells demonstrated that mPDC can enhance vasculogenesis by adapting a pericyte-like phenotype, in addition to their ability to stimulate blood vessel ingrowth from the host. In conclusion, these findings demonstrate that periosteal cells contribute to fracture repair, not only through their strong osteogenic potential but also through their proangiogenic features and thus provide an ideal cell source for bone regeneration therapies. STEM CELLS2012;30:2412–2422
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
To date, autologous bone transplantation remains the therapy of choice to treat nonhealing skeletal defects caused by traumatic injury, osteo-degenerative diseases, or cancer [1]. Nevertheless, the use of autografts is restricted by major drawbacks, including limited availability and donor site morbidity. In response to these limitations, tissue engineering strategies have emerged as a potential alternative.
A promising concept in the repair of bone defects is the use of osteogenic cells seeded on a degradable carrier [2]. Of great interest are mesenchymal stem cells (MSC), which can be isolated from several adult tissues and are capable of giving rise to cells of different lineages [3, 4]. MSC intended for bone tissue engineering applications have typically been isolated from bone marrow, but the periosteum might represent a more relevant cell source as it is intrinsically closer related to bone formation and repair [5, 6]. As known from orthopedic practice, destruction of the periosteum will lead to delayed bone healing or nonunion [7]. Several animal studies have further demonstrated that periosteal cells contribute to bone repair by recapitulating specific features of the bone development process [8–10]. Following the early inflammatory stage, periosteal cells undergo a strong proliferative burst, after which these cells differentiate toward chondrocytes and osteoblasts, initiating endochondral bone formation. The essential function of the periosteum is further underscored by the finding that periosteum-derived cells (PDC) contain a high number of MSC in comparison to bone marrow stromal cells (BMSC) [11, 12]. Furthermore, a number of animal studies have provided proof of concept using PDC for the repair of critically sized bone defects [13–15].
The search for a satisfactory cell source for bone tissue engineering has frequently been focused on the in vitro osteogenic properties of the cell types of interest. In vivo osteogenesis is however inherently coupled to angiogenesis [16] and successful bone regeneration thus requires the timely formation of blood vessels. During uncomplicated bone fracture healing, blood vessels present in the periosteum and adjacent tissues will guarantee the survival of the periosteal cells and allow their rapid proliferation, preventing the formation of fibrous tissue [17, 18]. The importance of adequate vascularization is underscored by the limited survival of cells in tissue engineering constructs upon implantation [19, 20]. Multiple approaches have been proposed to ensure sufficient vascularization of implants, including the use of angiogenic growth factors, microsurgery, or the addition of endothelial (progenitor) cells to the scaffold [21]. This last strategy is particularly interesting, given the close interplay between bone-forming osteoblasts and endothelial cells during bone development and repair [22–24]. Moreover, different studies have indicated that in order to engineer mature, long-lasting vascular structures, coimplantation of endothelial cells with perivascular cells is necessary [25, 26]. The choice of a cell source for bone tissue engineering applications should thus not only take into account their osteogenic properties but also other cues such as proangiogenic characteristics of the implanted cells [16].
In this study, we explored the proangiogenic potential of murine PDC (mPDC). We first developed a technique to isolate mPDC from long bones of adult mice and confirmed the presence of MSC. Subcutaneous implantation of the mPDC on a collagen-calcium phosphate carrier resulted in the formation of vascularized bone accompanied by the establishment of bone marrow. Further in vitro and in vivo analyses revealed that mPDC can contribute to multiple aspects of neovascularization by producing growth factors that enhance angiogenesis and by providing structural support as mural cells to the vascular networks formed by endothelial cells.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Animals and Human Tissues
C57BL/6 mice and NMRI nu/nu nude mice were purchased from the R. Janvier Breeding Center (Le Genest St. Isle, France). Transgenic mice ubiquitously expressing the enhanced green fluorescent protein (eGFP) under the control of the chicken β-actin promoter (C57BL/6-ACTb-eGFP) were obtained from A. Nagy (Samuel Lunenfeld Research Institute, Toronto, Canada). Human periosteal biopsies were obtained from the proximal medial tibia during orthopedic surgery with patient informed consent. A human fibular bone sample was obtained as discarded tissue during the course of orthopedic surgery. All experiments were conducted in accordance with the Declaration of Helsinki and with approval of the Medical and the Animal Ethics Committee of the KU Leuven.
Isolation of mPDC
To obtain mPDC, femurs and tibias isolated from 7–9-week-old male C57BL/6J or C57BL/6-ACTb-eGFP mice were dissected free of muscle and connective tissue under sterile conditions. Subsequently, the epiphyses were protected from digestion by submerging them in 5% low melting point agarose (SeaPlaque, Lonza, Verviers, Belgium, www.lonza.com). After solidification of the agarose, mPDC were isolated by a collagenase-dispase digest (3 mg/ml collagenase and 4 mg/ml dispase in α-minimal essential medium [α-MEM] with 2 mM glutaMAX-I; Gibco, Invitrogen, Carlsbad, CA, www.invitrogen.com). Cells from the first digest (10 minutes) were discarded as they contained cells from remaining muscle and connective tissue. mPDC were released by a subsequent 1 hour digest. The cell suspension was passed through a 70 μm nylon mesh (BD Falcon, BD Biosciences, Erembodegem, Belgium, www.bdbiosciences.com), washed twice, and plated at 1 × 104 cells per square centimeter in growth medium (α-MEM with 2 mM glutaMAX-I, containing 1% penicillin/streptomycin and 10% fetal bovine serum; all from Gibco, Invitrogen). mPDC were replated at a 1:3 ratio when reaching 80%–90% confluence.
In Vitro Cell Cultures
Growth curve analysis, in vitro differentiation of mPDC toward osteoblasts, chondrocytes and adipocytes, flow cytometry, fluorescence-activated cell sorting, and colony-forming unit fibroblast (CFU-F) assays were performed using standard techniques and are described in detail in Supporting Information.
The support of osteoclast formation by mPDC, mBMSC, or murine trabecular osteoblasts (mTOB) was determined by coculturing these cells with freshly isolated murine bone marrow cells (mBMC) in a 1:10 ratio in the presence of 20 nM 1,25-dihydroxyvitamin D3 as previously described [31]. After 6 days, cultures were stained for tartrate-resistant acid phosphatase (TRAP) to visualize osteoclasts.
When indicated mPDC, seeded at 3 × 104 cells per square centimeter, were cultured under hypoxia (1% oxygen) or normoxia (21% oxygen) for 24 hours, after which growth medium was collected to determine the secretion of vascular endothelial growth factor (VEGF; also known as VEGF-A) and total RNA or nuclear proteins were extracted from the cultured cells (described in detail in Supporting Information). To functionally assess whether mPDC secrete angiogenic factors, conditioned media from mPDC, grown under normoxia or hypoxia, or α-MEM was added to HUVEC plated on collagen type 1 (BD Biosciences). After 24 hours, proliferation was determined by BrdU incorporation (Biotrak cell proliferation ELISA system, GE Healthcare, Diegem, Belgium, www.gehealthcare.com).
Two in vitro coculture models were used to study the interactions between mPDC and endothelial cells. In the first assay, mPDC were seeded at 5 × 104 cells per square centimeter and after 24 hours human blood outgrowth endothelial cells (hBOEC) were added at 1 × 104 cells per square centimeter. Cells were cultured for 7 days in growth medium with or without the addition of anti-murine VEGF-164 antibody (anti-mVEGF164; 200 ng/ml; R&D systems, Minneapolis, MN, www.gehealthcare.com) or recombinant mVEGF receptor (mVEGFR)-1/fms-related tyrosine kinase(Flt)-1 Fc Chimera (smFlt-1; 100 ng/ml; R&D systems, Minneapolis, MN, www.rndsystems.com). After culture, the endothelial cells were stained with a mouse-anti-human CD31 primary antibody (Supporting Information Table S1) and visualized using the TSA Cyanine 3 System (NEN, PerkinElmer, Boston, MA, www.perkinelmer.com). For the Matrigel in vitro angiogenesis assay, mPDC and HUVEC/hBOEC were first labeled with, respectively, CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) and CellTracker CM-DiI (Molecular Probes, Invitrogen) according to the manufacturer's instructions. Subsequently, mPDC (1 × 104 cells per square centimeter) and HUVEC or hBOEC (2 × 104 cells per square centimeter) were resuspended alone or together in EGM-2 and seeded on top of growth factor-reduced Matrigel (BD Biosciences) and cultured for 24 hours.
In Vivo Bone Formation
To investigate the in vivo osteogenic potential, 1 × 106 mPDC or human PDC (hPDC) (passage 3) were seeded onto Collagraft (NeuColl Campbell, CA, www.neucoll.com) scaffolds (3 × 3 × 3 mm3), incubated overnight, and implanted subcutaneously on the back of female NMRI nu/nu mice, as described previously [32]. Scaffolds without cells were used as negative controls. Eight weeks after implantation, scaffolds were retrieved and processed for histology. For cell tracing, mPDC were labeled with CellTracker CM-DiI prior to seeding.
In Vivo Vasculogenesis/Angiogenesis Assay
The in vivo coimplantation of mPDC and HUVEC was adapted from Allen et al. [33]. Cells (mPDC, HUVEC or mPDC and HUVEC in a 60:40 ratio) were resuspended in collagen type 1 (5 mg/ml in EGM-2) at a density of 1 × 106 cells per milliliter and 200 μl was injected subcutaneously on the back of female NMRI nu/nu mice. After 7 days, the gels were retrieved and processed for histology.
(Immuno)Histochemistry and Histomorphometry
Isolated bones and scaffolds were fixed in 2% paraformaldehyde overnight and decalcified in EDTA for 14 days at 4°C prior to dehydration, embedding in paraffin, and sectioning at 4 μm (bone samples) or 10 μm (Collagraft). Collagen gels were fixed in 2% paraformaldehyde overnight, embedded in NEG-50 frozen section medium (Richard-Allen Scientific, Kalamazoo, MI), and sectioned with a cryostat at 7 μm.
All histochemical stainings (hematoxylin and eosin [H&E], Safranin O, Sirius Red, and TRAP) and immunohistochemical stainings (CD31, CD34, CD45, osteocalcin, Ulex europaeus agglutinin [UEA]-lectin, and α-smooth muscle actin [α-SMA]) have been reported previously [23, 28, 30, 34, 35] and were performed as described in Supporting Information. Images were taken on a Zeiss Axioplan 2 light microscope or a Zeiss LSM510-META NLO multiphoton confocal microscope. Collagen fibers were imaged by polarized light microscopy following Sirius Red staining as previously described [35]. Histomorphometry was performed using the ImageJ software (National Institutes of Health). Quantification of newly formed bone in Collagraft scaffolds was performed by manual outlining areas of bone and total tissue. Quantification of blood vessels was performed by counting or outlining CD31/CD34/UEA lectin-positive vessels with a clear lumen.
Statistical Analysis
Data are presented as means ± SE. Data were analyzed by two-sided two-sample Student's t test or one-way ANOVA using the NCSS statistical software. Differences were considered statistically significant at p < .05.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Generation of Primary Cell Cultures from Adult Mouse Periosteum
The isolation of human PDC is typically achieved by mechanically stripping off the periosteum from the diaphyseal bone, followed by an enzymatic digestion [36]. The periosteum from adult mice is however only a few cell layers thick and thus difficult to isolate by dissection (Fig. 1A, 1B). A possible solution is to perform an enzymatic digest directly on the long bones, but without releasing cells from the growth plate, articular cartilage, tendon, and connective tissue, thus avoiding the attainment of a heterogeneous cell population. To prevent the release of nonperiosteal cells, we embedded the epiphyses of the bones in low melting point agarose (Fig. 1C), prior to the enzymatic digest. Histological analysis showed that the isolation of the mPDC was not only efficient but also selective since joint and connective tissues surrounding the epiphysis were not removed by the enzymatic digest (Supporting Information Fig. S1). The isolated cells exhibited a fibroblast-like morphology (Fig. 1D) and possessed a high proliferative capacity.compared to mBMSC and mTOB, the growth curve of mPDC was characterized by a shorter lag time and the population doubling time was shorter compared to mTOB (Fig. 1E).
The periosteum is known to contain different cell populations including osteoprogenitor and chondroprogenitor cells and we therefore analyzed the expression of early osteogenic and chondrogenic genes in mPDC relative to primary mTOB and murine growth plate chondrocytes, which are more mature cells (Fig. 1F, 1G). mPDC expressed markers of osteogenic progenitor cells, including runt-related transcription factor 2 and osterix (Osx; also known as Sp7), while the expression of the mature osteoblast markers collagen type 1 alpha 1 and osteocalcin (Ocn; also known as Bglap) was low. Similarly, mPDC expressed sex-determining region Y-box containing gene 9, a marker of chondroprogenitor cells, whereas the levels of mature chondrocyte markers collagen type 2 alpha 1 (Col2a1) and aggrecan (Acan) and of the hypertrophic chondrocyte marker collagen type 10 alpha 1 were negligible.
mPDC Can Differentiate Toward the Osteoblastic, Chondrogenic, and Adipogenic Lineage
Next, the differentiation potential of mPDC toward different mesenchymal cell lineages was investigated. Osteoblastic differentiation was induced by culturing confluent mPDC in medium containing ascorbic acid and β-glycerophosphate and was analyzed at days 14 and 21. The positive alkaline phosphatase (ALP) staining at day 14 was indicative of osteogenic differentiation and quantification of ALP activity showed a threefold increase compared to day 0 (Fig. 2A). After 21 days of culture in osteogenic medium, the formation of mineralized nodules was evidently shown by Alizarin Red S staining (Fig. 2B). The marked upregulation of Osx, osteopontin (Opn; also known as Spp1), and Ocn expression confirmed the differentiation of mPDC toward mature osteoblasts (Fig. 2B).
To induce chondrogenic differentiation, mPDC were seeded as micromasses and treated for 7 days with transforming growth factor-β1. At the end of culture, the micromasses clearly stained positive with Alcian Blue, indicating that proteoglycans were produced and deposited in the matrix (Fig. 2C). Analysis of gene expression confirmed the differentiation of mPDC to mature chondrocytes, characterized by the increase in Col2a1 and Acan (Fig. 2C).
Finally, we induced adipogenic differentiation by culturing the cells in medium containing insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and indomethacin. After 14 days of culture, the presence of adipocytes was marked by the detection of oil droplets in the cytoplasm (Fig. 2D). The upregulation of peroxisome proliferator-activated receptor-gamma and fatty acid binding protein 4 gene expression confirmed the differentiation of mPDC to adipocytes (Fig. 2D). These data indicate that mPDC have trilineage differentiation potential, which is one of the hallmarks of MSC.
MSC Are Abundantly Present in the mPDC Population
To further confirm the presence of MSC in the mPDC population, we next analyzed the surface marker profile of these cells. Flow cytometric analysis revealed that more than 50% of the mPDC at passage 1 expressed one of the mesenchymal markers CD105, CD90.2, CD73, CD51, and stem cell antigen-1 (Sca-1) (Fig. 3A). Conversely, only very few cells expressed hematopoietic (CD11b and CD45) or endothelial (CD31, CD34, and VEGFR-2) markers. Analysis of mPDC at different passages (freshly isolated, passages 1, 3, and 6) showed that the cells expressing hematopoietic or endothelial markers disappeared after culture (<1% by passage 3), while the number of cells expressing mesenchymal markers further increased (Supporting Information Table S3). We next compared the abundance of putative MSC in the periosteum and the bone marrow stroma using the stem cell marker Sca-1, which has been described to identify MSC in murine bone marrow [37, 38]. To quantify the number of MSC relative to the stromal fraction, mPDC and mBMC cell populations were first gated for the CD45-negative fraction hereby excluding the hematopoietic cells that are abundantly present especially in the bone marrow. Flow cytometric analysis revealed that the percentage of putative MSC was considerably higher in periosteum than in the bone marrow stroma, even when mBMC were isolated by collagenase treatment of bone fragments, which is known to enrich the number of isolated MSC [38]. By performing a CFU-F assay, we showed that in periosteum, as has been described for bone marrow, the Sca-1+CD45− fraction contains the vast majority of cells with colony-forming potential (Fig. 3C, 3D). Taken together, these results confirm that MSC are abundantly present within the mPDC population.
In Vivo Transplanted mPDC Form Vascularized Bone with a Hematopoietic Compartment
To assess the in vivo osteogenic potential of mPDC, cells were seeded on Collagraft scaffolds and implanted subcutaneously in nude mice. hPDC were used as a positive control. Implants were retrieved 8 weeks postimplantation, and histomorphometrical analysis of H&E stained sections revealed that mPDC formed a substantial amount of bone tissue throughout the scaffolds, comparable to hPDC (9.2% ± 0.9% vs. 7.1% ± 0.5%; Fig. 4A–4C). The newly formed tissue displayed the typical features of bone. Indeed, polarized light microscopy of Sirius Red stained sections showed the presence of highly organized thick collagen fibers in the newly formed bone matrix (Fig. 4D–4F). In addition, osteocalcin staining revealed that differentiated osteoblasts were lining the bone surface (Fig. 4G), further confirming that mature bone was formed. By fluorescent labeling of the mPDC prior to implantation, we could show that several of the osteocytes embedded in the bone matrix were derived from the implanted cells (Fig. 4H). Bone formation was not detected when scaffolds without cells were implanted (data not shown). To assess whether a cartilage intermediate preceded the bone formation in these implants, sections were stained with Safranin O to detect proteoglycans. As seen in Figure 4I, implants were at 8 weeks predominantly devoid of chondrocytes, and only occasionally very small cartilaginous condensations, consisting of a few chondrocytes embedded in the calcium phosphate granules, could be detected (inset in Fig. 4I).
The presence of mPDC contributed not only to the formation of bone but also to the development of a hematopoietic compartment. Typical for bone marrow spaces, these compartments were packed with CD45-positive hematopoietic cells in close association with large CD31-positive sinusoid-like blood vessels (Fig. 4J, 4K). In addition, TRAP-positive osteoclasts were detected on the bone surfaces bordering the hematopoietic compartments, indicating active bone remodeling (Fig. 4L). Osteoclasts are indispensable for the remodeling of the newly formed immature bone into mature, lamellar bone [39], and are thus part of the global bone formation process. They are generally formed by close interaction with osteogenic cells and we therefore investigated whether mPDC can support osteoclast formation using an in vitro coculture system. As shown in Figure 4M, mPDC induced the formation of a large number of multinucleated TRAP-positive cells, comparable to cocultures with mTOB, and manifestly more than in conditions using mBMSC, which yielded only a limited number of osteoclasts.
Another prerequisite for adequate bone formation is the presence of functional blood vessels. The number of blood vessels as well as the area covered by the vessels was significantly higher in the mPDC-seeded implants compared to scaffolds without cells (Fig. 5A–5C). Detailed analysis revealed numerous blood vessels in close contact with the newly formed bone (Fig. 5D; arrow) or even enclosed by bone tissue (Fig. 5D; arrowhead). Costaining for the pericyte marker α-SMA and the endothelial cell marker CD31 (Fig. 5E, 5F) showed that many of the blood vessels near the bone surface were covered with mural cells (white arrows), which stabilize nascent vessels and promote vessel maturation. Blood vessels close to the hematopoietic sites on the other hand (white arrowhead) did not stain positive for α-SMA. Labeling of the mPDC with CM-DiI prior to implantation furthermore indicated that some of these perivascular cells were of donor origin (Fig. 5G). These results highlight an important role of the mPDC in promoting blood vessel ingrowth into the implants and possibly also in contributing to vessel stabilization and maturation.
Hypoxia Activates Metabolic and Proangiogenic Responses in mPDC
To further elucidate the proangiogenic properties of mPDC, we switched to in vitro cell cultures. Hypoxia is known to accompany the early stages of fracture healing [40] and is a major stimulus for angiogenesis. We therefore assessed the response of mPDC to low oxygen tensions (1% oxygen) by analyzing factors involved in the hypoxia-inducible pathway. Western blot analysis showed that hypoxia-inducible factor (HIF)-1α protein levels were noticeably elevated in nuclear extracts of mPDC cultured under hypoxia, whereas no differences in HIF-2α levels were detected (Fig. 6A). Based on these results, we analyzed the expression of known HIF-1α target genes Glut1 (glucose transporter 1; also known as Slc2a1) and Ldha (lactate dehydrogenase A), which are involved in cellular metabolism, and Vegf, a prime angiogenic factor. Hypoxia increased Glut1 and Ldha gene expression (Fig. 6B), suggesting a shift toward a more anaerobic energy metabolism. In addition, mRNA and protein levels of VEGF were almost threefold upregulated in hypoxia (Fig. 6B, 6C).
To assess whether these changes in VEGF expression were physiologically relevant, we cultured HUVEC, a well-studied endothelial cell model, in medium conditioned by mPDC. When medium from mPDC cultured for 24 hours in hypoxia was added, HUVEC displayed a significantly higher proliferative response in comparison to the addition of normoxia-conditioned medium (Fig. 6D, 6E). Furthermore, even normoxia-conditioned medium increased the proliferation of the endothelial cells in comparison to standard medium. These data indicate that mPDC produce angiogenic factors, like VEGF, and that this production is upregulated when oxygen levels decrease.
mPDC Promote the Survival of Endothelial Cells Through the Production of VEGF
We next investigated whether the proangiogenic activity of mPDC could be attributed to VEGF, as the survival of endothelial cells is known to depend on the presence of VEGF in the medium [41, 42]. We therefore assessed whether coculture of mPDC could substitute for the addition of exogenous angiogenic factors in cultures of hBOEC. The withdrawal of angiogenic factors induced cell death of hBOEC within 4 days (data not shown). The survival of hBOEC could however be rescued in a coculture with mPDC, as shown by hCD31 staining after 7 days of culture (Fig. 6F, 6G). The importance of mPDC-produced VEGF in the survival of endothelial cells was evidenced by the finding that inhibition of VEGF activity, by the addition of an anti-mVEGF164 antibody or a soluble receptor (smFlt-1), resulted in massive endothelial cell death. Taken together, these experiments illustrate that mPDC promote the survival of endothelial cells through the production of VEGF.
mPDC Possess Perivascular Cell Characteristics and Promote Both Vasculogenesis and Angiogenesis In Vivo
As vessel maturation is critical to the establishment of a long-lasting functional vasculature, we next investigated the pericytic characteristics of mPDC in more detail. Endothelial cells (HUVEC or hBOEC) and mPDC were seeded alone or together in a 2:1 ratio on Matrigel and their distribution, with respect to each other, was analyzed after 24 hours. Microscopic analysis revealed that the cocultures induced more robust networks than when either cell type was cultured alone (Supporting Information Fig. S2). Confocal microscopy showed that mPDC were localized perivascularly with cell-to-cell contact between the endothelial cells and the mPDC (Fig. 7A). Consistent with these observations, flow cytometric analysis revealed that numerous mPDC express the pericyte markers α-SMA (76.4% ± 5.7%) and platelet-derived growth factor receptor-β (PDGFR-β; 43.6% ± 0.2%) (Fig. 7B).
When mPDC were implanted together with endothelial cells in a collagen gel in vivo, the total number of formed blood vessels was significantly higher than when either cell type was injected alone (Fig. 7C, 7D). The addition of mPDC to HUVEC increased the number of blood vessels lined by human endothelial cells almost twofold (Fig. 7D), even though less endothelial cells were initially present in the coculture implants. The formed blood vessels were filled with erythrocytes, confirming their connection to the host vasculature (Supporting Information Fig. S3). Implants seeded with mPDC also contained numerous blood vessels derived from the host, confirming that mPDC have a strong proangiogenic potential. The higher number of host-derived blood vessels in gels containing only mPDC compared to cocultures can likely be explained by the higher number of mPDC initially seeded. By implanting mPDC derived from C57BL/6-ACTb-eGFP mice together with HUVEC, we could further demonstrate that numerous mPDC closely encircled the vessels formed by HUVEC (Fig. 7E), supporting our in vitro observations. In conclusion, our results show that mPDC can contribute to new blood vessel formation by the production of angiogenic growth factors and by obtaining a pericyte-like phenotype, contributing structurally to the new vessels.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
The progress in bone repair strategies critically depends on adequate in vitro and in vivo models to evaluate new therapies and obtain mechanistic insight in the bone healing process. Due to their unique role during bone fracture repair, PDC are a valuable candidate for tissue engineering strategies. However, periosteal cells and their in vivo mechanisms of action remain poorly characterized, which might be related to the lack of a robust technique to isolate PDC from mice in a manner comparable to human PDC. In this study, we developed a technique to isolate mPDC from long bones of adult mice and showed that these cells possess a strong osteogenic potential and marked proangiogenic features.
We were able to selectively isolate mPDC from the long bones of adult mice through the use of agarose to shield cells of the perichondrium and the articular surface from the enzymatic digest applied to the nonprotected diaphyseal region. The mPDC population contains an abundant number of MSC, as shown by means of trilineage differentiation studies and surface marker analysis. Our results further suggest that considerably more MSC are present in the periosteum in comparison to the bone marrow stroma, confirming earlier studies on mouse, rat, and human cells [11, 12, 43-45]. In addition, we show that the mPDC expressed markers of osteoprogenitor and chondroprogenitor cells, but not of more differentiated osteoblasts or chondrocytes, suggesting the presence of both very early cells and more committed skeletal progenitor cells in the mPDC population. The large number of MSC in PDC cultures emphasizes their suitability as a cell source for bone tissue engineering applications and favors their use over BMSC. Although rich in MSC, the mPDC population is heterogeneous in nature and the question remains whether all elucidated properties (bone formation, establishment of hematopoiesis, attraction of osteoclasts, and support of blood vessels) can be attributed to one single cell type or whether these different functions are carried out by specific subpopulations.
In this study, we used relatively young mice (7–9 weeks old). It is however known that with age, the thickness and cellularity of the periosteum decreases [6, 46], resulting in diminished periosteal bone formation and delayed fracture repair [47–49]. In addition, the angiogenic response during fracture healing may also decline with age [48, 50]. Conversely, the osteogenic and chondrogenic differentiation potential of human periosteal cells is maintained regardless of donor age [11, 36]. Future studies are thus required to investigate whether the proangiogenic properties of PDC decline with age and provide an explanation for the reduced fracture repair in the elderly.
When transplanted in vivo, mPDC formed not only mature bone but also a hematopoiesis-supportive stroma. As recently highlighted, the ability of transplanted cells to form marrow stroma at an ectopic site can be considered desirable and even essential for a bone tissue engineering cell source as it marks the presence of a very early, uncommitted skeletal stem cell (SSC) [51]. The presence of the SSC has generally been ascribed to BMSC [52–54], but this study and our previous work on hPDC [55] evidently proves that the SSC is also present in the periosteum. The presence of SSC at both locations can likely be explained by the common origin of the bone marrow stroma and periosteum, which both derive from the perichondrium during fetal bone development.
The marrow compartments were further characterized by the presence of TRAP-positive osteoclasts, resorbing the adjacent bone matrix. In vitro cocultures revealed that mPDC were very potent in supporting osteoclastogenesis and even more effective than mBMSC. These data suggest that during fracture repair periosteal cells can attract osteoclast precursors and promote their differentiation and activity, independent of MSC from the bone marrow, and hereby regulate the remodeling of the fracture callus into mature lamellar bone. These findings further underscore and clarify previous studies showing that removal of the bone marrow does not significantly influence fracture callus formation or remodeling [10, 56]. Osteoclasts are also crucial for the turnover of immature into mature bone in a tissue engineering construct [39], and the osteoclastogenic activities of PDC additionally favor their use in bone tissue engineering applications.
An important finding of this study is that mPDC have proangiogenic properties, and to our knowledge, this is the first report to describe this feature of periosteal cells. We demonstrated that mPDC significantly contributed to the attraction of new blood vessels into engineered constructs after in vivo implantation. Indeed, both in calcium phosphate-based scaffolds and in collagen gels together with endothelial cells, the presence of mPDC augmented the angiogenic response from the host. Bone fracture results in the rupture of the vasculature and during the first phases of fracture repair, cells in the periosteum experience hypoxia [40]. HIF-1α levels increased in mPDC exposed to hypoxia and resulted in increased expression of genes involved in anaerobic cell metabolism and angiogenesis. The VEGF produced by the (hypoxic) mPDC enhanced endothelial cell proliferation and was necessary for endothelial cell survival, indicating that VEGF plays a pivotal role in the communication between mPDC and endothelial cells.
In addition to angiogenic growth factor production, PDC may also structurally contribute to neovascularization. Indeed, several blood vessels in the engineered construct were mature, as they were covered with mural cells, and multiple of these perivascular cells were of donor origin. This finding was confirmed by in vitro coculture of mPDC and endothelial cells which resulted in the formation of robust pseudovascular networks, in which the mPDC obtained a pericyte-like phenotype. When coimplanted in vivo with HUVEC, mPDC substantially increased vasculogenesis by the HUVEC, suggesting that the combination of PDC and endothelial cells might be an ideal strategy to promote blood vessel formation in tissue engineering constructs. The capability of the mPDC to act as blood vessel-supporting cells was further emphasized by the perivascular localization of mPDC in the implants and by the expression of the pericyte markers α-SMA and PDGFR-β by a large subpopulation of the mPDC. The presence of these markers has also been described for MSC derived from other sources, such as the bone marrow [54, 57] and the expression of pericyte markers might be related to the perivascular origin of MSC in different tissues [58]. Whether the MSC in the periosteum reside in a perivascular location remains elusive, but evidence exists that periosteal pericytes possess MSC characteristics [59, 60]. As the periosteum is highly vascularized [61], this model could also explain the high number of MSC found in this tissue.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
In conclusion, we showed that periosteal cells can be isolated from the long bones of adult mice. mPDC are rich in MSC and fulfill all requirements of an ideal cell source for bone regeneration, including the potential to form mature bone and to stimulate blood vessel ingrowth. The proangiogenic potential of mPDC can be attributed to angiogenic growth factor production and to the structural support of blood vessels as perivascular cells. Finally, the straightforward isolation method of mPDC, their beneficial properties, and their in vivo bone formation potential, which is similar to hPDC, make mPDC a valuable preclinical cell model for bone tissue engineering research. Virtually, every available transgenic mouse strain, including reporter and knockout animals, can be used to isolate mPDC, which could help to further clarify the nature of the cells in the periosteum and understand by which mechanisms these cells orchestrate fracture repair, knowledge that is essential to bring periosteal cell-based tissue engineering constructs into the clinic.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
We thank Ingrid Stockmans, Riet Van Looveren, Carla Geeroms, and Petra Vandervoort for excellent technical assistance. We are also grateful to Kjell Laperre for the isolation of growth plate chondrocytes and to Steve Stegen for help with the isolation of bone marrow cells. This work was supported by grants from the Fund for Scientific Research Flanders (FWO; G.0500.08 and G.0982.11; G.C.), the Flemish Government (Long-term Structural Funding—Methusalem; P.C.), the European Commission (FP7-StG-IMAGINED 203291; A.L.), and the KU Leuven (IOF Knowledge Platform "Prometheus"; IOFKP/07/004; S.J.R., J.S., F.P.L. and Stem Cell Institute of Leuven; S.J.R.). N.v.G. is a fellow from the Agency for Innovation by Science and Technology in Flanders (IWT). This work is part of Prometheus, the Leuven Research & Development Division of Skeletal Tissue Engineering of the KU Leuven.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
The authors indicate no potential conflicts of interest.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
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Supporting Information
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Additional Supporting Information may be found in the online version of this article.
|Figure S1. Isolation of mPDC: embedding of epiphyses and enzymatic digest. (A-C): H&Estained sections showing murine tibias before digest (A), after enzymatic digest (B) and after embedding of the epiphyses in low melting point agarose and enzymatic digest (C). Black arrows indicate remaining tissue from the joint and tendon present on the epiphysis. (A1-C1): Magnification of the boxed areas in A-C showing release of perichondrial cells after enzymatic digest, which was precluded when epiphyses were first embedded in agarose (black arrow). (A2-C2): H&E-stained sections showing the periosteal surface of the diaphysis (black arrow) of the tibia before digest (A2) or after enzymatic digest without (B2) or with (C2) epiphyses embedded in low melting point agarose. Scale bars = 500μm in A, B, C; 200μm in A1, B1, C1, A2, B2, C2. Abbreviations: H&E: hematoxylin and eosin.
|Figure S2. mPDC stabilize networks formed by endothelial cells in vitro. (A, B): CMFDAlabeled mPDC were co-cultured with CM-DiI-labeled HUVEC or hBOEC on MatrigelTM for 24h. Brightfield (A) and fluorescent (B) microscopic analysis of cultures of endothelial cells alone (left), together with mPDC (middle) or of mPDC alone (right) showing formation of robust pseudovascular networks in the co-cultures, with mPDC localized predominantly at the outside of the structures, around the endothelial cells. Scale bar = 200μm. Abbreviations: hBOEC: human blood outgrowth endothelial cells; HUVEC: human umbilical vein endothelial cells; mPDC: murine periosteum-derived cells.
|Figure S3. Co-implantation of mPDC and HUVEC results in the formation of mature, perfused blood vessels. (A-B): In vivo co-implantation of mPDC and HUVEC in a 60:40 ratio in collagen gels for 7 days. H&E-stained sections (A) showing the presence of small (A1, A1′) and large (A2, A2′) blood vessels filled with red blood cells, confirming their connection to the host vasculature. Immunohistochemical staining of HUVEC with UEA-lectin in combination with green autofluorescence of blood erythrocytes (B) indicates that the blood vessels formed by the implanted HUVEC are functional. The section was counterstained with Hoechst to visualize cell nuclei. Scale bars = 500μm in A; 100μm in A1; 50μm in A1′, A2, B; 20μm in A2′. Abbreviations: H&E: hematoxylin and eosin, HUVEC: human umbilical vein endothelial cells; mPDC: murine periosteum-derived cells; UEA-lectin: Ulex europaeus agglutinin-lectin.
|Supplementary Table 1
|Supplementary Table 2
|Supplementary Table 3
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


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text:
The Elder Scrolls Online PC
- Publisher: Bethesda Softworks
- Release Date: Apr 4, 2014
- Critic score
- Publication
- By date
Pelit (Finland)May 9, 2014The Elder Scrolls Online has great potential, but wastes it on failed group gameplay and general bugginess of the game. Subscription fee and the stuff left exclusively to Imperial Edition feel a bit questionable too. [May 2014]
Games Master UKMay 4, 2014Great at telling stories, with a gargantuan world to feast upon, but its MMO failings let it down. [June 2014, p.60]
Jun 3, 2014If you're an Elder Scrolls fan who has always wanted to play one of these games in co-op, this is the next best thing, at least for the time being. However, if you're looking for Skyrim: The MMO, this will likely disappoint.
May 27, 2014The Elder Scrolls Online is offering time-proven game mechanics that are functioning and frequently entertaining, while following tasks dipped in narrative gravy create the impression of a living virtual world. At the same time, however, TESO does not bring anything exceptional that would let you stared open-mouthed.
May 19, 2014The Elder Scrolls Online is a good MMO, but it is not the masterpiece it could have been if ZeniMax dared more, and if they did not hurry to release it.
May 16, 2014Something good is being brewed here by Zenimax. The Elder Scrolls Online definitely needs a lot of work just to be able to reach other MMOs, but by playing this game you get a feeling that the online version of this franchise could become a milestone for the genre.
May 9, 2014All in all, The Elder Scrolls Online is a pretty good game. It's not outstanding, and it's not terrible.
Apr 30, 2014In the end, the feeling I have with Elder Scrolls Online is something strange, because I like it enough to want to play it, but not to the level of an MMO. I'm not compelled to make the necessary commitment.
Apr 28, 2014The Elder Scrolls Online retains most of the elements that made the series famous in a new astounding experience. The transition from offline to online is smooth and the game itself is fun. Unfortunately the monthly fee and lots of bugs partially ruin the experience.
Apr 24, 2014The Elder Scrolls Online needs a lot of work to become a competitive entry in the MMO market because right now it really does not know what to be. Is it a co-operative TES? Is it a single player game? Is it a MMO? Every time we found something good in it we had something else spoiling all the fun, such as lack of features, polishing or common sense.
Apr 22, 2014A few more months in development might have made a considerable difference. Still, this will probably make quite a splash when it arrives on consoles. On PC, though, TESO doesn't do enough to differentiate itself from the competition.
Apr 21, 2014Visually, the game is consistent, but that consistency is boring and drab. The game itself is punctuated with exciting moments, but overall it's just above average and I have a hard time recommending that in a subscription MMO. If you're a big Elder Scrolls fan, give it a try. If not, there are better choices out there.
Apr 17, 2014The main quality of The Elder Scrolls Online is the giving of a good answer to the fans asking for an MMO Elder Scrolls for years. But objectively, it's not the best MMORPG around, nor the best episode of the series. Consequently, the game has good moments but it never enters the unforgettable kind. And the subscription seems very high at the end of the day.
Apr 15, 2014Under the surface, not enough has changed to the formula that separates The Elder Scrolls Online from existing free or established properties, and I would find it genuinely hard to recommend to anyone seeking an experience outside of a cosy, well presented, box.
Apr 12, 2014Elder Scrolls Online presents an honest attempt to freshen up the MMO genre. It wants to offer fans a pure Elder Scrolls experience with a focus on story and characters, but with its fairly sloppy multiplayer mechanics the game gets a bit boring. Despite quite a few good ideas, Elder Scrolls Online sadly fails at several points. It's still a game that will really speak to a variety of players though.
Apr 12, 2014This is not the huge mess some people claim nor the masterpiece some others say. It's a good MMORPG with some issues that you can enjoy if you can forgive them.
Apr 4, 2014The Elder Scrolls Online is probably the weirdest MMO I've played in ages. It is constantly torn between its solo RPG origin and the need to provide the usual MMO package "a la WoW". In the end though, it is highly enjoyable to play, and nobody will be bored in embarking on this adventure.
Apr 25, 2014By their very nature, MMO's evovle - but as it stands right now, The Elder Scrolls online isn't a worthwhile investment. If you're an Elder Scrolls fan, you might want to give it a try but I do think you may be a teeny bit disappointed. If you're an MMORPG fan, there are some ideas here that you will certainly like.
Apr 14, 2014A few well-designed systems struggle to overcome lifeless presentation. Capable, but ultimately hard to recommend.
May 12, 2014Zenimax Online Studios was on an almost impossible task. Delivering an MMO experience on single player basis it's simply impossible, especially if you're sticking to traditional mechanics. Maybe, one day, reworked by patches and courageous changes, this will be a great MMO, but not today.
May 9, 2014I think right now, the most prudent thing to do is to wait for ZeniMax to iron out all of The Elder Scrolls Online's kinks (including how it handles post level 50 content to make it less grindy), and play the console version of the game. Not only will it arrive with all of the updates from the PC version in tow, but it'll also have full native controller support -- which feels more natural than a mouse and keyboard in this instance.
May 5, 2014The Elder Scrolls Online is a big game with a lot of work put into its creation. ZeniMax Online has created a large world with many quests and a cool PvP mode. The game tries to satisfy both solo gamers and MMO lovers, but the end result is short of expectations. TESO can be recommended only to the fans of the genre, who accept its flaws: repetitiveness, not very exciting combat and lots of simplifications. This "Skyrim online" is well made, but not very original.
Apr 29, 2014The parts I dislike are where they've tried to shoehorn an MMO into the mix. The combat, level grind, grouping, and people all discourage me from wanting to play. The MMO components feel forced and generic, and the game would be more enjoyable without them. This comes from an Elder Scrolls fan though.
CD-ActionJul 23, 2014Eight years of development, a team consisting of even 250 people at some points, budget reaching hundreds of millions of dollars. It's a pity that this 'Skyrim Online' disappoints both fans of previous Bethesda games and MMORPG veterans who expected some fresh air in the stale genre. [06/2014, p.44]
LEVEL (Czech Republic)Jul 8, 2014A well done MMO that can lure players not familiar with the genre. But if you've played MMOs before, this one doesn't offer anything new, nor anything exciting.
Hyper MagazineJun 7, 2014Gorgeous to look at and listen to, but rather forgettable to play. [Issue#249, p.65]
May 29, 2014Whether or not The Elder Scrolls Online is for you depends on what you are looking for. It is not a conversion of the single player series that many might have hoped for.
May 20, 2014With better MMORPGs already out there and seemingly more great ones coming, The Elder Scrolls Online is using the name to appeal to its large fan base, and I have no doubt some will enjoy, but for the rest, this game is an vacillating anomaly in a packed market.
Apr 25, 2014Elder Scrolls fans will be put off by its rigid structure and weak storytelling, while your average MMO player will tire of wading through the wan questing to get to the good stuff. Even the good stuff isn't outstanding, and the game doesn't represent good value compared to its competition.
Apr 24, 2014Hopefully, The Elder Scrolls Online will one day get out of its own way, and stop trying to stifle the very fun it's trying to provide.
Apr 24, 2014We approached Elder Scrolls Online as fans of the series and as MMO lovers, but it came up short from both perspectives. It's missing that spark of magic that enticed us to get lost in Skyrim or Morrowind for months, or that made us happy to fork over a monthly fee just to access our current favorite game. It seems like so much effort was put into forcibly translating Elder Scrolls' style into the genre's norms, but the payoff for that effort isn't there.
Apr 14, 2014The Elder Scrolls Online makes the fundamental mistake that most quests have been written as single player adventures. Other players disturb the illusion, instead of being the reason you'd want to keep playing. Good combat, very fun PvP and deep character development can't hide that ESO makes one too many compromises and estranges RPG- and MMO-lovers in the process.
Apr 14, 2014A lot of really great potential is held back by a huge amount of bugs.
Jun 16, 201460 days was more then enough time for me to determine that I would rather spend my time elsewhere.
Apr 29, 2014Players who seek the traditional fantasy MMOG experience may find something of value in TESO, because it has evidently been built with them in mind. But it is difficult to imagine many others investing hundreds of hours in a place this bland, in a formula this familiar, and in a game this demanding of both your time and your money.
Apr 18, 2014The Elder Scrolls Online is frustrating. It has moments of sheer class, but they're consistently tainted by bugs. I love the PvP, but it's pointless playing when the rewards are so slim. I like the questing, but huge numbers of quests are simply broken. I love the world, and the lore, and some of the systems they have produced. But my enjoyment of them was undermined by poor design.
Awards & Rankings
User Score
Mixed or average reviews- based on 2399 Ratings
User score distribution:
Positive: 390 out of 882
Mixed: 148 out of 882
Negative: 344 out of 882
Apr 4, 2014Did you ever play WoW and wished it wouldn't be so boring levelling up? Welcome to the Elder Scrolls Online. It plays very differently fromDid you ever play WoW and wished it wouldn't be so boring levelling up? Welcome to the Elder Scrolls Online. It plays very differently from traditional MMOs in the sense that there are not quest hubs. This means that it is up to you to find and explore and by doing so level up. The combat is fantastic, there are no cool down timers. It is up to you to dodge, block, interrupt, cast, heal. It's very hectic and very fun, but also true to the Elder Scrolls Combat formula. Being able to play in first person adds immersion. The graphics are fantastic. All quest texts are voiced. The list goes on and on, in short I couldn't be happier with this game.… Full Review »
Apr 4, 2014Its not terrible, its just very disappointing coming from the heritage of a game series that dared to be different from the crowd and defineIts not terrible, its just very disappointing coming from the heritage of a game series that dared to be different from the crowd and define its own genre of open sandbox rpg gaming. Elderscrolls online just leaves me feeling like the makers really just wanted to make a new RvR game in the same mould as DAoC or WAR. Its so standard MMO, so been there done that, so uninspired it just hurts. Where there is an attempt to incorporate some of the elements of the single player games it feels clunky or doesn't quite work because the fundamental starting point was your bog standard MMO the like of which we've seen time and again for the last 10 years. Take questing. full voice, lots of effort put into back story you say? Well yes, but because the questing and character advancement model is essentially the same as most MMOs (That being you advance by completing quests which give you xp to spend on level ups), the world is necessarily packed with quest givers, most of whom give 5 min quests which we've all done a million of before, kill 5 bears, pick 10 flowers, blah blah blah. The end result is all the fancy voice acting and back story just wastes your time because the fundamental activity is so mindless. I could go on and on about the disappointments in ESO, but it all boils down to one thing. Its nothing new, its not in the spirit of the single player games, its just another near enough carbon copy of a gameplay model that doesn't fit with the IP that's been forced into it. Come on Devs, can't you be a bit braver than this. If Bethesda had just copied the successful model of the time then Arena would of been a scripted on rails experience like Final Fantasy. The MMORPG world is crying out for something new, something different. Enough with the era of the WoW-a-like.… Full Review »
Apr 11, 2014Re-reviewing this game after a week of normal play and it is still disappointing.
As to be expected with the release of games these daysRe-reviewing this game after a week of normal play and it is still disappointing.
As to be expected with the release of games these days there are a ton of bugs, some that are seriously crippling, like everything in your bank disappearing or that really cool epic armor on your head just disappearing. Quests are broken and it seems that the higher level you get the less testing seems to have been done, which makes sense as they had a limited beta.
It doesn't even have the feel of a normal MMO because they gave it the base Elder Scrolls GUI but then cut back on its functionality. You have 7 slots attached to keyboard keys, 1-5, Q, R, 1 special R which is really lame as the power builds up while you fight and drops when you are not which means you rarely use it in PvE. The 7 slot Q is attached to a quick slot which means you can have several items on that, but can only use them one at a time and there is a cool-down timer on them. So that epic battle in Skyrim where you clicked through 30 potions, not going to happen in ESO.
The limited key functionality and simplicity that they have is clearly been done to make the titles transition to consoles easier. The greatness of the Elder Scrolls series is the graphics and how fantastic the game looks. ESO misses this mark completely, the graphics just are not there and seem like they maybe that way for console release.
The interesting thing about player and bank inventory space is that you can see where they could put a F2P or P2W system in place immediately without having to adjust game mechanics. It is almost like the expect this game to go F2P at some point.
With that in mind, there is no ground breaking systems here that are just the best thing ever, there are gold farmers, bots and spammers in the chat. With nothing really holding people other than the PvP which again is not ground breaking, and the hope of future releases of new lands I do not see a financial sustainability in the 15 dollars a month plan. I suspect before the one year mark either an announcement or the actually change to a F2P model. I hope that Bethesda sees this not as a title to build off of, but as a third part release for the MMO market.… Full Review »


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Luvu Brands (OTCQB: LUVU) is a Georgia-based manufacturer that produces a range of products in the wellness, lifestyle, and casual furniture categories. The company is made up of over 135 employees and emphasizes creativity throughout its business. LUVU products are sold in innovative eco-compressed vacuum packaging for all its retailers worldwide. In addition to offering products through retail stores, mass merchants, drug and Internet retailers, LUVU manages, markets, and distributes products through several of its own websites. The company's mission statement is set toward lowering carbon footprint, giving back to creatives, supporting local communities, and creating products that are simple, comfortable, and beautiful.
LUVU is committed to being resource-smart. This means that every time a new product is considered, the environment is the company's primary concern. A personal goal of every member of the staff is to reduce carbon footprint, eliminate forms of waste, use resources efficiently, and prevent pollution. The materials and components that are used are all tested to be non-toxic and in compliance with CE and RoHs standards. In addition to LUVU's serious efforts to minimize any impact on the environment, the company makes a point of always looking for economically sustainable opportunities to help communities grow.
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Since 2010, LUVU's net sales have risen 51%, and its inventory has grown 27%. LUVU has focused on moving its innovative packaging to the retail shelf, upgrading its e-commerce platform to add mobile capabilities, increasing its factory automation to reduce costs, developing sales and marketing tools, and expanding to European distribution. The company plans to continue to grow sales through various channels, establishing itself as a key "made-in-America" consumer products company and increasing shareholder communication.
For more information, visit www.luvubrands.com


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Social Media Professional who has a Google + account | Prefers coffee over protein shake | One who hogs, socializes and ties his shoelace frequently.
Face Tags
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Dinner at home.
So here is what my sister gifts me after appearing on #HOMP. Happiness!
Men, run for your lives! #ROFLMAO
@RichPurelyRich sexy no?
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Registration for 2016 Season
|Registration Dates:
| Saturday June 25
|9:00am - Noon
| Saturday July 9
|9:00am - Noon
| Saturday July 23
|9:00am - Noon
Final Registration Day
Indiana Girls Tackle Football League
The Indiana Girls Tackle Football League is open to all girls (grades 5-9) in surrounding communities as well as around the state of Indiana. The girls will be divided into two leagues — 5/6th grade and 7/8/9th grade.
Morris Cohen MJFL Junior Football League
Sign-up online or register on one of our registration days at the MJFL fields.
1. If you need equipment you must come on 1 of the registration dates at the field.
2. If you want to use your own personal equipment it must be checked by a Board Member.
Flag Football
Kids entering grades K–1, Age 5-6
Full Contact Football
2nd Grade Tackle Grade 2, Ages 6–8
American League, Grade 3–4, Ages 8–11
National League, Grade 5–6, Ages 11–12
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IMPORTANT NOTE: Field Housekeeping


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Arts and Music
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New Videos and Podcasts
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Arts and Music airing this week
La Jolla Symphony & Chorus presents a multimedia concert. The first half offers GOTHAM, a 2004 joint production by filmmaker Bill Morrison and composer Michael Gordon. Using archival footage of New York City, Gotham combines film, projectors, lighting, and the orchestra to create a haunting impression of that city. Stravinsky's opulent PERSEPHONE, which the composer described as a "melodrama," sets a text by André Gide that tells of the descent of the goddess Persephone into the underworld and her return. For this presentation the Symphony is joined by the Lux Boreal Dance Company, a narrator, a tenor soloist, and a children's choir.
San Diego Opera General Director David Bennett hosts beloved opera legend Frederica von Stade in a lively discussion about her career, and in particular her long-standing collaboration with composer Jake Heggie (Dead Man Walking, Moby-Dick. In addition to performing Heggie's songs, Von Stade starred in his operas Three Decembers and Great Scott.


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I am a scripting language connoisseur, regular expression aficionado, network geek, general lover of Linux and a frequent contributor to open source software. I transitioned to programming from other work because I was too busy automating my own work environment to actually do the other work. I have a hobby interest in cartography. For more see my personal site. Most importantly, my life is defined by the grace of God given to men through Jesus Christ. It is my ambition that everything I do would reflect His glory and point people towards Him.
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Iron Bartender finale this Sunday at Icehouse
The finals of the competition will be held on Sunday at Icehouse. We have all the details.
The event will be hosted by Ian Rans from TV's Drinking with Ian fame. Competing bartenders hail from Bradstreet Craftshouse, Cafe Maude Loring Park, Eat Street Social, and La Belle Vie.
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Fast Low Power ADC with Integrated Digital Data Processor
Award Information
Small Business Information
27 Via Porto Grande, Rancho Palos Verdes, CA, 90275-2049
Principal Investigator:
Vladimir Bratov
Principal Investigator
(310) 528-9118
Principal Investigator
(310) 528-9118
Business Contact:
Vladimir Katzman
Business Official
(310) 377-6029
Business Official
(310) 377-6029
Research Institution:
AbstractInnovative data measurement/acquisition systems are needed to support future Earth System Science measurements of the Earth's atmosphere and surface. An adequate system must employ a high-speed, extra low power, linear, analog-to-digital converter (ADC) with high input bandwidth and accuracy, followed by a digital signal processor that is usually implemented inside a field-programmable gate array (FPGA). Commercially available ADCs with input bandwidths larger than 1GHz feature high power consumption and latency, and poor linearity. With increasing ADC sampling rates, timing difficulties within the parallel interconnects between the ADC and the following FPGA become increasingly prominent. Therefore, a monolithic ADC incorporating a data converter that performs digital data demultiplexation and retiming is desired. To address these needs, the Advanced Science and Novel Technology Company proposes to develop a novel, extra low-power, extremely linear, under-sampling ADC featuring a high analog input bandwidth (>5GHz) that can easily interface to a following FPGA through a low-speed (<750Mb/s) parallel interface. To achieve this functionality, the proposed system-on-chip will utilize a proprietary sub-Nyquist front-end analog demultiplexer, a combinational ADC structure with proprietary gain-stabilization circuitry, and a digital signal processor implementing digital data demultiplexation and the company's proprietary data alignment scheme.
* information listed above is at the time of submission.
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Technology |
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As iPhone sales stagnate, services promise growth for Apple
Though Apple Inc is selling enough iPhones to keep investors happy for now, the world's most valuable publicly traded company is set increasingly to rely on its apps and services to drive growth. Full Article | Related Story
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