J. Kent Leach

Proangiogenic potential of a collagen/bioactive glass substrate.

PurposePrevious attempts to stimulate angiogenesis have focused on the delivery of growth factors and cytokines, genes encoding for specific angiogenic inductive proteins or transcription factors, or participating cells. While high concentrations of bioactive glasses have exhibited osteogenic potential, recent studies have demonstrated that low concentrations of particular bioactive glasses are angiogenic. We hypothesized that a well known bioactive glass (Bioglass® 45S5) possesses proangiogenic potential over a limited range of concentrations.Materials and MethodsVarying amounts of Bioglass were loaded into absorbable collagen sponges. The proangiogenic potential of Bioglass was determined by examining the capacity of the soluble products to induce endothelial cell proliferation, tubule formation in a co-culture, and upregulate vascular endothelial growth factor (VEGF) production.ResultsWe determined a range of Bioglass concentrations which exhibit proangiogenic potential. Furthermore, we demonstrated that the proangiogenic capacity of this material is related to the soluble dissolution products of Bioglass and the subsequent production of cell-secreted angiogenic factors by stimulated cells.ConclusionsThese studies suggest that this bioactive glass possesses a robust proangiogenic potential, and this strategy may provide an alternative to recombinant inductive growth factors.

Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue

OBJECTIVE To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells. ANIMALS 10 adult Quarter Horses and 8 newborn Thoroughbred foals. PROCEDURES eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated. RESULTS BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP activity and calcium deposition. There was no significant difference in BM-eMSC yield from the tuber coxae or sternum, but yield was significantly greater from the first 60-mL aspirate than from subsequent aspirates. The BM-eMSC expansion rate was significantly higher when cells were cultured in fetal bovine serum instead of autologous serum (AS). CONCLUSIONS AND CLINICAL RELEVANCE eMSCs from BM possessed the highest in vitro osteogenic potential; eMSCs from adipose tissue also had robust osteogenic potential. The tuber coxae and the sternum were viable sources of BM-eMSCs in yearlings, and 60 mL of BM aspirate was sufficient for culture and expansion. Expanding BM-eMSCs in AS to avoid potential immunologic reactions decreased the total yield because BM-eMSCs grew significantly slower in AS than in fetal bovine serum. Additional studies are needed to determine optimal ex vivo eMSC culture and expansion conditions, including the timing and use of growth factor—supplemented AS.

Angiogenic Response to Bioactive Glass Promotes Bone Healing in an Irradiated Calvarial Defect

Localized radiation is an effective treatment modality for carcinomas, yet the associated reduction of the host vasculature significantly inhibits the tissues regenerative capacity. Low concentrations of bioactive glass (BG) possess angiogenic potential, and we hypothesized that localized BG presentation would increase neovascularization and promote healing in an irradiated bone defect. An isolated calvarial region of Sprague-Dawley rats was irradiated 2 weeks before surgery. Bilateral critical-sized defects were created and immediately filled with a BG-loaded collagen sponge or an empty sponge as an internal control. Histological analysis of calvaria collected after 2 weeks demonstrated greater neovascularization within the defect in the presence of BG than with collagen alone. Noninvasive ultrasound imaging at 4 weeks detected less contrast agent in the brain below BG-treated defects than in the nearby untreated defects and images of treated defects acquired at 2 weeks. The reduced ability to detect contrast agent in BG-treated defects suggested greater attenuation of ultrasound signal due to early bone formation. Micro-computed tomography imaging at 12 weeks demonstrated significantly greater bone volume fraction within BG-treated defects than in controls. These results suggest that neovascularization induced by localized BG delivery promotes bone regeneration in this highly compromised model of bone healing and may offer an alternative approach to costly growth factors and their potential side-effects.

Differentiation-dependent secretion of proangiogenic factors by mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a promising cell population for cell-based bone repair due to their proliferative potential, ability to differentiate into bone-forming osteoblasts, and their secretion of potent trophic factors that stimulate angiogenesis and neovascularization. To promote bone healing, autogenous or allogeneic MSCs are transplanted into bone defects after differentiation to varying degrees down the osteogenic lineage. However, the contribution of the stage of osteogenic differentiation upon angiogenic factor secretion is unclear. We hypothesized that the proangiogenic potential of MSCs was dependent upon their stage of osteogenic differentiation. After 7 days of culture, we observed the greatest osteogenic differentiation of MSCs when cells were cultured with dexamethasone (OM+). Conversely, VEGF protein secretion and upregulation of angiogenic genes were greatest in MSCs cultured in growth media (GM). Using conditioned media from MSCs in each culture condition, GM-conditioned media maximized proliferation and enhanced chemotactic migration and tubule formation of endothelial colony forming cells (ECFCs). The addition of a neutralizing VEGF165/121 antibody to conditioned media attenuated ECFC proliferation and chemotactic migration. ECFCs seeded on microcarrier beads and co-cultured with MSCs previously cultured in GM in a fibrin gel exhibited superior sprouting compared to MSCs previously cultured in OM+. These results confirm that MSCs induced farther down the osteogenic lineage possess reduced proangiogenic potential, thereby providing important findings for consideration when using MSCs for bone repair.

The effect of sustained delivery of vascular endothelial growth factor on angiogenesis in tissue-engineered intestine.

Our group has previously created a functional neointestine that is capable of restoring absorptive function. However, the endogenous level of vascular endothelial growth factor (VEGF) is markedly reduced in the construct compared to native bowel. Therefore, we wanted to locally deliver VEGF in a sustained fashion to upregulate angiogenesis in the neointestine. Rat recombinant VEGF was encapsulated in poly(lactide-co-glycolide) microspheres by a double emulsion method. Release kinetics and bioactivity were determined in vitro. Tissue-engineered intestine was generated by seeding donor neonatal rat intestinal organoid units onto a biodegradable polyglycolic acid scaffold along with VEGF-containing or empty microspheres, and wrapped in the omentum of recipient rats. After 4 weeks, the neointestinal cysts were analyzed for morphometry, VEGF levels, epithelial proliferation, and capillary density. Sustained release of biologically active VEGF was confirmed by in vitro studies. Intestinal constructs with VEGF microspheres were significantly larger than those containing empty microspheres. Tissue VEGF levels were significantly higher in neointestine loaded with encapsulated VEGF compared to those without growth factor. Epithelial cellular proliferation and capillary density were significantly increased in the VEGF-containing neointestinal constructs compared to empty constructs. Tissue-engineered intestine responds to sustained delivery of VEGF by upregulating microvasculature and epithelial proliferation.

Concise Review: Optimizing Expansion of Bone Marrow Mesenchymal Stem/Stromal Cells for Clinical Applications

Bone marrow‐derived mesenchymal stem/stromal cells (MSCs) have demonstrated success in the clinical treatment of hematopoietic pathologies and cardiovascular disease and are the focus of treating other diseases of the musculoskeletal, digestive, integumentary, and nervous systems. However, during the requisite two‐dimensional (2D) expansion to achieve a clinically relevant number of cells, MSCs exhibit profound degeneration in progenitor potency. Proliferation, multilineage potential, and colony‐forming efficiency are fundamental progenitor properties that are abrogated by extensive monolayer culture. To harness the robust therapeutic potential of MSCs, a consistent, rapid, and minimally detrimental expansion method is necessary. Alternative expansion efforts have exhibited promise in the ability to preserve MSC progenitor potency better than the 2D paradigm by mimicking features of the native bone marrow niche. MSCs have been successfully expanded when stimulated by growth factors, under reduced oxygen tension, and in three‐dimensional bioreactors. MSC therapeutic value can be optimized for clinical applications by combining system inputs to tailor culture parameters for recapitulating the niche with probes that nondestructively monitor progenitor potency. The purpose of this review is to explore how modulations in the 2D paradigm affect MSC progenitor properties and to highlight recent efforts in alternative expansion techniques.

Increased Survival and Function of Mesenchymal Stem Cell Spheroids Entrapped in Instructive Alginate Hydrogels

Mesenchymal stem cell (MSC)‐based therapies are under broad investigation for applications in tissue repair but suffer from poor cell persistence and engraftment upon transplantation. MSC spheroids exhibit improved survival, anti‐inflammatory, and angiogenic potential in vitro, while also promoting vascularization when implanted in vivo. However, these benefits are lost once cells engage the tissue extracellular matrix and migrate from the aggregate. The efficacy of cell therapy is consistently improved when using engineered materials, motivating the need to investigate the role of biomaterials to instruct spheroid function. In order to assess the contribution of adhesivity on spheroid activity in engineered materials and promote the bone‐forming potential of MSCs, we compared the function of MSC spheroids when entrapped in Arg‐Gly‐Asp (RGD)‐modified alginate hydrogels to nonfouling unmodified alginate. Regardless of material, MSC spheroids exhibited reduced caspase activity and greater vascular endothelial growth factor (VEGF) secretion compared with equal numbers of dissociated cells. MSC spheroids in RGD‐modified hydrogels demonstrated significantly greater cell survival than spheroids in unmodified alginate. After 5 days in culture, spheroids in RGD‐modified gels had similar levels of apoptosis, but more than a twofold increase in VEGF secretion compared with spheroids in unmodified gels. All gels contained mineralized tissue 8 weeks after subcutaneous implantation, and cells entrapped in RGD‐modified alginate exhibited greater mineralization versus cells in unmodified gels. Immunohistochemistry confirmed more diffuse osteocalcin staining in gels containing spheroids compared with dissociated controls. This study demonstrates the promise of cell‐instructive biomaterials to direct survival and function of MSC spheroids for bone tissue engineering applications.

Oxygen tension differentially influences osteogenic differentiation of human adipose stem cells in 2D and 3D cultures

Skeletal defects commonly suffer from poor oxygen microenvironments resulting from compromised vascularization associated with injury or disease. Adipose stem cells (ASCs) represent a promising cell population for stimulating skeletal repair by differentiating toward the osteogenic lineage or by secreting trophic factors. However, the osteogenic or trophic response of ASCs to reduced oxygen microenvironments is poorly understood. Moreover, a direct comparison between 2D and 3D response of ASCs to hypoxia is lacking. Thus, we characterized the osteogenic and angiogenic potential of human ASCs under hypoxic (1%), normoxic (5%), and atmospheric (21%) oxygen tensions in both 2D and 3D over 4 weeks in culture. We detected greatest alkaline phosphatase activity and extracellular calcium deposition in cells cultured in both 2D and 3D under 21% oxygen, and reductions in enzyme activity corresponded to reductions in oxygen tension. ASCs cultured in 1% oxygen secreted more vascular endothelial growth factor (VEGF) over the 4‐week period than cells cultured in other conditions, with cells cultured in 2D secreting VEGF in a more sustained manner than those in 3D. Expression of osteogenic markers revealed temporal changes under different oxygen conditions with peak expression occurring earlier in 3D. In addition, the increase of most osteogenic markers was significantly higher in 2D compared to 3D cultures at 1% and 5% oxygen. These results suggest that oxygen, in conjunction with dimensionality, affects the timing of the differentiation program in ASCs. These findings offer new insights for the use of ASCs in bone repair while emphasizing the importance of the culture microenvironment. J. Cell. Biochem. 110: 87–96, 2010.

Noninvasive Multimodal Evaluation of Bioengineered Cartilage Constructs Combining Time-Resolved Fluorescence and Ultrasound Imaging

A multimodal diagnostic system that integrates time-resolved fluorescence spectroscopy, fluorescence lifetime imaging microscopy, and ultrasound backscatter microscopy is evaluated here as a potential tool for assessing changes in engineered tissue composition and microstructure nondestructively and noninvasively. The development of techniques capable of monitoring the quality of engineered tissue, determined by extracellular matrix (ECM) content, before implantation would alleviate the need for destructive assays over multiple time points and advance the widespread development and clinical application of engineered tissues. Using a prototype system combining time-resolved fluorescence spectroscopy, FLIM, and UBM, we measured changes in ECM content occurring during chondrogenic differentiation of equine adipose stem cells on 3D biodegradable matrices. The optical and ultrasound results were validated against those acquired via conventional techniques, including collagen II immunohistochemistry, picrosirius red staining, and measurement of construct stiffness. Current results confirm the ability of this multimodal approach to follow the progression of tissue maturation along the chondrogenic lineage by monitoring ECM production (namely, collagen type II) and by detecting resulting changes in mechanical properties of tissue constructs. Although this study was directed toward monitoring chondrogenic tissue maturation, these data demonstrate the feasibility of this approach for multiple applications toward engineering other tissues, including bone and vascular grafts.

The role of protein solubilization in antigen removal from xenogeneic tissue for heart valve tissue engineering.

Decellularization techniques have been developed in an attempt to reduce the antigenicity of xenogeneic biomaterials, a critical barrier in their use as tissue engineering scaffolds. However, numerous studies have demonstrated inadequate removal and subsequent persistence of antigens in the biomaterial following decellularization, resulting in an immune response upon implantation. Thus, methods to enhance antigen removal (AR) are critical for the use of xenogeneic biomaterials in tissue engineering and regenerative medicine. In the present study, AR methods incorporating protein solubilization principles were investigated for their ability to reduce antigenicity of bovine pericardium (BP) for heart valve tissue engineering. Bovine pericardium following AR (BP-AR) was assessed for residual antigenicity, tensile properties, and extracellular matrix composition. Increasing protein solubility during AR significantly decreased the residual antigenicity of BP-AR-by an additional 80% compared to hypotonic solution or 60% compared to 0.1% (w/v) SDS decellularization methods. Moreover, solubilizing agents have a dominant effect on reducing the level of residual antigenicity of BP-AR beyond that achieved by AR additives alone. Tested AR methods did not compromise the tensile properties of BP-AR compared to native BP. Furthermore, residual cell nuclei did not correlate to residual antigenicity, demonstrating that residual nuclei counts may not be an appropriate indicator of successful AR. In conclusion, AR strategies promoting protein solubilization significantly reduced residual antigens compared to decellularization methods without compromising biomaterial functional properties. This study demonstrates the importance of solubilizing protein antigens for their removal in the generation of xenogeneic scaffolds.