RESUMO
Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone grafts for enhancement of scaffold remodeling and fusion. Marrow-derived cytokines induce host neovascularization at the graft surface, but hypoxic conditions cause cell death at the core. Addition of cellular components that generate an extensive primitive plexus-like vascular network that would perfuse the entire scaffold upon anastomosis could potentially yield significantly higher-quality grafts. We used a mouse model to develop a two-stage protocol for generating vascularized bone grafts using mesenchymal stem cells (hMSCs) from human bone marrow and umbilical cord-derived endothelial cells. The endothelial cells formed tube-like structures and subsequently networks throughout the bone scaffold 4-7 days after implantation. hMSCs were essential for stable vasculature both in vitro and in vivo; however, contrary to expectations, vasculature derived from hMSCs briefly cultured in medium designed to maintain a proliferative, nondifferentiated state was more extensive and stable than that with hMSCs with a TGF-beta-induced smooth muscle cell phenotype. Anastomosis occurred by day 11, with most hMSCs associating closely with the network. Although initially immature and highly permeable, at 4 weeks the network was mature. Initiation of scaffold mineralization had also occurred by this period. Some human-derived vessels were still present at 5 months, but the majority of the graft vasculature had been functionally remodeled with host cells. In conclusion, clinically relevant progenitor sources for pericytes and endothelial cells can serve to generate highly functional microvascular networks for tissue engineered bone grafts.
Assuntos
Vasos Sanguíneos/fisiologia , Osso e Ossos/irrigação sanguínea , Células-Tronco Mesenquimais/fisiologia , Neovascularização Fisiológica , Pericitos/citologia , Engenharia Tecidual/métodos , Transplantes , Animais , Vasos Sanguíneos/citologia , Transplante Ósseo , Osso e Ossos/citologia , Linhagem da Célula , Humanos , Camundongos , Camundongos Endogâmicos , Modelos Animais , Osteogênese , Alicerces TeciduaisRESUMO
Mesothelium tissues such as peritoneum and pleura have a thin and strong layer of extracellular matrix that supports mesothelial cells capable of rapid healing. Decellularized porcine mesothelium was characterized for strength, composition of the matrix and biological activity. The tensile strength of the material was 40.65 +/- 21.65 N/cm. Extracellular matrix proteins collagen IV, fibronectin, and laminin as well as glycosaminoglycans were present in the material. Cytokines inherent in the extracellular matrix were preserved. Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and transforming growth factor beta (TGF-beta) were retained and the levels of VEGF and TGF-beta in the decellularized mesothelium were higher than those found in decellularized small intestinal submucosa (SIS). The decellularized mesothelium also stimulated human fibroblasts to produce more VEGF than fibroblasts grown on tissue culture plastic. Decellularized mesothelium is a sheet material with a combination of strength and biological activity that may have many potential applications in surgical repair and regenerative medicine.
Assuntos
Epitélio/metabolismo , Matriz Extracelular/metabolismo , Animais , Materiais Biocompatíveis/metabolismo , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Cães , Ensaio de Imunoadsorção Enzimática , Epitélio/ultraestrutura , Matriz Extracelular/ultraestrutura , Glicosaminoglicanos/metabolismo , Humanos , Microscopia Eletrônica de Varredura , SuínosRESUMO
Decellularized dermis materials demonstrate considerable utility in surgical procedures including hernia repair and breast reconstruction. A new decellularized porcine dermis material has been developed that retains many native extracellular matrix (ECM) proteins and cytokines. This material has substantial mechanical strength with maximum tensile strength of 141.7 +/- 85.4 (N/cm) and suture pull through strength of 47.0 +/- 14.0 (N). After processing, many ECM proteins remained in the material including collagen III, collagen IV, collagen VII, laminin and fibronectin. Glycosaminoglycans, including hyaluronic acid, were also preserved. Among several cytokines whose levels were quantified, more vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-beta) were retained within this material than in comparable decellularized dermis materials. The retention of bioactivity was demonstrated in a cell culture assay. Because this decellularized porcine dermis material both retains significant strength and has substantial biological activity, it may promote rapid integration and repair in clinical applications.
Assuntos
Indutores da Angiogênese/metabolismo , Citocinas/metabolismo , Derme/citologia , Derme/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Mitógenos/metabolismo , Animais , Células Cultivadas , Meios de Cultivo Condicionados , DNA/metabolismo , Derme/ultraestrutura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Glicosaminoglicanos/metabolismo , Humanos , Teste de Materiais , Compostos Orgânicos/metabolismo , Coloração e Rotulagem , Sus scrofa , Suturas , Resistência à TraçãoRESUMO
BACKGROUND: The objectives of this study were to evaluate the efficacy of poly(glycerol) sebacate (PGS) films for the prevention of visceroparietal peritoneal (VP) adhesions and demonstrate the ease of laparoscopic PGS film placement. Peritoneal adhesions occur in nearly 95% of all abdominal operations. VP adhesions can cause serious postoperative complications. The interposition of a barrier between damaged peritoneal areas during re-epithelialization has been shown to prevent adhesion formation. Current barrier products have serious drawbacks, including poor degradability, variable efficacy, and difficult handling characteristics. METHODS: The efficacy of PGS films to prevent VP adhesions was evaluated in a rat peritoneal adhesion model. The animals were evaluated for the presence of VP adhesions at 3, 5, and 8 weeks. The laparoscopic applicability of PGS films was demonstrated by placement into a juvenile porcine abdomen using standard laparoscopic equipment and techniques. RESULTS: A statistically significant 94% reduction in VP adhesion formation rate was observed between control animals (75%) and animals with a PGS film barrier (4.8%). PGS films were easily placed in the juvenile porcine abdomen and could be readily repositioned without material loss or tissue damage. CONCLUSION: PGS films possess a unique combination of properties, including biocompatibility, resorbability, and ease of handling. PGS barrier films were shown to be efficacious in reducing VP adhesions in the rat model. They also can be placed using standard laparoscopic techniques. These promising results suggest that PGS films will be effective barriers to adhesion formation for patients undergoing open and laparoscopic abdominal operations.