Development of an Osteoinductive Bone Graft




Sule, Anupam A.


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Bone is a unique tissue that serves multiple functions. One of its unique features is the ability to heal by formation of new bone, whereas most other tissues undergo the process of scar formation. When a large amount of bone is lost the only treatment available is the use of bone grafts. Multiple bone graft substitutes are being developed to address the shortage of autologous bone graft. 3-D models are being developed to further our understanding of the cellular processes taking place in vivo. In this study I examined the strategy of designing a 3-Dmodel of hard tissue and a potential bone graft substitute using collagen type I and several different porous scaffolds. Factors influencing collagen gel contraction by human mesenchymal stem cells (hMSC) during the process of osteogenic differentiation were studied and it was shown that collagen type I gels prepared in accordance with our patented technology contract far less than any other collagen gels reported in literature. The validity of MTT assay to track proliferation of hMSC in various 3-D matrices was established and allowed me to show that human mesenchymal stem cells (hMSC) proliferated, differentiated along an osteogenic lineage and mineralized the extracellular matrix (ECM). Higher cell seeding density and greater serum concentration in the culture medium, caused increased collagen type I gel contraction. Late passage cells and osteoblasts caused a greater collagen type I gel contraction than undifferentiated early passage hMSC. hMSC that had been transduced to constitutively express human telomerase reverse transcriptase (hTERT), and which had thereby acquired an extended in vitro life span (telomerized hMSC or TMSC), contracted the collagen gel lesser than hMSC. A Collagen type I Gel - Collagen type I foam Scaffold combination (CGCS) was investigated as a 3-D in vitro model to allow extrapolation of soft tissue results to those characteristic of hard tissue. Deep penetration of MSC into the CGCS with uniform distribution was achieved by the use of collagen type I gel, as the cell carrier. Collagen type I gel improved seeding efficiency and facilitated retention of cells that penetrated deep into the scaffold. Longterm survival, proliferation, viability and in situ osteogenic differentiation within the CGCS were demonstrated. A model that demonstrated migration of cells in and out of CGCS was assembled and tested. A need for the presence of fibrillar collagen gel for mineralization process to take place highlighted the benefit of adding collagen gel to the 3-D models. Porous Beta-tricalcium phosphate (-TCP) was used as the scaffold and impregnated with collagen gel to generate Collagen Gel Impregnated Porous Scaffolds (CGIPS). Highly efficient seeding of the cells throughout the porous scaffold was attained with collagen gel. hMSC proliferated in CGIPS without contracting the collagen gel. Cells could migrate into CGIPS and mineralized the ECM when cultured in vitro under osteogenic differentiation conditions. CGIPS allowed the application of pressure and hMSC responded to mechanical force by a change in proliferation. hMSC xenotransplanted into immunocompetent rats survived for a month and expressed markers of osteogenic differentiation. While cells alone improved vascularization of the implants, they did not improve mineralization. Presence of collagen gel alone allowed for faster invasion of cells into the implanted TCP and improved radiodensity but did not affect vascularization. A combination of cell and gel within the TCP (CGIPS) was necessary to improve all the measured varialbes (tissue invasion, vascularization, mineralization and radioopacity). Thus biocompatibility, greater vascularization and enhanced mineralization of CGIPS implants established the foundation to proceed with large animal bone defect model studies utilizing CGIPS in the future. I established that CGIPS could deliver small molecules into the surrounding milieu by a process of simple diffusion. A rapid intital burst followed by a slow sustained release was observed when collagen gel containing EphrinB2-Fc clusters was incorporated ointo CGIPS. The released EphrinB2-Fc was physiologically functional and increased hMSC proliferation and chemotaxis. CGIPS inhibited the growth of Methicillin resistant Staphylococcus aureus when vancomycin was incorporated into the CGIPS. Thus the potential of CGIPS to serve as a drug delivery device was demonstrated. This work has provided the scientific foundation for use of CGIPS as bone graft substitute and 3-D model of osteogenesis. In this research study, a number of challenges were solved and questions answered, and the applications of the proposed strategy formulated. However, as is frequently the case many more avenues of future research have been exposed and a variety of new questions posed to be pursued and answered in future.studies.