Bone tissue engineering utilizing adult stem cells in biologically functionalized hydrogels
Dosier, Christopher R.
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Repair of large bone defects remains a clinical challenge for orthopedic surgeons. Current treatment strategies such as autograft and allograft are limited by the amount of available tissue in the case of the former, and failure of revascularization effecting engraftment in the case of the latter. Tissue engineering offers an alternative approach to this challenging clinical problem. The general principle of tissue engineering for bone regeneration prescribes delivery of osteoinductive factors to induce an endogenous response within the host to repair a defect that will not normally heal. One such tissue engineering approach is cell based therapy and this is attractive in the cases of patients with a lack of endogenous osteoprogenitors cells due to volumetric loss of tissue/ageing. Stem cell therapy has emerged as a possible alternative to current treatment modalities, however many challenges to clinical translation remain. Central to these challenges for bone tissue engineering are lingering questions of which cells to use and how to effectively deliver those cells. The goal of this thesis was to elucidate more effective ways to enhance bone repair utilizing adult stem cells. First, we investigated adipose derived stem cells (ADSCs) as a viable cell source for bone tissue engineering. Upon isolation, adipose derived stem cells are a heterogeneous population of multipotent cells predisposed to adipogenic differentiation. We developed an enrichment protocol that demonstrated the osteogenic potential of ADSCs can be enhanced in a dose dependent manner with resveratrol, which had been demonstrated to up-regulate Runx-2 expression. This enrichment strategy produced an effective method to enhance the osteogenic potential of ADSCs while avoiding cell sorting and gene therapy techniques, thus bypassing the use of xenogenic factors to obtain an enriched source of osteoprogenitor cells. This protocol was also used to investigate differences between human and rat ADSCs and demonstrated that rat ADSCs have a higher osteogenic potential than human ADSCs in vitro. The second major thrust of this thesis was to develop an injectable hydrogel system to facilitate bone formation in vivo. Both a synthetic and a naturally based polymer system was investigated, the results of which demonstrated that the naturally based alginate hydrogel was a more effective vehicle for both cell viability in vitro and bone formation in vivo. Our results also demonstrated that despite the ability to increase the osteogenic potential of ADSCs in vitro with resveratrol treatment, this was insufficient to induce bone formation in vivo. However, the inclusion of bone marrow mesenchymal stem cells (BMMSCs) in BMP-2 functionalized alginate hydrogels resulted in significantly greater mineralization than acellular hydrogels. Finally, the effect of timing of delivery of therapeutics to a non-healing segmental bone defect in the femur was investigated. We hypothesized that delivery of biologics after the initial inflammation response caused by injury to the host tissue would result in greater regeneration of tissue in terms of newly formed bone. Contrary to our initial hypothesis, these experiments demonstrated that delayed implantation of therapeutics has a detrimental effect on the overall healing response. It was, however, demonstrated that the inclusion of BMMSCs results in greater bone volume regenerated in the defect site over acellular hydrogels. In conclusion, this work has rigorously investigated the use of adipose derived stem cells for bone tissue engineering, and further produced an injectable hydrogel system for stem cell based bone tissue engineering. This work also demonstrated that the inclusion of adult stem cells, specifically BMMSCs, can enhance the regeneration response in a non-healing bone defect model relative to acellular hydrogel.