Human bone marrow and adipose derived adult stem cells have shown great promise as a source of expandable and differentiable cells for tissue engineering and regenerative medicine. However, a more complete understanding of the optimal in vitro culture conditions is required to create functional engineered tissue. Especially key in the context of musculoskeletal tissue is the in vitro generation of nascent tissue with appropriate material properties for withstanding in vivo loading are the combined mechanical and chemical stimuli used to culture adult stem cells. Previous studies have shown that mechanical loading induces differentiation of human mesenchymal stem cells (hMSCs) and mesenchymal tissue into tissues such as bone, fibrous tissue, cartilage, and smooth muscle cells. More recently, human adipose derived adult stem cells (hASCs) have shown promise for similar tissue engineering applications. Fluid shear stress is believed to be one of the primary stimuli for osteocytes in the maintenance of mature bone, and tensile strain has been shown to induce the formation and repair of bone from mesenchymal tissue. However, the proper range of local stresses and strains required for stem cell-induced bone formation in vitro have yet to be determined.This body of work examined the application of different methods of external loading on human adult stem cells to induce their osteogenic differentiation. Both experimental and computational analyses were completed in order to provide a detailed understanding of cellular response to loading, and the local stresses and strains placed on cells during loading. Fluid shear stresses were applied to hASCs on and within a porous 3D scaffold to measure upregulation of osteogenic genetic markers. Cyclic tensile strain was applied to hASCs in 2D culture and calcium deposition was quantified as a measure of mRNA expression of osteogenic differentiation. Computational models were created to determine the range and location of different strains applied to the 2D substrate. Three dimensional finite element models were created for hMSC-seeded collagen gels subjected to cyclic tensile strain for bone tissue engineering, in order to determine the local strains most effective in inducing hMSC osteogenesis. Finally, in order to optimize the rapid creation of computational models of bone, different methods of automated finite element mesh generation were studied and validated via mechanical testing by four-point bending. The results of this research show promising initial results for application of 3 dynes/cm2 fluid shear stress on hASCs cultured on a novel three dimensional scaffold. In two-dimensional monolayer culture, local cyclic tensile strains of 7.7% to 20.4% were shown to induce highest calcium deposition by hASCs after 14 days. Human MSCs in three-dimensional collagen gel culture were modeled with finite element analysis and local tensile strains of 16.8% were calculated to upregulate BMP2 mRNA. Additionally, strains of 21.8% were shown to disrupt actin cytoskeletal alignment. Finally, a nonuniform voxel-based finite element mesh generation was shown to accurately predict physiological strains in a long bone.This body of work demonstrates the significance of the chemical and mechanical stimuli placed on adult stem cells during in vitro culture. It examines the mechanical forces necessary to induce osteogenesis of human adipose and bone marrow derived stem cells, and suggests ranges of mechanical stimuli that show promise for bone tissue engineering.
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Computational and Experimental Analyses of Bone and Adult Stem Cell Mechanobiology
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