Browsing by Author "Elizabeth G. Loboa, Committee Chair"

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Computational and Experimental Analyses of Bone and Adult Stem Cell Mechanobiology
(2010-03-07) Pfeiler, Terry Wayne; Elizabeth G. Loboa, Committee Chair
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.
Functional Bone Tissue Engineering using Human Mesenchymal Stem Cells and Polymeric Scaffolds
(2007-12-08) Sumanasinghe, Ruwan Deepal; Behnam Pourdeyhimi, Committee Member; Elizabeth G. Loboa, Committee Chair; Martin W. King, Committee Co-Chair; Nancy Monteiro-Riviere, Committee Member; Alan E. Tonelli, Committee Member
Microarray Analysis of Human Adipose-derived Adult Stem Cells Undergoing Osteogenic differentiation in the Presence and Absence of 10% Uniaxial Cyclic Tensile Strain
(2010-04-20) Charoenpanich, Adisri; Elizabeth G. Loboa, Committee Chair; Albert J. Banes, Committee Member; Greg McCarty, Committee Member
Human adipose derived stem cells (hASCs) have shown great potential for bone tissue engineering. However, the molecular mechanisms underlying this potential are not yet known, in particular the effects of mechanical loading which is known to play a pivotal role in bone remodeling and bone fracture repair. This study aimed to develop understanding of osteogenic differentiation and mechanical response properties of hASCs for potential use in optimizing and controlling hASCs for bone tissue engineering or cell based therapy. Gene expression profiles of hASCs were examined using cDNA microarray with 47,000 gene identifiers. Three dimension collagen type I matrix was used as a culture system for mimicking the collagenous micro-environment of bone tissue. To investigate the regulation of osteogenesis of hASCs, gene expression profiles of hASCs cultured in osteogenic induction media were compared to proliferating hASCs in complete growth media for 14 days. The effect of mechanical loading on hASCs during osteogenesis was determined by comparing between hASCs cultured in osteogenic induction media in the presence and absence of 10% uniaxial cyclic tensile strain for up to 14 days. 847 genes were significantly modulated by osteogenic induction media, and 147 genes were significantly modulated by 10% uniaxial cyclic tensile strain. For osteogenesis of hASCs, 95 canonical pathways were identified as affected with the details of upregulated and downregulated genes for continuing molecular characterization. Some pathways were examined and showed the potential role in osteogenesis of hASCs including Wnt/Î²-catenin signaling, transforming growth factor- Î² (TGF- Î²) signaling, platelet derived growth factor (PDGF) signaling, and insulin-like growth factor 1 (IGF-1) signaling. To validate the microarray data, RT-PCR was performed to confirm changes in corin mRNA expression levels. To identify the effect of 10% uniaxial cyclic tensile strain, canonical pathway, function, and network analyses were performed. The function analysis showed the potential role of 10% uniaxial cyclic tensile strain in angiogenic induction on hASCs during osteogenesis. Twelve canonical pathways were provided with details for further analysis. Network analysis indicated interleukin 1 receptor antagonist (IL1RN), and suppressor of cytokine signaling 3 (SOCS3) as the potential key factors in response to mechanical loading in hASCs. The results of this study indicate multiple candidate genes and pathways that may play a role in the response of hASCs to osteogenesis and cyclic tensile strain, thus providing further understanding for the development of tissue-engineered bone using these relatively accessible and abundant adult stem cells.