Type of Document	Dissertation
Author	Grayson, Warren L
Author's Email Address	wgrayson@eng.fsu.edu
URN	etd-06302005-171755
Title	Reconstructing the In Vivo Environment for the Development of Tissue-Engineered Constructs from Human Mesenchymal Stem Cells
Degree	Doctor of Philosophy
Department	Chemical Engineering, Department of
Advisory Committee	Advisor Name Title Teng Ma Committee Chair Bruce Locke Committee Member Soonjo Kwon Committee Member Thomas Keller III Committee Member
Keywords	Tissue Engineering Mesenchymal Stem Cells Hypoxia 3D
Date of Defense	2005-05-23
Availability	unrestricted
Abstract Human mesenchymal stem cells (hMSC) are obtained primarily from adult bone marrow and hold tremendous promise for use in a number of clinical applications. One approach combines hMSC with 3D scaffolds to develop tissue constructs, which can be directly implanted into the pathological site. However, little work has been done to understand the developmental patterns of stem cells in 3D constructs. Previous research has shown that cells cultured in 3D substrates exhibit markedly different contact structures and regulatory controls than cells grown on conventional 2D culture dishes. Furthermore, the spatial organization of cells has significant effects on their growth patterns and tissue formation. In this study, poly(ethylene terephthalate) (PET) matrices were treated to achieve similar surface properties to conventional 2D tissue culture plastics. In addition to comparing hMSC behavior in 2D and 3D systems, different 3D culture conditions were obtained by varying oxygen concentrations or applying media flow. HMSC cultured in 3D scaffolds proliferated significantly slower than 2D controls. However, they secreted and embedded themselves in an extensive ECM network, and demonstrated significant ability to organize their ECM proteins into aligned fibrils. They also regulated their expression of adhesion proteins consistent with the ECM structure indicating that cells in 3D can reciprocally interact with their surroundings and are capable of remodeling their microenvironment. Constructs grown at 2% oxygen exhibit a prolonged proliferation phase. This gives rise to constructs with similar ECM organization but higher cell-densities. HMSC also demonstrated improved ability to retain stem-cell characteristics and exhibited enhanced differentiation ability when cultured under low oxygen conditions. Continuously perfusing hMSC constructs with fresh media allowed these cells to proliferate exponentially. However, the ECM in perfusion constructs was disorganized, there was considerably less protein production, and there was a drastic change in the nuclear morphology from elongated to spherical. Hence perfusion systems facilitated superior cell growth, but affected cell phenotype and the physical and biological properties of the resulting constructs. These studies approximate various elements of the native hMSC environment and emphasize the need for careful consideration of culture parameters in order to develop functional tissue-engineered constructs for clinical use.
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