: The proposed tissue engineering studies are motivated by the medical need for biologically based, functional tissues for transplantation. An important issue with respect to engineering tissues in vitro is to understand the role of environmental factors on the process of tissue formation. Controlled in vitro studies of tissue development in three-dimensional culture can improve our fundamental understanding of regulatory signals directing progenitor cell differentiation, affecting the structure and function of engineered tissues formed, such as Anterior Cruciate Ligaments (ACL). In addition, bioreactor systems which allow the stimulation of the cells growing on these matrices by physical forces that are physiological in nature (e.g., tension and torsion in the case of the ACL) could be extended to other skeletal tissues and become a valuable tool for basic biomedical research. Specifically, we propose to test the hypothesis that mechanical forces that are physiological in nature, intensity and frequency for native ACLs will direct human bone marrow stem cell differentiation into ligament-forming cells and result in the in vitro formation of functional equivalents of native ACLs. Our objectives are to (a) gain fundamental insight into the relationships between mechanical and biochemical stimulation and the differentiation of human bone marrow stem cells (hBMSCs) into ligament fibroblasts expressing biochemical and genetic markers characteristic for cells in native ligaments, and (b) to engineer ligament-like structures with appropriate mechanical properties starting from hBMSCs. In our experimental plan we will study the culture hBMCSs on 3-dimensional crosslinked collagen fiber scaffolds in a bioreactor designed to provide a highly controlled biochemical and mechanical environment. The study will focus on elucidating the relationships between specific biochemical factors oxygen tension, mechanical regulatory signals and growth period, related to (1) cellular differentiation and (2) ligament tissue structure and function. We will build upon our Preliminary Data that demonstrate: (a) hBMSCs undergo selective differentiation to ligament cells due to mechanical forces in the absence of specific exogenous growth factors, and (b) ligament structures are formed during the process. We expect that different combinations of biochemical factors and applied mechanical stresses will influence the rate and extent of differentiation of hBMSCs into ligament-like cells leading to ligament structures in a manner dependent not only on the presence of the specific individual factors, but also on their interactions. The evaluation of the responses will be based on statistical analysis of time-dependent (0, 7, 14 and 28 day) changes in cell proliferation (DNA content), upregulation of the ligament-specific mRNA transcripts (real time RT-PCR). production of collagen type I via western blot and immunohistochemical analysis of structural features, and the mechanical properties
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