Title page for ETD etd-11082010-123437

Type of Document Dissertation
Author Shaffer, Robyn Denise
Author's Email Address robyns@vt.edu
URN etd-11082010-123437
Title Complementary Strategies to Promote Mesenchymal Stem Cell Differentiation for Ligament Tissue Engineering
Degree PhD
Department Biomedical Engineering
Advisory Committee
Advisor Name Title
Dahlgren, Linda A. Committee Co-Chair
Goldstein, Aaron S. Committee Co-Chair
Huckle, William R. Committee Member
Van Dyke, Mark E. Committee Member
Yoo, James Y. Committee Member
  • tendon
  • regenerative medicine
  • electrospun
  • nanofibers
  • topography
  • nucleofection
Date of Defense 2010-11-01
Availability restricted
Anterior cruciate ligament (ACL) ruptures and tears are significant orthopedic problems that result in discomfort and limited mobility. Fully functional tissue engineered ligament replacements are promising alternatives to current graft choices for repair of ACL disruptions. The cell-based approach to construct engineered ligament grafts presented herein involves the culture of mesenchymal stem cells (MSC) on biodegradable, fibrous polymeric scaffolds to promote tissue formation. Multipotent MSCs are advantageous because of their in vitro proliferative capacity and ease of harvest; however; the promotion of MSC differentiation into mature fibroblasts and subsequent extracellular matrix (ECM) development is unknown. The proposed studies utilized three complementary methods to promote differentiation of MSCs: scaffold architecture, mechanical stretch and over-expression of the transcription factor, scleraxis. First, elastomeric scaffolds were fabricated by electrospinning a segmented poly(esterurethane urea) with variations in fiber diameter and fiber alignment. Primary mesenchymal stem cells and the mesenchymal stem cell line, C3H10T1/2, were seeded on these scaffolds and assumed spindle-shaped morphologies and oriented with the direction of fiber alignment. Fiber diameter affected cellular responses, including the expression of ECM genes (e.g. collagen type 1 and decorin) which were elevated on smaller mean fiber diameter scaffolds initially. However, scleraxis gene expression was greatest on larger mean fiber diameter scaffolds at the end of two weeks. Second, cyclic stretch was applied to C3H10T1/2 cells on semi-aligned scaffolds using a novel bioreactor. Cell attachment was verified during and after the application of mechanical stress by confocal microscopy. Cyclic stretch induced cells to assume a highly elongated morphology; however ECM gene expression changes were moderate. Third, forced constitutive expression of scleraxis was accomplished by nucleofection of C3H10T1/2 cells. Transient mRNA expression, accumulation of the gene product in the cell nucleus, and cell death were observed. Future work will seek to refine the experimental methods, including the development and testing of an inducible scleraxis transgene and the application of longer periods of mechanical stimulation. Finally, these complementary approaches may be combined to further extend this work in pursuit of directed differentiation of stem cells and the ensuing generation of a robust tissue graft.
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