Tendon Regeneration: Roles of Growth Factors and Phenotypic Diversity in Tendon Stem Cells
Rajpar, Ibtesam Mohamed Husein
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Tendon injuries significantly impact quality of life and are often career ending. Mesenchymal stem cell (MSC) therapy is known to augment intrinsic tendon healing, however, little is known of the stem cells endogenous to tendon, the microenvironmental cues that induce tendon differentiation, and whether individual cells in an inflammatory milieu respond differently to these cues. To address these questions, a three-dimensional tenogenesis assay was developed as an efficient and reproducible metric of cellular capacity to differentiate toward tendon. In contrast to more complex assays of tenogenesis, this design incorporates a simple apparatus using commercially available plasticware for the application of uniaxial static strain in in a type I collagen cell-seeded hydrogel construct. Tendon-related gene expression, glycosaminoglycan levels, elongated cell morphologies and parallel cell alignments are enhanced with BMP-12 induction over ten days of culture. This dissertation provides novel insight to the roles of growth factors in MSC tenogenesis. Tendon healing in vivo is dependent on endogenous tendon stem cells (TSC) that mediate the inflammatory response to injury and promote synthesis of collagen and matrix remodeling, among other extracellular processes. Recent evidence suggests that these cells exist on a spectrum of differentiation potencies, and may be differently committed to the tendon fate. Individual stem cells were isolated from the tendon, and their capacities for proliferation, tri-lineage differentiation and tenogenesis were evaluated. Three distinct TSC phenotypes were revealed, and significant, positive correlations were found in quadra-differentiation potency (toward four lineages) and the expression of a strong, composite tendon phenotype. These studies have important implications in the current standard-of-care in regenerative therapies for tendon. Our benchtop tenogenesis assay can be used to determine the therapeutic potential of allogeneic MSC lines and MSCs from novel sources for 'off-the-shelf' treatments. Our study of TSCs lends valuable insight to the diversity of cell phenotypes found in tendon, and the potential contributions of each phenotype to tendon healing and homeostasis. These results further strengthen the status of tendon as a superior source of stem cells for tendon repair.
General Audience Abstract
Tendons are fibrous, elastic bands of collagen that connect muscles to bones and are essential to movement and proper functioning of the skeletal system. Weight-bearing tendons like the Achilles in humans and superficial digital flexor tendons in horses are particularly prone to damage and degeneration with overuse and/or aging. Bone marrow-derived stem cell treatments have shown promise in the reduction of pain and inflammation, and restoration of native tendon structure and function in cases of severe tendon injuries. However, the roles of stem cells in tendon healing, particularly their ability to transition to cell types native to tendon and integrate with an environment distinct from their own is unknown. Culturing of stem cells in three dimensional (3D) environments has enabled us to identify and understand the biochemical and mechanical signals that trigger stem cell transitions to tendon cells in tendons, but currently available 3D culture systems are complex and inefficient. In this dissertation we have developed a cost-effective and high throughput 3D culture system to assay the potential of stem cells to form tendon cells and composite tendon-like tissues. Toward this, we have also optimized the effects of known tendon proteins on the tendon fate in 3D culture of stem cells. Like most adult tissues, the tendon encompasses an in-house repository of stem cells. Tendon stem cells (TSCs) are primarily responsible for the inflammatory and reparative responses to tendon injury. Recent evidence suggests that TSCs are diverse in character, and differ from each other in their ability to form cells and tissues of fat, bone and cartilage. In this work, we provide evidence that TSCs are also differently committed to forming tendon tissue, and moreover that significant inter-relationships among gene expression patterns in these cells directly contribute to cultural diversity. In sum, our results provide novel insight to the roles of stem cells in tendon healing, particularly their response to subtle changes in their biochemical environment, and the contributions of individual cells in a milieu to a holistic reparative response.
- Doctoral Dissertations