Characterization of Tissue Development 'in Vivo' and Elucidation of the Potential of Embryonic Developmental Factors to Guide Tenogenesis 'in Vitro

Tendons and ligaments (TL) are structurally and functionally similar tissues, critical to musculoskeletal function, and are frequently injured with a poor innate healing ability. Current treatments for injured TL have several shortcomings and this has led to efforts to engineer replacement TL in vitro. Previous attempts have had limited success, and our approach is to inform tissue engineering strategies with embryonic TL development. We hypothesized that this approach would be enhanced by furthering our understanding of the in vivo development of TL, by characterizing how phenotypic aspects of differentiating TL cells vary by developmental stage and anatomic origin, and by investigating the potential utility of putative tenogenic factors to differentiate MSCs in vitro. To further our understanding of in vivo TL development, we characterized the spatiotemporal distribution of elastogenic proteins and fibers in a spinal ligament in the mouse from embryonic development through postnatal maturation. This work provided new insight into the elaboration of elastic matrix constituents in a ligament and significantly enhanced our knowledge of the temporal aspects of elastogenesis. To characterize aspects of the developing TL cell phenotype in vitro, we harvested primary limb and axial embryonic TL progenitor cells (TLPC) from transgenic mice from different stages of embryonic development and treated them with developmental factors (mechanically loading, TGFbeta2 or FGF4). We then compared cellular responses by analyzing the expression of marker genes (Scx, Tnmd, TGFbeta2, Col I, and Eln). This characterization profiled phenotypic variations by stage and anatomic origin and provided benchmarks for tenogenically differentiating cells in vitro. Moreover, the resolution of the discrete effects of factors imputed to guide TL development in vivo elucidated their tenogenic potential in vitro. Lastly, we advanced a TL engineering strategy by investigating the potential of developmental factors to differentiate MSCs and TLPCs by comparing their expression and regulation of marker genes. We found that substantially inducing Scx expression in MSCs may induce differentiation toward an TLPC lineage, we identified potential benchmarks for differentiation and concluded that TGFbeta2 and mechanical loading are potentially tenogenic factors. Furthermore, we speculate that MSCs may undergo tenogenesis when treated with embryonically tenogenic factors.