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Skeletal shape depends on the transmission of contractile muscle forces from tendon to bone across the enthesis. Loss of muscle loading impairs enthesis development, yet little is known if and how the postnatal enthesis adapts to increased loading. Here, we studied adaptations in enthesis structure and function in response to increased loading, using optogenetically induced muscle contraction in young (i.e., growth) and adult (i.e., mature) mice. Daily bouts of unilateral optogenetic loading in young mice led to radial calcaneal expansion and warping. This also led to a weaker enthesis with increased collagen damage in young tendon and enthisis, with little change in adult mice. We then used RNA sequencing to identify the pathways associated with increased mechanical loading during growth. In tendon, we found enrichment of glycolysis, focal adhesion, and cell-matrix interactions. In bone, we found enrichment of inflammation and cell cycle. Together, we demonstrate the utility of optogenetic-induced muscle contraction to elicit in vivo adaptation of the enthesis.

Effective tendon regeneration following injury is contingent on appropriate differentiation of recruited cells and deposition of mature, aligned, collagenous extracellular matrix that can withstand the extreme mechanical demands placed on the tissue. As such, myriad biomaterial approaches have been explored to provide biochemical and physical cues that encourage tenogenesis and template aligned matrix deposition in lieu of dysfunctional scar tissue formation. Fiber‐reinforced hydrogels present an ideal biomaterial system toward this end given their transdermal injectability, tunable stiffness over a range amenable to tenogenic differentiation of progenitors, and capacity for modular inclusion of biochemical cues. Here, tunable and modular, fiber‐reinforced, synthetic hydrogels are employed to elucidate salient microenvironmental determinants of tenogenesis and aligned collagen deposition by tendon progenitor cells. Transforming growth factor β3 drives a cell fate switch toward pro‐regenerative or pro‐fibrotic phenotypes, which can be biased toward the former by culture in softer microenvironments or inhibition of the RhoA/ROCK activity. Furthermore, studies demonstrate that topographical anisotropy in fiber‐reinforced hydrogels critically mediates the alignment ofde novocollagen fibrils, reflecting native tendon architecture. These findings inform the design of cell‐free, injectable, synthetic hydrogels for tendon tissue regeneration and, likely, that of a range of load‐bearing connective tissues.

Synthetic hydrogels represent an exciting avenue in the field of regenerative biomaterials given their injectability, orthogonally tunable mechanical properties, and potential for modular inclusion of cellular cues. Separately, recent advances in soluble factor release technology have facilitated control over the soluble milieu in cell microenvironments via tunable microparticles. A composite hydrogel incorporating both of these components can robustly mediate tendon healing following a single injection. Here, a synthetic hydrogel system with encapsulated electrospun fiber segments and a novel microgel‐based soluble factor delivery system achieves precise control over topographical and soluble features of an engineered microenvironment, respectively. It is demonstrated that three‐dimensional migration of tendon progenitor cells can be enhanced via combined mechanical, topographical, and microparticle‐delivered soluble cues in both a tendon progenitor cell spheroid model and an ex vivo murine Achilles tendon model. These results indicate that fiber reinforced hydrogels can drive the recruitment of endogenous progenitor cells relevant to the regeneration of tendon and, likely, a broad range of connective tissues.