Acute tendon rupture or degeneration is a common clinical problem, with tendinopathy listed as the fourth leading cause of missed work in the United States among non-fatal disease. While surgical repair remains the gold standard treatment, long-term outcomes remain poor due to the non-regenerative healing capacity of tendon. The main limitation impeding adequate tendon repair is the formation of fibrous scar tissue with no regeneration. To determine the cellular and molecular mechanisms of tendon regeneration, our lab has established the neonatal mouse as a model of tendon regeneration. In this dissertation, I show that regeneration is mediated by recruitment and differentiation of tenocytes to the wound site with full restoration of functional properties;​ these features are absent in adults, which instead heal by fibrotic scar. While there are likely intrinsic (tenocyte) and extrinsic (environment) differences that impact the regenerative vs. scar healing programs, I propose that neonatal immunity is one major factor that directly establishes a pro-regenerative environment during tendon healing. In this dissertation I test this hypothesis using mouse genetics, next generation sequencing, and heterochronic/homochronic cell transfer approaches.

Shortly after injury, I observe that neonatal mice exhibit a rapid transition to a type 2 anti-inflammatory response while adult mice exhibit a pro-inflammatory type 1 response in the injured tendon. These differences manifest in macrophage polarization, where the contribution of inflammatory (type 1) and anti-inflammatory (type 2) macrophages are known drivers of fibrotic versus regenerative healing, respectively. Among lymphocytes, the recruitment of regulatory T-cells (Tregs) was notably increased in injured neonatal versus adult tendon. Since Tregs have emerged as non-conventional regulators of wound healing and can direct both immune polarization as well as orchestrate tissue repair, I hypothesize that neonatal Tregs are key drivers of neonatal immune polarization that permit successful tendon regeneration. I further hypothesize that adult inflammation is restrictive of tendon regeneration and that rejuvenation of adult inflammation with neonatal Tregs can restore tendon regeneration.

Using the neonatal mouse as a regenerative model of tendon healing, I show that ablation of Tregs following tendon injury impairs functional recovery with limited recruitment of tenocytes and robust infiltration of αSMA+ cells, indicative of fibrotic scar. I next performed transcriptional profiling of Tregs recruited to the injured tendon in neonatal and adult mice to identify gene signatures that may explain differences in neonatal and adult Treg response. Surprisingly, neonatal Tregs express a type 2 antiinflammatory signature that was absent in adult Tregs. Using heterochronic and homochronic adoptive transfer experiments of human and mouse Tregs into immunodeficient neonatal and adult hosts, I show that neonatal Tregs are uniquely able to permit tendon regeneration and macrophage polarization. Furthermore, I demonstrate that adoptive transfer of neonatal Tregs can improve tendon repair in adult mice.

Collectively, the data presented in this dissertation demonstrate a critical role for the immune environment in tendon repair that can be both permissive (neonates) or restrictive (adults) of tendon regeneration. From these approaches I demonstrate an age-dependent role for Tregs in supporting reparative inflammation, and identify a neonatal Treg signature that can be used to select hits that can be screened using FDAapproved drug libraries. Moreover, these approaches highlight the importance of the immune response in tendon repair, and the need for further studies to elucidate reparative inflammation. Success in these endeavors will undoubtedly lead to improved tendon therapies and quality of life for patients.