Tendons are dense connective tissues that play an integral role in transmitting mechanical forces from the muscle to the bone, stabilizing joints, and enabling locomotion of the skeleton. In adults, tendon tears and ruptures heal poorly by forming disorganized scar tissue that rarely recovers the original tissue structure and mechanical properties. Current clinical treatment options, including surgical repair and orthobiologics, are unable to attenuate this scarmediated/canonical healing process and consequently lead to diminished function, mobility, and quality of life for patients. A major hurdle to the development of effective therapeutics for tendon injuries is that the biological mechanisms underlying regenerative, or scarless healing, are unknown.

To address this challenge, the super-healer Murphy Roths Large (MRL/MpJ) mouse strain has emerged as a promising tool to interrogate the cellular and molecular drivers of mammalian tendon regeneration. Mounting evidence implicates the local tissue environment consisting of the resident tendon cells and provisional extracellular matrix (ECM), a temporary bioactive matrix produced upon injury, as the primary driver of the vastly improved healing in MRL/MpJ tendons following an acute laceration injury. However, it remains unclear whether the scarless healing capacity unique to the MRL/MpJ tendon environment possesses the therapeutic utility to ameliorate otherwise canonical healing outcomes. Accordingly, the overarching goal of this dissertation was to harness MRL/MpJ tendon-derived biological cues and assess their capability to promote a regenerative MRL/MpJ-like cellular phenotype in canonical healing tendon cells.

To assess the influence of MRL/MpJ tendon-derived ECM constituents on canonical healing tendon cell behavior, Chapter 2 outlines the development of a tissue decellularization method to enable in vitro functional evaluation. Our findings indicate that canonical healing tendon cells cultured on decellularized MRL/MpJ provisional ECM-coated substrates display increased cell elongation, cellular protrusion formation, and proliferation that are consistent with scarless healing observations in vivo. Taken together, these results illustrate the promising therapeutic potential of natural MRL/MpJ tendon-derived biochemical signal for effective healing outcomes.

Building on Chapter 2 and the tools and data outlined, Chapter 3 establishes the capability of MRL/MpJ tendon cell-secreted soluble factors, or the secretome, in combination with the decellularized MRL/MpJ provisional ECM to phenotypically reprogram canonical healing cells towards regenerative behavior. Moreover, we show that a recombinant panel of MRL/MpJ-enriched structural and soluble proteins recapitulate the effects elicited by MRL/MpJ-derived components on rodent tendon cells of different species.

Lastly, Chapter 4 investigates the sex-dependent behavior of MRL/MpJ tendon cells using 2D and 3D culture systems to elucidate cellular processes universal to scarless tendon healing, thus informing the design of more equitable therapeutic approaches.

Collectively, the studies in this dissertation illustrate the untapped mechanistic and therapeutic of natural MRL/MpJ tendon- and cell-derived biological cues in promoting effective healing outcomes, thereby setting the foundation to systematically interrogate the role of key protein regulators in mammalian tendon regeneration.