The tendon healing process is regulated by the coordinated interaction of multiple cell types and molecular processes. However, these processes are not well-defined leading to a paucity of therapeutic approaches to enhance tendon healing. Scleraxis-lineage (ScxLin) cells are the major cellular component of adult tendon and make time-dependent contributions to the healing process. Prior work from our lab and others suggests heterogeneity within the broader ScxLin population over the course of tendon healing; therefore delineating the temporal and spatial contributions of these cells is critical to understanding and improving the healing process. In the present study we utilize lineage tracing of the adult ScxLin population to determine whether these cells undergo cellular activation and subsequent myofibroblast differentiation, which is associated with both proper healing and fibrotic progression in many tissues. We show that adult ScxLin cells undergo transient activation in the organized cellular bridge at the tendon repair site, contribute to the formation of an organized neo-tendon, and contribute to a persistent myofibroblast population in the native tendon stubs. The mechanisms dictating this highly specialized spatial response are unknown. We therefore utilized spatial transcriptomics to better define the spatio-molecular program of tendon healing. Integrated transcriptomic analyses across the healing time-course identifies five distinct molecular regions, including key interactions between the inflammatory bridging tissue and highly reactive tendon tissue at the repair site, with adult ScxLin cells being a central player in the transition from native tendon to reactive, remodeling tendon. Collectively, these data provide important insights into both the role of adult ScxLin cells during healing as well as the molecular mechanisms that underpin and coordinate the temporal and spatial healing phenotype, which can be leveraged to enhance the healing process.

Obesity and type II Diabetes Mellitus (T2DM) have substantial pathological effects on tendon homeostasis, including loss of collagen organization and increased risk of tendon rupture. Moreover, following rupture or acute injury, the healing process is impaired by T2DM. We have previously demonstrating that restoring normal metabolic function in a murine model of obesity/ T2DM is insufficient to blunt or reverse the progression of diabetic tendinopathy, indicating the need for identification of novel therapeutic approaches to both maintain tendon homeostasis, and to improve the healing process. RAGE, the Receptor for Advanced Glycation Endproducts has been implicated as a key driver of several diabetic pathologies. We have demonstrated that pharmacological antagonism of RAGE is sufficient to partially improve tendon healing in non-diabetic animals. Therefore, in the current study we tested the efficacy of blunted RAGE signaling, via treatment with a RAGE Antagonist Peptide (RAP), to improve tendon healing in the context of T2DM. While our study did not find a beneficial effect of short-term RAP treatment on the healing process of T2DM mice, we did identify several important challenges brought about by this model of diet-induced obesity and T2DM. Both high fat (HFD) and low fat diet (LFD) feeding shifted the temporal molecular profile of healing compared to standard laboratory chow fed mice. Moreover, RAP treatment resulted in a transient disruption in homeostasis in the contralateral control tendons of both HFD and LFD mice, and this was due to a potential interaction with the systemic response to tendon injury as this response was not observed in HFD and LFD fed mice that did not undergo tendon repair surgery. Collectively, these data highlight the complications associated with models of diet induced obesity, and the lean control diets that should be considered in future studies.