Nelly Andarawis‐Puri

Tendinopathies are prevalent musculoskeletal conditions that have no effective therapies to attenuate scar formation. In contrast to other adult mammals, the tendons of Murphy Roths Large (MRL/MpJ) mice possess a superior healing capacity following acute and overuse injuries. Here, we hypothesized that the application of biological cues derived from the local MRL/MpJ tendon environment would direct otherwise scar-mediated tenocytes towards a pro-regenerative MRL/MpJ-like phenotype. We identified soluble factors enriched in the secretome of MRL/MpJ tenocytes using bioreactor systems and quantitative proteomics. We then demonstrated that the combined administration of structural and soluble constituents isolated from decellularized MRL/MpJ tendon provisional ECM (dPECM) and the secretome stimulate scar-mediated rodent tenocytes towards enhanced mechanosensitivity, proliferation, intercellular communication, and ECM deposition associated with MRL/MpJ cell behavior. Our findings highlight key biological mechanisms that drive MRL/MpJ tenocyte activity and their interspecies utility to be harnessed for therapeutic strategies that promote pro-regenerative healing outcomes. Teaser: Proteins enriched in a super-healer mouse strain elicit interspecies utility in promoting pro-regenerative tenocyte behavior.

Murphy Roths Large mice (MRL) exhibit improved tendon healing and are often described as a "super-healer" strain. The underlying mechanisms that drive the superior healing response of MRL remain a controversial subject. We utilized a tendon transplantation model between MRL and "normal-healer" B6-mice to differentiate between the contribution of MRL's innate tendon and systemic environment to its improved healing capacity. Patellar tendons with a midsubstance punch injury were transplanted back into the same animal (autograft) or into an animal of the other strain (allograft). Findings at 4 weeks showed that the innate MRL tendon environment drives its improved healing capacity as demonstrated by improved stiffness and maximum load in MRL-grafts-in-B6-host-allografts compared to B6-autografts, and higher modulus in MRL-autografts compared to B6-graft-in-MRL-host-allografts. Groups with an MRL component showed an increase in pro-inflammatory cytokines in the 3 days after injury, suggesting an early enhanced inflammatory profile in MRL that ultimately resolves. A preserved range of motion of the knee joint in all MRL animals suggests a systemic "shielding effect" of MRL in regard to joint adhesiveness. Our findings 4-weeks post injury are consistent with previous studies showing tissue-driven improved healing and suggest that the systemic environment contributes to the overall healing process.

Tendons are unique dense connective tissues with discrete zones having specific structure and function. They are juxtaposed with other tissues (e.g., bone, muscle, and fat) with different compositional, structural, and mechanical properties. Additionally, tendon properties change drastically with growth and development, disease, aging, and injury. Consequently, there are unique challenges to performing high quality histological assessment of this tissue. To address this need, histological assessment was one of the breakout session topics at the 2022 Orthopaedic Research Society (ORS) Tendon Conference hosted at the University of Pennsylvania. The purpose of the breakout session was to discuss needs from members of the ORS Tendon Section related to histological procedures, data presentation, knowledge dissemination, and guidelines for future work. Therefore, this review provides a brief overview of the outcomes of this discussion and provides a set of guidelines, based on the perspectives from our laboratories, for histological assessment to assist researchers in their quest to utilize these techniques to enhance the outcomes and interpretations of their studies.

Tendinopathies are common injuries that typically occur from overuse and fatigue. Treatments target late-stage symptoms with limited success, leading to high rates of reinjury. Early intervention could halt tendinopathy progression to rupture but requires a better understanding of the biomechanical environment associated with early-stage disease. While fatigue injured tendons are further damaged by exercise that is initiated immediately after onset of injury, exercise that is initiated after a brief delay promotes repair. Similar macroscale mechanical properties and collagen damage throughout this delay period suggests that microscale, non-collagenous matrix changes after fatigue injury modulate tendon mechanotransduction and shifts the exercise response from detrimental to reparative. Glycosaminoglycans (GAGs) and proteoglycans (PGs) are increased during chronic tendinopathy, but their role in early-stage disease is unknown. We hypothesized that increased GAGs from fatigue injury modulate viscoelasticity and microscale strains to enable repair from exercise. Various GAG types were increased in the weeks after onset of fatigue injury in the extracellular and pericellular matrices of rat patellar tendons. Enzymatic removal of GAGs from these fatigued tendons increased microscale shear strain, suggesting that GAGs modulate the cell microenvironment after fatigue injury. GAG removal decreased dynamic modulus in the toe region and decreased loss tangent in the linear region of the stress-strain curve in fatigued tendons, suggesting the GAG increase modulates tendon multiscale mechanics and viscoelasticity during fiber uncrimping and fibril sliding and strain transfer. GAGs may influence repair in response to exercise and could serve as a therapeutic target for tendinopathy.

Clinical and animal studies have reported the influence of sex on the incidence and progression of tendinopathy, which results in disparate structural and biomechanical outcomes. However, there remains a paucity in our understanding of the sex-specific biological mechanisms underlying effective tendon healing. To overcome this hurdle, our group has investigated the impact of sex on tendon regeneration using the super-healer Murphy Roths Large (MRL/MpJ) mouse strain. We have previously shown that the scarless healing capacity of MRL/MpJ patellar tendons is associated with sexually dimorphic regulation of gene expression for pathways involved in fibrosis, cell migration, adhesion, and extracellular matrix (ECM) remodeling following an acute mid-substance injury. Thus, we hypothesized that MRL/MpJ scarless tendon healing is mediated by sex-specific and temporally distinct orchestration of cell-ECM interactions. Accordingly, the present study comparatively evaluated MRL/MpJ tendon cells on two-dimensional (2D; glass) and scaffold platforms to examine cell behavior under biochemical and topographical cues associated with tendon homeostasis and healing. Female MRL/MpJ cells showed reduced 2D migration and spreading area accompanied by enhanced mechanosensing, ECM alignment, and fibronectin-mediated cell proliferation compared to male MRL/MpJ cells. Interestingly, female MRL/MpJ cells cultured on isotropic scaffolds showed diminished cell-ECM organization compared to male MRL/MpJ cells. Lastly, MRL/MpJ cells elicited enhanced cytoskeletal elongation and alignment, ECM deposition and organization, and connexin 43-mediated intercellular communication compared to male B6 cells, regardless of culture condition or sex. These results provide insight into the cellular features conserved within the MRL/MpJ phenotype and potential sex-specific targets for the development of more equitable therapeutics.