The superior healing capacity of MRL tendons is minimally influenced by the systemic environment of the MRL mouse.

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.

Histological and immunohistochemical guide to tendon tissue.

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.

Regenerative MRL/MpJ tendon cells exhibit sex differences in morphology, proliferation, mechanosensitivity, and cell-ECM organization.

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.

Glycosaminoglycans modulate microscale mechanics and viscoelasticity in fatigue injured tendons.

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.

Detergent-Free Decellularization Preserves the Mechanical and Biological Integrity of Murine Tendon.

Tissue decellularization has demonstrated widespread applications across numerous organ systems for tissue engineering and regenerative medicine applications. Decellularized tissues are expected to retain structural and/or compositional features of the natural extracellular matrix (ECM), enabling investigation of biochemical factors and cell-ECM interactions that drive tissue homeostasis, healing, and disease. However, the dense collagenous tendon matrix has limited the efficacy of traditional decellularization strategies without the aid of harsh chemical detergents and/or physical agitation that disrupt tissue integrity and denature proteins involved in regulating cell behavior. In this study, we adapted and established the advantages of a detergent-free decellularization method that relies on latrunculin B actin destabilization, alternating hypertonic-hypotonic salt and water incubations, nuclease-assisted elimination of cellular material, and protease inhibitor supplementation under aseptic conditions. Our method maintained the collagen molecular structure (i.e., minimal extent of denaturation), while adequately removing cells and preserving bulk mechanical properties. Furthermore, we demonstrated that decellularized tendon ECM-derived coatings isolated from different mouse strains, injury states (i.e., naive and acutely injured/"provisional"), and anatomical sites harness distinct biochemical cues and robustly maintain tendon cell viability in vitro. Together, our work provides a simple and scalable decellularization method to facilitate mechanistic studies that will expand our fundamental understanding of tendon ECM and cell biology. Impact statement In this study, we present a decellularization method for tendon that does not rely on any detergent or physical processing techniques. We assessed the impact of detergent-free decellularization using tissue, cellular, and molecular level analyses and validated the preservation of gross fiber architecture, collagen molecular structure, and extracellular matrix (ECM)-associated biological cues that are essential for studying physiological cell-ECM interactions. Finally, we demonstrated the applicability of this method for healthy and injured tendon environments, across mouse strains, and for different types of tendons, illustrating the utility of this approach for isolating the contributions of biochemical cues within unique tendon ECM microenvironments.

Enhanced healing outcomes in MRL/MpJ mouse tissues conserved in insertion site following surgical repair.

BACKGROUND: Surgical repair of supraspinatus tendons (SSTs) has a high failure rate at the insertion site. A significant hurdle to therapeutic development is that effective intrinsic healing mechanisms are unknown. The MRL/MpJ (MRL) mouse exhibits tissue-specific enhanced healing; however, these tissues exhibit disparate properties from the complex SST. The extent of SST healing in the complex environment of the rotator cuff is unknown. We hypothesized that MRL mice would exhibit enhanced restoration of the structurally complex insertion site, resulting in functional improvements. METHODS: B6 and MRL mice underwent SST detachment and immediate surgical repair. Mice were analyzed for gait assessment after either 2 or 6 weeks and were then killed humanely for immunohistologic analysis. RESULTS: MRL SSTs demonstrated enhanced recovery of zonal architecture and bone structure compared with B6 SSTs. MRL SSTs exhibited decreased levels of type III collagen at 2 weeks and increased levels of type I procollagen at 6 weeks compared with B6 SSTs. MRL mice experienced initial gait deficits at 2 weeks that had recovered by 6 weeks. DISCUSSION: The temporal balance of collagen in MRL mice suggests recovery toward naive composition. Initial gait deficits in MRL mice may provide a protective loading environment that is ultimately beneficial. The mechanisms of enhanced healing observed previously in MRL mice may be conserved in the complex SST, providing a platform to interrogate specific aspects of improved healing.

The role of the tendon ECM in mechanotransduction: disruption and repair following overuse.

Purpose: Tendon overuse injuries are prevalent conditions with limited therapeutic options to halt disease progression. The specialized extracellular matrix (ECM) both enables joint function and mediates mechanical signals to tendon cells, driving biological responses to exercise or injury. With overuse, tendon ECM composition and structure changes at multiple scales, disrupting mechanotransduction and resulting in inadequate repair and disease progression. This review highlights the multiscale ECM changes that occur with tendon overuse and corresponding effects on cell-matrix interactions and cellular response to load.Results: Different functional joint requirements and tendon types experience a wide range of loading profiles, creating varied downstream mechanical stimuli. Distinct ECM structure and mechanical properties within the fascicle matrix, interfascicle matrix, and enthesis and their varied disruption with overuse are considered. The pericellular matrix (PCM) comprising the microscale tendon cell environment has a unique composition that changes with overuse injury and exercise, suggesting an important role in mechanotransduction and promoting repair. Cell-matrix interactions are mediated by structures including cilia, integrins, connexins and cytoskeleton that signal downstream homeostasis, adaptation, or repair. ECM disruption with tendon overuse may cause altered mechanical loading and cell-matrix interactions, resulting in mechanobiological understimulation, apoptosis, and ineffective repair. Current interventions to promote repair of tendon overuse injuries including exercise, targeting cell signaling, and modulating inflammation are considered.Conclusion: Future therapeutics should be assessed with regard of their effects on multiscale mechanotransduction in addition to joint function, with consideration of the central role of ECM.

The Role of the Non-Collagenous Extracellular Matrix in Tendon and Ligament Mechanical Behavior: A Review.

Tendon is a connective tissue that transmits loads from muscle to bone, while ligament is a similar tissue that stabilizes joint articulation by connecting bone to bone. The 70-90% of tendon and ligament's extracellular matrix (ECM) is composed of a hierarchical collagen structure that provides resistance to deformation primarily in the fiber direction, and the remaining fraction consists of a variety of non-collagenous proteins, proteoglycans, and glycosaminoglycans (GAGs) whose mechanical roles are not well characterized. ECM constituents such as elastin, the proteoglycans decorin, biglycan, lumican, fibromodulin, lubricin, and aggrecan and their associated GAGs, and cartilage oligomeric matrix protein (COMP) have been suggested to contribute to tendon and ligament's characteristic quasi-static and viscoelastic mechanical behavior in tension, shear, and compression. The purpose of this review is to summarize existing literature regarding the contribution of the non-collagenous ECM to tendon and ligament mechanics, and to highlight key gaps in knowledge that future studies may address. Using insights from theoretical mechanics and biology, we discuss the role of the non-collagenous ECM in quasi-static and viscoelastic tensile, compressive, and shear behavior in the fiber direction and orthogonal to the fiber direction. We also address the efficacy of tools that are commonly used to assess these relationships, including enzymatic degradation, mouse knockout models, and computational models. Further work in this field will foster a better understanding of tendon and ligament damage and healing as well as inform strategies for tissue repair and regeneration.

Outpatient Surgical Fixation of Proximal Humerus Fractures Can Be Performed Without Increased Rates of Short-Term Complications or Readmissions.

OBJECTIVE: To delineate differences in short-term complications between outpatient versus inpatient open reduction and internal fixation (ORIF) of proximal humerus fractures. DESIGN: Retrospective database review. SETTING: Hospitals participating in the American College of Surgeons National Surgical Quality Improvement Program database. PATIENTS: Patients in the National Surgical Quality Improvement Program database with proximal humerus fractures from 2005 to 2017. INTERVENTION: Proximal humerus fracture ORIF. MAIN OUTCOME MEASUREMENTS: Thirty-day readmission, reoperation, thromboembolic events, and other complications. RESULTS: In total, 920 outpatient and 2490 inpatient ORIF cases were identified. The proportion of outpatient proximal humerus fracture ORIF increased throughout the years from 6.67% in 2007 to 34.89% in 2017. Each outpatient case was propensity-score-matched with one inpatient case by age, sex, functional status, American Society of Anesthesiologists classification, smoking status, diabetes mellitus type, hypertension, chronic obstructive pulmonary disease, and dyspnea on exertion. After matching, there were 920 outpatient and 920 inpatient cases. Statistical analysis revealed no significant difference in complications including reoperation (1.63% vs. 2.50%), thromboembolic events (0.65% vs. 0.65%), and 30-day readmissions (2.93% vs. 2.69%) between outpatient versus matched inpatient procedures (all P > 0.05). The only significant finding was a lower rate of blood transfusion in outpatient procedures (0.54%) compared with inpatient procedures (4.02%) (P < 0.001). CONCLUSIONS: The perioperative outcomes assessed here support the conclusion that ORIF for proximal humerus fractures can be performed in the outpatient setting without increased rates of 30-day perioperative complications or readmissions compared with inpatient procedures. However, it is worth noting that the majority of outpatient cases were younger than the average geriatric proximal humerus fracture patient. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

MRL/MpJ tendon matrix-derived therapeutic promotes improved healing outcomes in scar-mediated canonical tendon healing.

Tendons are commonly injured connective soft tissues characterized by an ineffective healing response that results in scar formation and loss of functional and structural properties. Naturally occurring extracellular matrix (ECM) constructs have become a promising therapeutic for tendon injuries due to their capacity to harness a complex biological environment. However, in tendon, the ECM properties needed for improved healing remain unknown. Interestingly, we have determined that the improved tendon healing response of the scarless-healing MRL/MpJ is driven by intrinsic properties with therapeutic potential to modulate the proliferative and morphological behavior of cells derived from a canonically healing model in vitro. We hypothesize that a distinct composition of ECM deposited during the early healing response of the MRL/MpJ will harnesses the biological cues to stimulate improved structure and function in vivo of canonically healing B6 mice. Accordingly, MRL/MpJ and B6 patellar tendons were injured via midsubstance punch defects. Healing tendons were isolated after 3 or 7 days and encapsulated in PEG-4MAL hydrogels to develop ECM-derived therapeutic constructs. Constructs were then introduced into B6 mice as a treatment following full thickness midsubstance-punch injuries. Treatment with ECM-derived constructs from MRL/MpJ tendons after 7-days post-injury (M7) resulted in improved matrix alignment, tissue stiffness, decreased collagen III content and improved cell morphology in B6 tendons after 6 weeks post-injury. Furthermore, proteomic analysis showed that M7 contained a unique compositional profile rich in glycoproteins, thereby elucidating a valuable naturally-derived platform for the treatment of tendon injuries. Overall this work highlights promising targets for future therapeutic development and tissue engineering applications.

Superior mechanical recovery in male and female MRL/MpJ tendons is associated with a unique genetic profile.

Tendon ruptures heal by forming a mechanically inferior scar. We have shown that male Murphy Roths large (MRL/MpJ) mice exhibit improved tendon healing, suggesting that they can inform biological mechanisms that lead to effective tendon healing. As sex impacts healing, we assessed the effect of sex on tendon healing in MRL/MpJ and normal healer C57BL/6 (B6) mice and compared the associated biological environment with identify genes that may be integral to the improved healing outcome. We hypothesized that (a) male MRL/MpJ mice will heal with improved mechanical properties compared to females; and (b) that regenerative tendon healing will be associated with decreased fibrotic pathways, decreased inflammation, and increased activity of matrix metalloproteinases (MMPs). A midsubstance punch was introduced, and tendons were harvested after (a) 1 or 7 days for profiling of 84 genes; (b) 7 or 14 days for the assessment of MMP-2 and MMP-9 activity; and (c) 6 weeks for mechanical assessment. MRL/MpJ tendons healed with the better restoration of mechanical properties than B6 tendons. Sex did not affect the mechanical properties of healing B6 or MRL/MpJ tendons. Comparison of the gene expression profiles in the context of the mechanical outcome revealed several differences between MRL/MpJ and B6 tendon healing, including, lower inflammation, an earlier higher expression of TGF-ß-related genes that diminish by 7 days, and genes associated with enhanced cell migration in MRL/MpJ in comparison to B6 tendons. We expect that the timecourse and expression levels of these genes in scarless MRL/MpJ tendon healing represent the balanced environment that leads to improved tendon healing.

Innate tissue properties drive improved tendon healing in MRL/MpJ and harness cues that enhance behavior of canonical healing cells.

Development of tendon therapeutics has been hindered by the lack of informative adult mammalian models of regeneration. Murphy Roth's Large (MRL/MpJ) mice exhibit improved healing following acute tendon injuries, but the driver of this regenerative healing response remains unknown. The tissue-specific attributes of this healing response, despite a shared systemic environment within the mouse, support the hypothesis of a tissue-driven mechanism for scarless healing. Our objective was to investigate the potential of MRL/MpJ tendon extracellular matrix (ECM)-derived coatings to regulate scar-mediated healing. We found that deviations in the composition of key structural proteins within MRL/MpJ vs C57Bl/6 tendons occur synergistically to mediate the improvements in structure and mechanics following a 1-mm midsubstance injury. Improvement in mechanical properties of healing MRL/MpJ vs C57Bl/6 tendons that were isolated from systemic contributions via organ culture, highlighted the innate tendon environment as the driver of scarless healing. Finally, we established that decellularized coatings derived from early-deposited MRL/MpJ tendon provisional extracellular matrix (provisional-ECM), can modulate canonical healing B6 tendon cell behavior by inducing morphological changes and increasing proliferation in vitro. This study supports that the unique compositional cues in MRL/MpJ provisional-ECM have the therapeutic capability to motivate canonically healing cells toward improved behavior; enhancing our ability to develop effective therapeutics.

Murine patellar tendon transplantation requires transosseous cerclage augmentation - development of a transplantation model for investigation of systemic and local drivers to healing.

BACKGROUND: Tendon injuries are common musculoskeletal injuries that heal with scar tissue formation, often achieving reduced biomechanical and functional properties. The murine patellar tendon is a research tool that holds potential for investigating tendon healing and can be useful for exploring therapeutic strategies. Since healing is a complex process that results from the collaboration between the systemic and local tissue environment, a murine tendon transplantation model that can be applied to transgenic mice and genetic mutants would allow isolation of systemic versus local tendon factors in driving effective tendon healing. Preliminary studies have shown that transplantation with simple tendon sutures results in a proximalization of the patellar bone due to the involuntary quadriceps muscle force leading to tearing of the graft and failure of the knee extensor mechanism. To avoid this elongation of the graft, two cerclage techniques for murine patellar tendon transplantation were introduced and validated. METHODS: Three developed surgical techniques (no-cerclage-augmentation (NCA)), transfascial suture cerclage with encirclement of the patellar tendon (TFSC), and dual-cerclage-augmentation with a transosseous bone-to-bone cerclage through the patella bone and an additional musculotendinous cerclage (DCA)) were compared at 4 and 8 weeks macroscopically in regards to graft continuity, cerclage integrity, gap formation, and radiologically by measuring the patello-tibial distance and using a patella bone position grading system. RESULTS: The NCA group showed complete failure at 5-7 days after surgery. The TFSC has led to 69% functional failure of the cerclage. In contrast, the DCA with a has led to 78% success with improvement in patellar bone position and a similar patello-tibial distance to the naïve contralateral murine knees over the time period of 8 weeks. CONCLUSIONS: This study shows that a bone-to-bone cerclage is necessary to maintain a desired graft length in murine patellar tendon models. This surgery technique can serve for future graft trans- and implantations in the murine patellar tendon.

Multiscale mechanisms of tendon fatigue damage progression and severity are strain and cycle dependent.

Tendinopathies are common chronic injuries that occur when damage accumulation caused by sub-rupture fatigue loading outpaces repair. Studies have linked fatigue loading with various mechanical, structural, and biological changes associated with pathology. However, the multiscale progression of damage accumulation with respect to area, severity and the distinct contributions of strain level and number of cycles has not been fully elucidated. The objective of this study was to investigate multiscale mechanisms underlying fatigue damage accumulation and their effect on the cellular environment. Using an in situ model in rat tail tendon (RTT), fatigue loading was applied at various strains and cycle numbers to induce fatigue damage. Pre- and post- fatigue diagnostic mechanical testing, second harmonic generation (SHG) imaging, and transmission electron microscope (TEM) imaging were used to investigate extracellular and cellular damage modes at multiple scales. Fatigue loading at strains at or below 1.0% resulted in no significant changes in SHG damage area or severity and no changes in collagen fibril or cell morphology compared with controls. Fatigue loading at strains above 1.5% resulted in greater mechanical changes correlated with increased damage area measured by SHG and collagenous damage observed by TEM. Increased cycles at high strain further altered mechanical properties, increased structural damage severity (but not area), and altered TEM collagen rupture patterns. Cell morphology was similarly progressively affected with increased strain and cycle number. These damage mechanisms that may trigger degenerative changes characteristic of tendinopathy could be targeted as a part of prevention or therapy.

Promoting effective tendon healing and remodeling.

Daily activities subject our tendons to accumulation of sub-rupture fatigue injury which can lead to tendon rupture. Consequently, tendinopathies account for over 30% of musculoskeletal consultations. We adopted a multidisciplinary approach to determine the role of the extracellular matrix (ECM) in the pathogenesis of tendinopathy and impaired healing of ruptured tendons. We have been investigating three main areas: (i) the pathogenesis of tendon degeneration; (ii) approaches to promoting remodeling of sub-rupture fatigue injuries; and the (iii) role of the ECM in promoting scarless tendon healing. In this Kappa Delta Young Investigator award paper, we describe the key discoveries made in each of our three research areas of focus. Briefly, we discovered that sub-rupture fatigue damage can accumulate from just one bout of fatigue loading. Furthermore, any attempt to repair the fatigue damage diminishes as the severity of induced damage increases. We have utilized exercise to develop animal models of exercise-led degeneration and exercise-led repair of sub-rupture fatigue damage injuries, wherein underlying mechanisms can be uncovered, thereby overcoming a major hurdle to development of therapeutics. Since damage accumulation ultimately leads to rupture that is characterized by formation of a mechanically inferior scar, we have used the MRL/MpJ mouse to evaluate the role of the systemic environment and the local tendon environment in driving regeneration to identify new therapeutic pathways to promote scarless healing. Our data suggests that the therapeutic potential of the MRL/MpJ provisional ECM should be further explored as it may harness biological and structural mechanisms to promote scarless healing. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3115-3124, 2018.

A potential new role for myofibroblasts in remodeling of sub-rupture fatigue tendon injuries by exercise.

Tendons are ineffective at repairing sub-rupture fatigue injuries. Accordingly, we evaluated whether an exercise protocol that we have previously found to decrease structural damage kinks in fatigue damaged tendons, leads to improvement in mechanical properties. We hypothesized that exercise that promotes repair of fatigue damage will decrease apoptosis and increase the population of myofibroblasts. Rat patellar tendons underwent in vivo fatigue loading for 500 or 7200 cycles. Animals resumed cage activity for 2-weeks, then either remained cage active or began treadmill running until sacrifice at 4- or 10-weeks post-fatigue loading. Exercise following fatigue damage increased the stiffness back towards naïve levels, decreased apoptosis and increased the population of myofibroblasts. Next, proteins associated with inhibition of apoptosis (Collagen VI) or activation of myofibroblast (pSmad 2/3, fibrillin, integrin subunits αV and α5) were evaluated. Data suggests that collagen VI may not be integral to inhibition of apoptosis in this context. Exercise increased pSmad 2/3 and fibrillin in the insertion region for the 7200-cycles group. In addition, exercise decreased integrin αV and increased integrin α5 in fatigue damaged tendons. Data suggests that a decrease in apoptosis and an increase in population of myofibroblasts may be integral to remodeling of fatigue damaged tendons.

Uncorrelated healing response of tendon and ear injuries in MRL highlight a role for the local tendon environment in driving scarless healing.

PURPOSE: Tendon tears are common injuries that heal with scar formation. Interestingly, MRL/MpJ mice heal without scar in several tissues, including tendon. Most hypotheses regarding scarless healing implicate the systemic environment. However, the tissue-specificity of this regenerative response and our previous findings showing regeneration of sub-rupture tendon injuries, which lack an overt systemic response, motivate a tissue-driven hypothesis. Our objective is to investigate the potential of the local tendon environment in driving scarless healing (1) by comparing the systemic response and the healing capacity associated with ear and tendon injuries in MRL/MpJ mice, and (2) by comparing intrinsic healing properties between MRL/MpJ and normal healer C57Bl/6 tendons. METHODS: We examined the systemic inflammatory and local structural environments of ear and tendon punch injuries in MRL/MpJ and C57Bl/6 mice. Systemic differences were analyzed to assess effects of different injuries on the inflammatory response. Correlations were assessed between MRL/MpJ ear and tendon injuries to compare the extent of healing between regenerative tissues. RESULTS: Analysis showed similarities between the systemic environment in MRL/MpJ post ear or tendon injuries. However, comparable inflammatory responses did not translate into analogous healing between tissues, suggesting that the systemic environment is not the driver of regeneration. Supporting the regenerative role of the local environment, healing MRL/MpJ tendons exhibited improved matrix and cell alignment and a distinct composition of growth factors and Hyaluronan from C57Bl/6. CONCLUSION: These findings support the tissue-driven hypothesis for MRL/MpJ tendon regeneration and motivate further investigation regarding specific roles of extracellular factors in scarless healing.

Novel image analysis methods for quantification of in situ 3-D tendon cell and matrix strain.

Macroscopic tendon loads modulate the cellular microenvironment leading to biological outcomes such as degeneration or repair. Previous studies have shown that damage accumulation and the phases of tendon healing are marked by significant changes in the extracellular matrix, but it remains unknown how mechanical forces of the extracellular matrix are translated to mechanotransduction pathways that ultimately drive the biological response. Our overarching hypothesis is that the unique relationship between extracellular matrix strain and cell deformation will dictate biological outcomes, prompting the need for quantitative methods to characterize the local strain environment. While 2-D methods have successfully calculated matrix strain and cell deformation, 3-D methods are necessary to capture the increased complexity that can arise due to high levels of anisotropy and out-of-plane motion, particularly in the disorganized, highly cellular, injured state. In this study, we validated the use of digital volume correlation methods to quantify 3-D matrix strain using images of naïve tendon cells, the collagen fiber matrix, and injured tendon cells. Additionally, naïve tendon cell images were used to develop novel methods for 3-D cell deformation and 3-D cell-matrix strain, which is defined as a quantitative measure of the relationship between matrix strain and cell deformation. The results support that these methods can be used to detect strains with high accuracy and can be further extended to an in vivo setting for observing temporal changes in cell and matrix mechanics during degeneration and healing.

Novel Model of Tendon Regeneration Reveals Distinct Cell Mechanisms Underlying Regenerative and Fibrotic Tendon Healing.

To date, the cell and molecular mechanisms regulating tendon healing are poorly understood. Here, we establish a novel model of tendon regeneration using neonatal mice and show that neonates heal via formation of a 'neo-tendon' that differentiates along the tendon specific lineage with functional restoration of gait and mechanical properties. In contrast, adults heal via fibrovascular scar, aberrant differentiation toward cartilage and bone, with persistently impaired function. Lineage tracing identified intrinsic recruitment of Scx-lineage cells as a key cellular mechanism of neonatal healing that is absent in adults. Instead, adult Scx-lineage tenocytes are not recruited into the defect but transdifferentiate into ectopic cartilage; in the absence of tenogenic cells, extrinsic αSMA-expressing cells persist to form a permanent scar. Collectively, these results establish an exciting model of tendon regeneration and uncover a novel cellular mechanism underlying regenerative vs non-regenerative tendon healing.

Tendinopathy: Investigating the Intersection of Clinical and Animal Research to Identify Progress and Hurdles in the Field.

Biological treatments, surgical interventions, and rehabilitation exercises have been successfully used to treat tendinopathy, but the development of effective treatments has been hindered by the lack of mechanistic data regarding the pathogenesis of the disease. While insightful, clinical studies are limited in their capacity to provide data regarding the pathogenesis of tendinopathies, emphasizing the value of animal models and cell culture studies to fill this essential gap in knowledge. Clinical pathological findings from imaging studies or histological analysis are not universal across patients with tendinopathy and have not been clearly associated with the onset of symptoms. There are several unresolved controversies, including the cellular changes that accompany the tendinopathic disease state and the role of inflammation. Additional research is needed to correlate the manifestations of the disease with its pathogenesis, with the goal of reaching a field-wide consensus on the pathology of the disease state. Such a consensus will allow standardized clinical practices to more effectively diagnose and treat tendinopathy.