Pharmacological Therapies for Connective Tissue Fibrosis in Orthopaedics.

Fibrosis is a common and debilitating pathological process that affects many organ systems and contributes to connective tissue disorders in orthopaedics. Tendons heal after acute and chronic injury through a process of fibrovascular scar tissue formation, and soft tissue joint capsules can be affected after traumatic joint injury, leading to arthrofibrosis. Although the precise underlying mechanisms are still being elucidated, fibrosis is thought to be a consequence of dysregulated immune and cytokine signaling that leads to myofibroblast activation and proliferation and subsequent excessive collagen deposition. Current treatments for connective tissue fibrosis include physical therapy and surgery, but there are no therapies that directly target the underlying cellular and molecular mechanisms of fibrosis. Many pharmacological agents have been used to successfully target fibrosis in other tissues and organ systems and thus are a promising treatment option to fill this gap. However, limited evidence is available to guide the use of these agents in musculoskeletal connective tissues. This article provides an overview of pharmacological therapies that have potential to treat connective tissue fibrosis in patients with musculoskeletal conditions, along with the current supporting evidence and future uses of each therapy.

Achilles Tendons Display Region-Specific Transcriptomic Signatures Associated With Distinct Mechanical Properties.

BACKGROUND: Previous studies have examined the transcriptomes and mechanical properties of whole tendons in different regions of the body. However, less is known about these characteristics within a single tendon. PURPOSE: To develop a regional transcriptomic atlas and evaluate the region-specific mechanical properties of Achilles tendons. STUDY DESIGN: Descriptive laboratory study. METHODS: Achilles tendons from 2-month-old male Sprague Dawley rats were used. Tendons were isolated and divided into proximal, middle, and distal thirds for RNA sequencing (n = 5). For mechanical testing, the Achilles muscle-tendon-calcaneus unit was mounted in a custom-designed materials testing system with the unit clamped over the musculotendinous junction (MTJ) and the calcaneus secured at 90° of dorsiflexion (n = 9). Tendons were stretched to 20 N at a constant speed of 0.0167 mm/s. Cross-sectional area, strain, stress, and Young modulus were determined in each tendon region. RESULTS: An open-access, interactive transcriptional atlas was generated that revealed distinct gene expression signatures in each tendon region. The proximal and distal regions had the largest differences in gene expression, with 2596 genes significantly differentially regulated at least 1.5-fold (q < .01). The proximal tendon displayed increased expression of genes resembling a tendon phenotype and increased expression of nerve cell markers. The distal region displayed increases in genes involved in extracellular matrix synthesis and remodeling, immune cell regulation, and a phenotype similar to cartilage and bone. There was a 3.72-fold increase in Young modulus from the proximal to middle region (P < .01) and an additional 1.34-fold increase from the middle to distal region (P = .027). CONCLUSION: Within a single tendon, there are region-specific transcriptomic signatures and mechanical properties, and there is likely a gradient in the biological and functional phenotype from the proximal origin at the MTJ to the distal insertion at the enthesis. CLINICAL RELEVANCE: These findings improve our understanding of the underlying biological heterogeneity of tendon tissue and will help inform the future targeted use of regenerative medicine and tissue engineering strategies for patients with tendon disorders.

Multiomics analysis of the mdx/mTR mouse model of Duchenne muscular dystrophy.

PURPOSE/AIM: Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease characterized by extensive muscle weakness. Patients with DMD lack a functional dystrophin protein, which transmits force and organizes the cytoskeleton of skeletal muscle. Multiomic studies have been proposed as a way to obtain novel insight about disease processes from preclinical models, and we used this approach to study pathological changes in dystrophic muscles. MATERIALS AND METHODS: We evaluated hindlimb muscles of male mdx/mTR mice, which lack a functional dystrophin protein and have deficits in satellite cell abundance and proliferative capacity. Wild type (WT) C57BL/6 J mice served as controls. Muscle fiber contractility was measured, along with changes in the transcriptome using RNA sequencing, and in the proteome, metabolome, and lipidome using mass spectrometry. RESULTS: While mdx/mTR mice displayed gross pathological changes and continued cycles of degeneration and regeneration, we found no differences in permeabilized fiber contractility between strains. However, there were numerous changes in the transcriptome and proteome related to protein balance, contractile elements, extracellular matrix, and metabolism. There was only a 53% agreement in fold-change data between the proteome and transcriptome. Numerous changes in markers of skeletal muscle metabolism were observed, with dystrophic muscles exhibiting elevated glycolytic metabolites such as 6-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate, fructose bisphosphate, phosphorylated hexoses, and phosphoenolpyruvate. CONCLUSIONS: These findings highlight the utility of multiomics in studying muscle disease, and provide additional insight into the pathological changes in dystrophic muscles that might help to indirectly guide evidence-based nutritional or exercise prescription in DMD patients.

Single-cell transcriptomic analysis identifies extensive heterogeneity in the cellular composition of mouse Achilles tendons.

Tendon is a dense connective tissue that stores and transmits forces between muscles and bones. Cellular heterogeneity is increasingly recognized as an important factor in the biological basis of tissue homeostasis and disease, yet little is known about the diversity of cell types that populate tendon. To address this, we determined the heterogeneity of cell populations within mouse Achilles tendons using single-cell RNA sequencing. In assembling a transcriptomic atlas of Achilles tendons, we identified 11 distinct types of cells, including three previously undescribed populations of tendon fibroblasts. Prior studies have indicated that pericytes, which are found in the vasculature of tendons, could serve as a potential source of progenitor cells for adult tendon fibroblasts. Using trajectory inference analysis, we provide additional support for the notion that pericytes are likely to be at least one of the progenitor cell populations for the fibroblasts that compose adult tendons. We also modeled cell-cell interactions and identified previously undescribed ligand-receptor signaling interactions involved in tendon homeostasis. Our novel and interactive tendon atlas highlights previously underappreciated heterogeneity between and within tendon cell populations. The atlas also serves as a resource to further the understanding of tendon extracellular matrix assembly and maintenance and in the design of therapies for tendinopathies.

Musculoskeletal Consequences of COVID-19.

Coronavirus disease 2019 (COVID-19) is an emerging pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the majority of patients who become infected with SARS-CoV-2 are asymptomatic or have mild symptoms, some patients develop severe symptoms that can permanently detract from their quality of life. SARS-CoV-2 is closely related to SARS-CoV-1, which causes severe acute respiratory syndrome (SARS). Both viruses infect the respiratory system, and there are direct and indirect effects of this infection on multiple organ systems, including the musculoskeletal system. Epidemiological data from the SARS pandemic of 2002 to 2004 identified myalgias, muscle dysfunction, osteoporosis, and osteonecrosis as common sequelae in patients with moderate and severe forms of this disease. Early studies have indicated that there is also considerable musculoskeletal dysfunction in some patients with COVID-19, although long-term follow-up studies have not yet been conducted. The purpose of this article was to summarize the known musculoskeletal pathologies in patients with SARS or COVID-19 and to combine this with computational modeling and biochemical signaling studies to predict musculoskeletal cellular targets and long-term consequences of the SARS-CoV-2 infection.

Widespread diversity in the transcriptomes of functionally divergent limb tendons.

KEY POINTS: Tendon is a hypocellular, matrix-rich tissue that has been excluded from comparative transcriptional atlases. These atlases have provided important knowledge about biological heterogeneity between tissues, and our study addresses this important gap. We performed measures on four of the most studied tendons, the Achilles, forepaw flexor, patellar and supraspinatus tendons of both mice and rats. These tendons are functionally distinct and are also among the most commonly injured, and therefore of important translational interest. Approximately one-third of the filtered transcriptome was differentially regulated between Achilles, forepaw flexor, patellar and supraspinatus tendons within either mice or rats. Nearly two-thirds of the transcripts that are expressed in anatomically similar tendons were different between mice and rats. The overall findings from this study identified that although tendons across the body share a common anatomical definition based on their physical location between skeletal muscle and bone, tendon is a surprisingly genetically heterogeneous tissue. ABSTRACT: Tendon is a functionally important connective tissue that transmits force between skeletal muscle and bone. Previous studies have evaluated the architectural designs and mechanical properties of different tendons throughout the body. However, less is known about the underlying transcriptional differences between tendons that may dictate their designs and properties. Therefore, our objective was to develop a comprehensive atlas of the transcriptome of limb tendons in adult mice and rats using systems biology techniques. We selected the Achilles, forepaw digit flexor, patellar, and supraspinatus tendons due to their divergent functions and high rates of injury and tendinopathies in patients. Using RNA sequencing data, we generated the Comparative Tendon Transcriptional Database (CTTDb) that identified substantial diversity in the transcriptomes of tendons both within and across species. Approximately 30% of filtered transcripts were differentially regulated between tendons of a given species, and nearly 60% of the filtered transcripts present in anatomically similar tendons were different between species. Many of the genes that differed between tendons and across species are important in tissue specification and limb morphogenesis, tendon cell biology and tenogenesis, growth factor signalling, and production and maintenance of the extracellular matrix. This study indicates that tendon is a surprisingly heterogenous tissue with substantial genetic variation based on anatomical location and species.

Adaptive and innate immune cell responses in tendons and lymph nodes after tendon injury and repair.

Tendon injuries are a common clinical condition with limited treatment options. The cellular components of the innate immune system, such as neutrophils and macrophages, have been studied in tendon injuries. However, the adaptive immune system, comprising specialized lymphocytes, plays an important role in orchestrating the healing of numerous tissues, but less is known about these cells in tendon healing. To gain a greater understanding of the biological processes that regulate tendon healing, we determined how the cellular components of the adaptive and innate immune system respond to a tendon injury using two-month-old male mice. We observed that lymphatic vasculature is present in the epitenon and superficial regions of Achilles tendons, and that the lymphatics drain into the popliteal lymph node. We then created an acute Achilles tenotomy followed by repair, and collected tendons and popliteal lymph nodes 1, 2, and 4 wk after injury. Tendon injury resulted in a robust adaptive immune cell response that followed an initial innate immune cell response in tendons and lymph nodes. Monocytes, neutrophils, and macrophages initially accumulated at 1 wk after injury in tendons, while dendritic cells and CD4+ T cells peaked at 2 wk after injury. B cells and CD8+ T cells progressively increased over time. In parallel, immune cells of the popliteal lymph node demonstrated a similarly coordinated response to the injury. These results suggest that there is an adaptive immune response to tendon injury, and adaptive immune cells may play a role in regulating tendon healing.NEW & NOTEWORTHY While the innate immune system, consisting of macrophages and related hematopoietic cells, has been studied in tendon injury, less is known about the adaptive immune system. Using a mouse model of Achilles tendon tenotomy and repair, we observed an adaptive immune cell response, consisting of CD4+ and CD8+ T cells, and B cells, which occur through 4 wk after tendon injury. This response appeared to be coordinated by the draining popliteal lymph node.

Insulin-like growth factor 1 signaling in tenocytes is required for adult tendon growth.

Tenocytes serve to synthesize and maintain collagen fibrils and other extracellular matrix proteins in tendon. Despite the high prevalence of tendon injury, the underlying biologic mechanisms of postnatal tendon growth and repair are not well understood. IGF1 plays an important role in the growth and remodeling of numerous tissues but less is known about IGF1 in tendon. We hypothesized that IGF1 signaling is required for proper tendon growth in response to mechanical loading through regulation of collagen synthesis and cell proliferation. To test this hypothesis, we conditionally deleted the IGF1 receptor (IGF1R) in scleraxis (Scx)-expressing tenocytes using a tamoxifen-inducible Cre-recombinase system and caused tendon growth in adult mice via mechanical overload of the plantaris tendon. Compared with control Scx-expressing IGF1R-positive (Scx:IGF1R+) mice, in which IGF1R is present in tenocytes, mice that lacked IGF1R in their tenocytes [Scx-expressing IGF1R-negative (Scx:IGF1RΔ) mice] demonstrated reduced cell proliferation and smaller tendons in response to mechanical loading. Additionally, we identified that both the PI3K/protein kinase B and ERK pathways are activated downstream of IGF1 and interact in a coordinated manner to regulate cell proliferation and protein synthesis. These studies indicate that IGF1 signaling is required for proper postnatal tendon growth and support the potential use of IGF1 in the treatment of tendon disorders.-Disser, N. P., Sugg, K. B., Talarek, J. R., Sarver, D. C., Rourke, B. J., Mendias, C. L. Insulin-like growth factor 1 signaling in tenocytes is required for adult tendon growth.

Postnatal tendon growth and remodeling require platelet-derived growth factor receptor signaling.

Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in the fundamental biological activities of many cells that compose musculoskeletal tissues. However, little is known about the role of PDGFR signaling during tendon growth and remodeling in adult animals. Using the hindlimb synergist ablation model of tendon growth, our objectives were to determine the role of PDGFR signaling in the adaptation of tendons subjected to a mechanical growth stimulus, as well as to investigate the biological mechanisms behind this response. We demonstrate that both PDGFRs, PDGFRα and PDGFRß, are expressed in tendon fibroblasts and that the inhibition of PDGFR signaling suppresses the normal growth of tendon tissue in response to mechanical growth cues due to defects in fibroblast proliferation and migration. We also identify membrane type-1 matrix metalloproteinase (MT1-MMP) as an essential proteinase for the migration of tendon fibroblasts through their extracellular matrix. Furthermore, we report that MT1-MMP translation is regulated by phosphoinositide 3-kinase/Akt signaling, while ERK1/2 controls posttranslational trafficking of MT1-MMP to the plasma membrane of tendon fibroblasts. Taken together, these findings demonstrate that PDGFR signaling is necessary for postnatal tendon growth and remodeling and that MT1-MMP is a critical mediator of tendon fibroblast migration and a potential target for the treatment of tendon injuries and diseases.

Local cryotherapy minimally impacts the metabolome and transcriptome of human skeletal muscle.

Cryotherapy is commonly used in the treatment of skeletal muscle injuries. However, the data to support the use of cryotherapy is inconclusive, and the biochemical etiology of cryotherapy in human skeletal muscle remains largely unknown. We therefore sought to determine how a clinically-relevant dose of cryotherapy would impact the transcriptome and metabolome of skeletal muscle. Eight healthy male subjects (age 24.7 ± 4.5 years, BMI 22.2 ± 1.6) received a 15 minute bout of local cryotherapy, delivered via ice cup massage over the anterolateral thigh. This resulted in an 85% decrease in skin temperature and a predicted 27% reduction in intramuscular temperature. The contralateral side served as a non-treated control. Two hours after cryotherapy, muscle biopsies were obtained to analyze changes in the transcriptome, metabolome, and activation of p38 MAPK, ERK1/2, Akt, and p70S6K proteins. No changes were detected in the transcriptome between control and cooled muscles. Cryotherapy reduced levels of hexose sugars and hypoxanthine by 1.3%, but no statistically different changes were observed in 60 additional metabolites. Overall, no differences in phosphorylated p38 MAPK, ERK1/2, Akt, and p70S6K were observed. A clinically relevant dose of cryotherapy produced negligible acute biochemical and molecular changes in the skeletal muscle of human subjects.