The subacromial bursa modulates tendon healing after rotator cuff injury in rats.

Rotator cuff injuries result in more than 500,000 surgeries annually in the United States, many of which fail. These surgeries typically involve repair of the injured tendon and removal of the subacromial bursa, a synovial-like tissue that sits between the rotator cuff and the acromion. The subacromial bursa has been implicated in rotator cuff pathogenesis and healing. Using proteomic profiling of bursa samples from nine patients with rotator cuff injury, we show that the bursa responds to injury in the underlying tendon. In a rat model of supraspinatus tenotomy, we evaluated the bursa's effect on the injured supraspinatus tendon, the uninjured infraspinatus tendon, and the underlying humeral head. The bursa protected the intact infraspinatus tendon adjacent to the injured supraspinatus tendon by maintaining its mechanical properties and protected the underlying humeral head by maintaining bone morphometry. The bursa promoted an inflammatory response in injured rat tendon, initiating expression of genes associated with wound healing, including Cox2 and Il6. These results were confirmed in rat bursa organ cultures. To evaluate the potential of the bursa as a therapeutic target, polymer microspheres loaded with dexamethasone were delivered to the intact bursae of rats after tenotomy. Dexamethasone released from the bursa reduced Il1b expression in injured rat supraspinatus tendon, suggesting that the bursa could be used for drug delivery to reduce inflammation in the healing tendon. Our findings indicate that the subacromial bursa contributes to healing in underlying tissues of the shoulder joint, suggesting that its removal during rotator cuff surgery should be reconsidered.

A role for TGFß signaling in Gli1+ tendon and enthesis cells.

The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGFß signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGFß signaling in Gli1+ cell function, the receptor for TGFß, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGFß signaling in these cells led to defects in tendon enthesis formation by P56, including defective bone morphometry underlying the enthesis and decreased mechanical properties. Immunohistochemical staining of these Gli1+ cells showed that loss of TGFß signaling reduced proliferation and increased apoptosis. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGFß controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGFß directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.

Hedgehog Activation for Enhanced Rotator Cuff Tendon-to-Bone Healing.

BACKGROUND: Rotator cuff repair is a common orthopaedic procedure, yet the rate of failure to heal after surgery is high. Repair site rupture is due to poor tendon-to-bone healing and lack of regeneration of the native fibrocartilaginous enthesis. During development, the enthesis is formed and mineralized by a pool of progenitors activated by hedgehog signaling. Furthermore, hedgehog signaling drives regenerative enthesis healing in young animals, in contrast to older animals, in which enthesis injuries heal via fibrovascular scar and without participation of hedgehog signaling. HYPOTHESIS: Hedgehog activation improves tendon-to-bone healing in an animal model of rotator cuff repair. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 78 adult Sprague-Dawley rats were used. Supraspinatus tendon injury and repair were completed bilaterally, with microsphere-encapsulated hedgehog agonist administered to right shoulders and control microspheres administered to left shoulders. Animals were sacrificed after 3, 14, 28, or 56 days. Gene expression and histological, biomechanical, and bone morphometric analyses were conducted. RESULTS: At 3 days, hedgehog signaling pathway genes Gli1 (1.70; P = .029) and Smo (2.06; P = .0173), as well as Runx2 (1.69; P = .0386), a transcription factor of osteogenesis, were upregulated in treated relative to control repairs. At 14 days, transcription factors of tenogenesis, Scx (4.00; P = .041), and chondrogenesis, Sox9 (2.95; P = .010), and mineralized fibrocartilage genes Col2 (3.18; P = .031) and Colx (1.85; P = .006), were upregulated in treated relative to control repairs. Treatment promoted fibrocartilage formation at the healing interface by 28 days, with improvements in tendon-bone maturity, organization, and continuity. Treatment led to improved biomechanical properties. The material property strength (2.43 vs 1.89 N/m2; P = .046) and the structural property work to failure (29.01 vs 18.09 mJ; P = .030) were increased in treated relative to control repairs at 28 days and 56 days, respectively. Treatment had a marginal effect on bone morphometry underlying the repair. Trabecular thickness (0.08 vs 0.07 mm; P = .035) was increased at 28 days. CONCLUSION: Hedgehog agonist treatment activated hedgehog signaling at the tendon-to-bone repair site and prompted increased mineralized fibrocartilage production. This extracellular matrix production and mineralization resulted in improved biomechanical properties, demonstrating the therapeutic potential of hedgehog agonism for improving tendon-to-bone healing after rotator cuff repair. CLINICAL RELEVANCE: This study demonstrates the therapeutic potential of hedgehog agonist treatment for improving tendon-to-bone healing after rotator cuff injury and repair.

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how).

Several tendon and ligament animal models were presented at the 2022 Orthopaedic Research Society Tendon Section Conference held at the University of Pennsylvania, May 5 to 7, 2022. A key objective of the breakout sessions at this meeting was to develop guidelines for the field, including for preclinical tendon and ligament animal models. This review summarizes the perspectives of experts for eight surgical small and large animal models of rotator cuff tear, flexor tendon transection, anterior cruciate ligament tear, and Achilles tendon injury using the framework: "Why, Who, What, Where, When, and How" (5W1H). A notable conclusion is that the perfect tendon model does not exist; there is no single gold standard animal model that represents the totality of tendon and ligament disease. Each model has advantages and disadvantages and should be carefully considered in light of the specific research question. There are also circumstances when an animal model is not the best approach. The wide variety of tendon and ligament pathologies necessitates choices between small and large animal models, different anatomic sites, and a range of factors associated with each model during the planning phase. Attendees agreed on some guiding principles including: providing clear justification for the model selected, providing animal model details at publication, encouraging sharing of protocols and expertise, improving training of research personnel, and considering greater collaboration with veterinarians. A clear path for translating from animal models to clinical practice was also considered as a critical next step for accelerating progress in the tendon and ligament field.

The subacromial bursa is a key regulator of the rotator cuff and a new therapeutic target for improving repair.

Rotator cuff injuries result in over 500,000 surgeries performed annually, an alarmingly high number of which fail. These procedures typically involve repair of the injured tendon and removal of the subacromial bursa. However, recent identification of a resident population of mesenchymal stem cells and inflammatory responsiveness of the bursa to tendinopathy indicate an unexplored biological role of the bursa in the context of rotator cuff disease. Therefore, we aimed to understand the clinical relevance of bursa-tendon crosstalk, characterize the biologic role of the bursa within the shoulder, and test the therapeutic potential for targeting the bursa. Proteomic profiling of patient bursa and tendon samples demonstrated that the bursa is activated by tendon injury. Using a rat to model rotator cuff injury and repair, tenotomy-activated bursa protected the intact tendon adjacent to the injured tendon and maintained the morphology of the underlying bone. The bursa also promoted an early inflammatory response in the injured tendon, initiating key players in wound healing. In vivo results were supported by targeted organ culture studies of the bursa. To examine the potential to therapeutically target the bursa, dexamethasone was delivered to the bursa, prompting a shift in cellular signaling towards resolution of inflammation in the healing tendon. In conclusion, contrary to current clinical practice, the bursa should be retained to the greatest extent possible and provides a new therapeutically target for improving tendon healing outcomes. One Sentence Summary: The subacromial bursa is activated by rotator cuff injury and regulates the paracrine environment of the shoulder to maintain the properties of the underlying tendon and bone.

Guidelines for ex vivo mechanical testing of tendon.

Tendons are critical for the biomechanical function of joints. Tendons connect muscles to bones and allow for the transmission of muscle forces to facilitate joint motion. Therefore, characterizing the tensile mechanical properties of tendons is important for the assessment of functional tendon health and efficacy of treatments for acute and chronic injuries. In this guidelines paper, we review methodological considerations, testing protocols, and key outcome measures for mechanical testing of tendons. The goal of the paper is to present a simple set of guidelines to the nonexpert seeking to perform tendon mechanical tests. The suggested approaches provide rigorous and consistent methodologies for standardized biomechanical characterization of tendon and reporting requirements across laboratories.

The effects of NF-κB suppression on the early healing response following intrasynovial tendon repair in a canine model.

The highly variable clinical outcomes noted after intrasynovial tendon repair have been associated with an early inflammatory response leading to the development of fibrovascular adhesions. Prior efforts to broadly suppress this inflammatory response have been largely unsuccessful. Recent studies have shown that selective inhibition of IkappaB kinase beta (IKK-ß), an upstream activator of nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) signaling, mitigates the early inflammatory response and leads to improved tendon healing outcomes. In the current study, we test the hypothesis that oral treatment with the IKK-ß inhibitor ACHP (2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinenitrile an inhibitor) will modulate the postoperative inflammatory response and improve intrasynovial flexor tendon healing. To test this hypothesis, the flexor digitorum profundus tendon of 21 canines was transected and repaired within the intrasynovial region and assessed after 3 and 14 days. Histomorphometry, gene expression analyses, immunohistochemistry, and quantitative polarized light imaging were used to examine ACHP-mediated changes. ACHP led to reduction in phosphorylated p-65, indicating that NF-κB activity was suppressed. ACHP enhanced expression of inflammation-related genes at 3 days and suppressed expression of these genes at 14 days. Histomorphometry revealed enhanced cellular proliferation and neovascularization in ACHP-treated tendons compared with time-matched controls. These findings demonstrate that ACHP effectively suppressed NF-κB signaling and modulated early inflammation, leading to increased cellular proliferation and neovascularization without stimulating the formation of fibrovascular adhesions. Together, these data suggest that ACHP treatment accelerated the inflammatory and proliferative phases of tendon healing following intrasynovial flexor tendon repair. Clinical Significance: Using a clinically relevant large-animal model, this study revealed that targeted inhibition of nuclear factor kappa-light chain enhancer of activated B cells signaling with ACHP provides a new therapeutic strategy for enhancing the repair of sutured intrasynovial tendons.

Nonlinear time-dependent mechanical behavior of mammalian collagen fibrils.

The viscoelastic mechanical behavior of collagenous tissues has been studied extensively at the macroscale, yet a thorough quantitative understanding of the time-dependent mechanics of the basic building blocks of tissues, the collagen fibrils, is still missing. In order to address this knowledge gap, stress relaxation and creep tests at various stress (5-35 MPa) and strain (5-20%) levels were performed with individual collagen fibrils (average diameter of fully hydrated fibrils: 253 ± 21 nm) in phosphate buffered saline (PBS). The experimental results showed that the time-dependent mechanical behavior of fully hydrated individual collagen fibrils reconstituted from Type I calf skin collagen, is described by strain-dependent stress relaxation and stress-dependent creep functions in both the heel-toe and the linear regimes of deformation in monotonic stress-strain curves. The adaptive quasilinear viscoelastic (QLV) model, originally developed to capture the nonlinear viscoelastic response of collagenous tissues, provided a very good description of the nonlinear stress relaxation and creep behavior of the collagen fibrils. On the other hand, the nonlinear superposition (NSP) model fitted well the creep but not the stress relaxation data. The time constants and rates extracted from the adaptive QLV and the NSP models, respectively, pointed to a faster rate for stress relaxation than creep. This nonlinear viscoelastic behavior of individual collagen fibrils agrees with prior studies of macroscale collagenous tissues, thus demonstrating consistent time-dependent behavior across length scales and tissue hierarchies. STATEMENT OF SIGNIFICANCE: Pure stress relaxation and creep experiments were conducted for the first time with fully hydrated individual collagen fibrils. It is shown that collagen nanofibrils have a nonlinear time-dependent behavior which agrees with prior studies on macroscale collagenous tissues, thus demonstrating consistent time-dependent behavior across length scales and tissue hierarchies. This new insight into the non-linear viscoelastic behavior of the building blocks of mammalian collagenous tissues may serve as the foundation for improved macroscale tissue models that capture the mechanical behavior across length scales.

Metabolic regulation of intrasynovial flexor tendon repair: The effects of dichloroacetate administration on early tendon healing in a canine model.

Enriched in glycolytic enzymes, paucicellular and hypovascular intrasynovial flexor tendons fail to mount an effective healing response after injury and repair. In contrast, well-vascularized extrasynovial flexor tendons possess high levels of oxidative phosphorylation (OXPHOS) enzymes and have a markedly improved healing capacity. This study was designed to compare the metabolic profiles of the two types of tendons and to evaluate the impact of metabolic reprogramming on early intrasynovial tendon healing in a clinically relevant canine model. Results showed that healthy intrasynovial tendons expressed higher levels of PDK1 and GAPDH and lower levels of SCX and IGF1 than did extrasynovial tendons. PDK1 encodes a subtype of pyruvate dehydrogenase kinase (PDK) that inhibits OXPHOS. Consistently, ATP production via glycolysis was favored in intrasynovial tendon cells whereas OXPHOS was the preferred pathway in extrasynovial tendon cells. Inhibition of glycolysis in vitro increased SCX expression in intrasynovial tendon cells. Therefore, dichloroacetate (DCA), a PDK1 inhibitor, was used in vivo to shift intrasynovial tendon ATP production from glycolysis to OXPHOS. Oral DCA administration reduced serum lactate concentration and increased acetyl-CoA content in repaired intrasynovial tendons and led to reduced TLR4 and IL1B and increased IGF1, SCX, and TGFB3 expressions in treated intrasynovial tendons compared to controls. Immunohistochemistry staining with anti-Ki67 and anti-CD31 antibodies revealed marked increases in cellularity and neovascularization in treated intrasynovial tendons. Clinical significance: The findings of this experiment indicate that improved gene expression and histological outcomes can be achieved by regulating glucose metabolism in the early stages following intrasynovial tendon repair.

Tenotomy-induced muscle atrophy is sex-specific and independent of NFκB.

The nuclear factor-κB (NFκB) pathway is a major thoroughfare for skeletal muscle atrophy and is driven by diverse stimuli. Targeted inhibition of NFκB through its canonical mediator IKKß effectively mitigates loss of muscle mass across many conditions, from denervation to unloading to cancer. In this study, we used gain- and loss-of-function mouse models to examine the role of NFκB in muscle atrophy following rotator cuff tenotomy - a model of chronic rotator cuff tear. IKKß was knocked down or constitutively activated in muscle-specific inducible transgenic mice to elicit a twofold gain or loss of NFκB signaling. Surprisingly, neither knockdown of IKKß nor overexpression of caIKKß significantly altered the loss of muscle mass following tenotomy. This finding was consistent across measures of morphological adaptation (fiber cross-sectional area, fiber length, fiber number), tissue pathology (fibrosis and fatty infiltration), and intracellular signaling (ubiquitin-proteasome, autophagy). Intriguingly, late-stage tenotomy-induced atrophy was exacerbated in male mice compared with female mice. This sex specificity was driven by ongoing decreases in fiber cross-sectional area, which paralleled the accumulation of large autophagic vesicles in male, but not female muscle. These findings suggest that tenotomy-induced atrophy is not dependent on NFκB and instead may be regulated by autophagy in a sex-specific manner.

Muscle atrophy ­ the gradual loss of muscle mass ­ follows injuries to our muscles, tendons, or joints. During atrophy, muscles shrink and become weaker, which can interfere with everyday activities and, ultimately, decrease quality of life. Rotator cuff tears are a common example of such injuries. A rotator cuff is group of four muscles that come together as tendons to form a cuff that normally stabilises our shoulders and allows us to lift and move our arms over our heads. Rotator cuff tears can result from an injury or may be caused by ageing-related wear and tear of the tendon. A signalling protein, called NFκB, is thought to be involved in muscle atrophy. When the NFκB signal is switched on, it interacts with genes that are thought to speed up the loss of muscle mass. However, NFκB's precise role in atrophy and recovery after muscle injury is still poorly understood, particularly following injuries where a tendon is cut or torn. Meyer et al. therefore set out to determine whether or not NFκB played a role in the muscle atrophy following rotator cuff tears. Meyer et al. used genetically engineered mice in which NFκB's signal could be turned off at the time of rotator cuff injury, and specifically in muscle cells (but not other parts of the body). The experiments revealed that stopping NFκß signalling in these mice did not reduce muscle atrophy after a rotator cuff injury: the levels of atrophy, muscle performance, and muscle composition were the same regardless of whether the NFκß signal was active. The sex of the mice did, however, affect muscle atrophy, specifically the way in which they lost muscle mass. In male mice, the size of muscle cells decreased, while in female mice, the number of muscle cells decreased. Muscle cells in male mice (but not in females) also accumulated abnormally high amounts of protein, which is an indication of a mechanism of muscle breakdown called autophagy. These results shed new light on the way that we lose muscle mass after injury, and how that could vary depending on the individual. Meyer et al. hope that this study will help guide the development of new, more effective treatments for muscle atrophy, and ultimately contribute to therapies tailored to the characteristics of the patient and the type of injury.

A mineralizing pool of Gli1-expressing progenitors builds the tendon enthesis and demonstrates therapeutic potential.

The enthesis, a fibrocartilaginous transition between tendon and bone, is necessary for force transfer from muscle to bone to produce joint motion. The enthesis is prone to injury due to mechanical demands, and it cannot regenerate. A better understanding of how the enthesis develops will lead to more effective therapies to prevent pathology and promote regeneration. Here, we used single-cell RNA sequencing to define the developmental transcriptome of the mouse entheses over postnatal stages. Six resident cell types, including enthesis progenitors and mineralizing chondrocytes, were identified along with their transcription factor regulons and temporal regulation. Following the prior discovery of the necessity of Gli1-lineage cells for mouse enthesis development and healing, we then examined their transcriptomes at single-cell resolution and demonstrated clonogenicity and multipotency of the Gli1-expressing progenitors. Transplantation of Gli1-lineage cells to mouse enthesis injuries improved healing, demonstrating their therapeutic potential for enthesis regeneration.

Neonatal Enthesis Healing Involves Noninflammatory Acellular Scar Formation through Extracellular Matrix Secretion by Resident Cells.

Wound healing typically recruits the immune and vascular systems to restore tissue structure and function. However, injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Herein, this study established an injury model to the Achilles entheses of neonatal mice to study the effectiveness of early-age enthesis healing. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including endoplasmic reticulum stress, autophagy, and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. These findings reveal a novel healing mechanism in neonatal entheses, whereby local extracellular matrix secretion by resident cells forms an acellular extracellular matrix deposit without inflammation, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.

Pegylated insulin-like growth factor-1 biotherapeutic delivery promotes rotator cuff regeneration in a rat model.

Tears in the rotator cuff are challenging to repair because of the complex, hypocellular, hypovascular, and movement-active nature of the tendon and its enthesis. Insulin-like Growth Factor-1 (IGF-1) is a promising therapeutic for this repair. However, its unstable nature, short half-life, and ability to disrupt homeostasis has limited its clinical translation. Pegylation has been shown to improve the stability and sustain IGF-1 levels in the systemic circulation without disrupting homeostasis. To provide localized delivery of IGF-1 in the repaired tendons, we encapsulated pegylated IGF-1 mimic and its controls (unpegylated IGF-1 mimic and recombinant human IGF-1) in polycaprolactone-based matrices and evaluated them in a pre-clinical rodent model of rotator cuff repair. Pegylated-IGF-1 mimic delivery reestablished the characteristic tendon-to-bone enthesis structure and improved tendon tensile properties within 8 weeks of repair compared to controls, signifying the importance of pegylation in this complex tissue regeneration. These results demonstrate a simple and scalable biologic delivery technology alternative to tissue-derived grafts for soft tissue repair.

The use of connective tissue growth factor mimics for flexor tendon repair.

Intrasynovial flexor tendon lacerations of the hand are clinically problematic, typically requiring operative repair and extensive rehabilitation. The small-molecule connective tissue growth factor (CTGF) mimics, oxotremorine M (Oxo-M) and 4-PPBP maleate (4-PPBP), have been shown to improve tendon healing in small animal models by stimulating the expansion and differentiation of perivascular CD146+ cells. To enhance intrasynovial flexor tendon healing, small-molecule CTGF mimics were delivered to repaired canine flexor tendons via porous sutures. In vitro studies demonstrated that Oxo-M and 4-PPBP retained their bioactivity and could be released from porous sutures in a sustained manner. However, in vivo delivery of the CTGF mimics did not improve intrasynovial tendon healing. Histologic analyses and expression of tenogenic, extracellular matrix, inflammation, and remodeling genes showed similar outcomes in treated and untreated repairs across two time points. Although in vitro experiments revealed that CTGF mimics stimulated robust responses in extrasynovial tendon cells, there was no response in intrasynovial tendon cells, explaining the lack of in vivo effects. The results of the current study indicate that therapeutic strategies for tendon repair must carefully consider the environment and cellular makeup of the particular tendon for improving the healing response.

Mechanically Competent Chitosan-Based Bioadhesive for Tendon-to-Bone Repair.

Current suture-based surgical techniques used to repair torn rotator cuff tendons do not result in mechanically competent tendon-to-bone attachments, leading to high postoperative failure rates. Although adhesives have been proposed to protect against sutures tearing through tendon during healing, no currently available adhesive meets the clinical needs of adhesive strength, biocompatibility, and promotion of healing. Here, a biocompatible, graded, 3,4-dihydroxy phenyl chitosan (BGC) bioadhesive designed to meet these needs is presented. Although 3,4-dihydroxy phenyl chitosan (DP-chitosan) bioadhesives are biocompatible, their adhesion strength is low; soluble oxidants or cross-linking agents can be added for higher bonding strength, but this sacrifices biocompatibility. These challenges are overcome by developing a periodate-modified ion exchange resin-bead filtration system that oxidizes catechol moieties to quinones and filters off the activating agent and resin. The resulting BGC bioadhesive exhibited sixfold higher strength compared to commercially available tissue adhesives, with strength in the range necessary to improve tendon-to-bone repair (≈1MPa, ≈20% of current suture repair strength). The bioadhesive is biocompatible and promoted tenogenesis; cells exposed to the bioadhesive demonstrated enhanced expression of collagen I and the tenogenic marker Scx. Results demonstrated that the bioadhesive has the potential to improve the strength of a tendon-to-bone repair and promote healing.

Augmentation of Rotator Cuff Healing With Orthobiologics.

The limited regenerative capacity of the tendon-bone enthesis after surgical repair poses a significant challenge to achieving desired clinical outcomes. Biologic augmentation of the repair site has the potential to enhance the biomechanical and histological integrity of the enthesis, leading to lower retear rates and greater patient satisfaction. Platelet-rich plasma, stem cells and bone marrow aspirate concentrate, growth factors, biodegradable or biomimetic scaffolds, and amniotic products have been investigated in preclinical and, in some cases, clinical studies aimed at augmenting tendon-bone healing. Although many of these therapies have achieved some degree of success in improving structural, histological, and clinical outcomes after surgical tendon-bone enthesis repair, none have reliably and consistently lead to clinical improvement. High-quality randomized controlled clinical studies are needed to definitively evaluate the efficacy of these biologic therapies and ultimately determine which, if any, are capable of achieving a tendon-bone repair that is structurally noninferior to the native enthesis before injury.

Hedgehog signaling underlying tendon and enthesis development and pathology.

Hedgehog (Hh) signaling has been widely acknowledged to play essential roles in many developmental processes, including endochondral ossification and growth plate maintenance. Furthermore, a rising number of studies have shown that Hh signaling is necessary for tendon enthesis development. Specifically, the well-tuned regulation of Hh signaling during development drives the formation of a mineral gradient across the tendon enthesis fibrocartilage. However, aberrant Hh signaling can also lead to pathologic heterotopic ossification in tendon or osteophyte formation at the enthesis. Therefore, the therapeutic potential of Hh signaling modulation for treating tendon and enthesis diseases remains uncertain. For example, increased Hh signaling may enhance tendon-to-bone healing by promoting the formation of mineralized fibrocartilage at the healing interface, but pathologic heterotopic ossification may also be triggered in the adjacent tendon. Further work is needed to elucidate the distinct functions of Hh signaling in the tendon and enthesis to support the development of therapies that target the pathway.

Transient neonatal shoulder paralysis causes early osteoarthritis in a mouse model.

Neonatal brachial plexus palsy (NBPP) occurs in approximately 1.5 of every 1,000 live births. The majority of children with NBPP recover function of the shoulder. However, the long-term risk of osteoarthritis (OA) in this population is unknown. The purpose of this study was to investigate the development of OA in a mouse model of transient neonatal shoulder paralysis. Neonatal mice were injected twice per week for 4 weeks with saline in the right supraspinatus muscle (Saline, control) and botulinum toxin A (BtxA, transient paralysis) in the left supraspinatus muscle, and then allowed to recover for 20 or 36 weeks. Control mice received no injections, and all mice were sacrificed at 24 or 40 weeks. BtxA mice exhibited abnormalities in gait compared to controls through 10 weeks of age, but these differences did not persist into adulthood. BtxA shoulders had decreased bone volume (-9%) and abnormal trabecular microstructure compared to controls. Histomorphometry analysis demonstrated that BtxA shoulders had higher murine shoulder arthritis scale scores (+30%), and therefore more shoulder OA compared to controls. Articular cartilage of BtxA shoulders demonstrated stiffening of the tissue. Compared with controls, articular cartilage from BtxA shoulders had 2-fold and 10-fold decreases in Dkk1 and BMP2 expression, respectively, and 3-fold and 14-fold increases in Col10A1 and BGLAP expression, respectively, consistent with established models of OA. In summary, a brief period of paralysis of the neonatal mouse shoulder was sufficient to generate early signs of OA in adult cartilage and bone.

The role of loading in murine models of rotator cuff disease.

Rotator cuff disease pathogenesis is associated with intrinsic (e.g., age, joint laxity, muscle weakness) and extrinsic (e.g., mechanical load, fatigue) factors that lead to chronic degeneration of the cuff tissues. However, etiological studies are difficult to perform in patients due to the long duration of disease onset and progression. Therefore, the purpose of this study was to determine the effects of altered joint loading on the rotator cuff. Mice were subjected to one of three load-dependent rotator cuff tendinopathy models: underuse loading, achieved by injecting botulinum toxin-A into the supraspinatus muscle; overuse loading, achieved using downhill treadmill running; destabilization loading, achieved by surgical excision of the infraspinatus tendon. All models were compared to cage activity animals. Whole joint function was assessed longitudinally using gait analysis. Tissue-scale structure and function were determined using microCT, tensile testing, and histology. The molecular response of the supraspinatus tendon and enthesis was determined by measuring the expression of 84 wound healing-associated genes. Underuse and destabilization altered forepaw weight-bearing, decreased tendon-to-bone attachment strength, decreased mineral density of the humeral epiphysis, and reduced tendon strength. Transcriptional activity of the underuse group returned to baseline levels by 4 weeks, while destabilization had significant upregulation of inflammation, growth factors, and extracellular matrix remodeling genes. Surprisingly, overuse activity caused changes in walking patterns, increased tendon stiffness, and primarily suppressed expression of wound healing-related genes. In summary, the tendinopathy models demonstrated how divergent muscle loading can result in clinically relevant alterations in rotator cuff structure, function, and gene expression.