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.

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.

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.

Red blood cell exposure increases chondrocyte susceptibility to oxidative stress following hemarthrosis.

OBJECTIVE: The detrimental effects of blood exposure on articular tissues are well characterized, but the individual contributions of specific whole blood components are yet to be fully elucidated. Better understanding of mechanisms that drive cell and tissue damage in hemophilic arthropathy will inform novel therapeutic strategies. The studies here aimed to identify the specific contributions of intact and lysed red blood cells (RBCs) on cartilage and the therapeutic potential of Ferrostatin-1 in the context of lipid changes, oxidative stress, and ferroptosis. METHODS: Changes to biochemical and mechanical properties following intact RBC treatment were assessed in human chondrocyte-based tissue-engineered cartilage constructs and validated against human cartilage explants. Chondrocyte monolayers were assayed for changes to intracellular lipid profiles and the presence of oxidative and ferroptotic mechanisms. RESULTS: Markers of tissue breakdown were observed in cartilage constructs without parallel losses in DNA (control: 786.3 (102.2) ng/mg; RBCINT: 751 (126.4) ng/mg; P = 0.6279), implicating nonlethal chondrocyte responses to intact RBCs. Dose-dependent loss of viability in response to intact and lysed RBCs was observed in chondrocyte monolayers, with greater toxicity observed with lysates. Intact RBCs induced changes to chondrocyte lipid profiles, upregulating highly oxidizable fatty acids (e.g., FA 18:2) and matrix disrupting ceramides. RBC lysates induced cell death via oxidative mechanisms that resemble ferroptosis. CONCLUSIONS: Intact RBCs induce intracellular phenotypic changes to chondrocytes that increase vulnerability to tissue damage while lysed RBCs have a more direct influence on chondrocyte death by mechanisms that are representative of ferroptosis.

Blocking toll-like receptor 4 mitigates static loading induced pro-inflammatory expression in intervertebral disc motion segments.

While the anabolic effects of mechanical loading on the intervertebral disc (IVD) have been extensively studied, inflammatory responses to loading have not been as well characterized. Recent studies have highlighted a significant role of innate immune activation, particularly that of toll-like receptors (TLRs), in IVD degeneration. Biological responses of intervertebral disc cells to loading depend on many factors that include magnitude and frequency. The goals of this study were to characterize the inflammatory signaling changes in response to static and dynamic loading of IVD and investigate the contributions of TLR4 signaling in response to mechanical loading. Rat bone-disc-bone motion segments were loaded for 3 hr under a static load (20 % strain, 0 Hz) with or without an additional low-dynamic (4 % dynamic strain, 0.5 Hz) or high-dynamic (8 % dynamic strain, 3 Hz) strain, and results were compared to unloaded controls. Some samples were also loaded with or without TAK-242, an inhibitor of TLR4 signaling. The magnitude of NO release into the loading media (LM) was correlated with the applied frequency and strain magnitudes across different loading groups. Injurious loading profiles, such as static and high-dynamic, significantly increased Tlr4 and Hmgb1 expression while this result was not observed in the more physiologically relevant low-dynamic loading group. TAK-242 co-treatment decreased pro-inflammatory expression in static but not dynamic loaded groups, suggesting that TLR4 plays a direct role in mediating inflammatory responses of IVD to static compression. Overall, the microenvironment induced by dynamic loading diminished the protective effects of the TAK-242, suggesting that TLR4 plays a direct role in mediating inflammatory responses of IVD to static loading injury.

Toward defining the role of the synovium in mitigating normal articular cartilage wear and tear.

Cartilage repair has been studied extensively in the context of injury and disease, but the joint's management of regular sub-injurious damage to cartilage, or 'wear and tear,' which occurs due to normal activity, is poorly understood. We hypothesize that this cartilage maintenance is mediated in part by cells derived from the synovium that migrate to the worn articular surface. Here, we demonstrate in vitro that the early steps required for such a process can occur. First, we show that under physiologic mechanical loads, chondrocyte death occurs in the cartilage superficial zone along with changes to the cartilage surface topography. Second, we show that synoviocytes are released from the synovial lining under physiologic loads and attach to worn cartilage. Third, we show that synoviocytes parachuted onto a simulated or native cartilage surface will modify their behavior. Specifically, we show that synoviocyte interactions with chondrocytes lead to changes in synoviocyte mechanosensitivity, and we demonstrate that cartilage-attached synoviocytes can express COL2A1, a hallmark of the chondrogenic phenotype. Our findings suggest that synoviocyte-mediated repair of cartilage 'wear and tear' as a component of joint homeostasis is feasible and is deserving of future study.

Toward Development of a Diabetic Synovium Culture Model.

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation and inflammation of synovium, the specialized connective tissue that envelops the diarthrodial joint. Type 2 diabetes mellitus (DM) is often found in OA patients, with nearly double the incidence of arthritis reported in patients with diabetes (52%) than those without it (27%). The correlation between OA and DM has been attributed to similar risk factors, namely increasing age and joint loading due to obesity. However, a potential causative link is not well understood due to comorbidities involved with treating diabetic patients, such as high infection rates and poor healing response caused by hyperglycemia and insulin resistance. The purpose of this study was to investigate the effect of hyperglycemic and insulin culture conditions on synovium properties. It was hypothesized that modeling hyperglycemia-induced insulin resistance in synovium would provide novel insights of OA pathogenesis in DM patients. To simulate DM in the synovial joint, healthy synovium was preconditioned in either euglycemic (EG) or hyperglycemic (HG) glucose concentrations with insulin in order to induce the biological response of the diseased phenotype. Synovium biochemical composition was evaluated to determine ECM remodeling under hyperglycemic culture conditions. Concurrent changes in AKT phosphorylation, a signaling pathway implicated in insulin resistance, were measured along with gene expression data for insulin receptors, glucose transporters, and specific glycolysis markers involved in glucose regulation. Since fluid shear stress arising during joint articulation is a relevant upstream stimulus for fibroblast-like synoviocytes (FLS), the predominant cell type in synovium, FLS mechanotransduction was evaluated via intracellular calcium ([Ca2+]i). Incidence and length of primary cilia, a critical effector of cell mechanosensing, were measured as potential mechanisms to support differences in [Ca2+]i responses. Hyperglycemic culture conditions decreased collagen and GAG content compared to EG groups, while insulin recovered ECM constituents. FLS mechanosensitivity was significantly greater in EG and insulin conditions compared to HG and non-insulin treated groups. Hyperglycemic treatment led to decreased incidence and length of primary cilia and decreased AKT phosphorylation, providing possible links to the mechanosensing response and suggesting a potential correlation between glycemic culture conditions, diabetic insulin resistance, and OA development.

Sustained Delivery of SB-431542, a Type I Transforming Growth Factor Beta-1 Receptor Inhibitor, to Prevent Arthrofibrosis.

Fibrosis of the knee is a common disorder resulting from an aberrant wound healing response and is characterized by extracellular matrix deposition, joint contraction, and scar tissue formation. The principal regulator of the fibrotic cascade is transforming growth factor beta-1 (TGF-ß1), a factor that induces rapid proliferation and differentiation of resident fibroblasts. In this study, we demonstrate successful inhibition of TGF-ß1-driven myofibroblastic differentiation in human fibroblast-like synoviocytes using a small molecule TGF-ß1 receptor inhibitor, SB-431542. We also demonstrate successful encapsulation of SB-431542 in poly(D,L-lactide-co-glycolide) (PLGA) as a potential prophylactic treatment for arthrofibrosis and characterize drug release and bioactivity in a three-dimensional collagen gel contraction assay. We assessed the effects of TGF-ß1 and SB-431542 on cell proliferation and viability in monolayer cultures. Opposing dose-dependent trends were observed in cell proliferation, which increased in TGF-ß1-treated cultures and decreased in SB-431542-treated cultures relative to control (p < 0.05). SB-431542 was not cytotoxic at the concentrations studied (0-50 µM) and inhibited TGF-ß1-induced collagen gel contraction in a dose-dependent manner. Specifically, TGF-ß1-treated gels contracted to 18% ± 1% of their initial surface area, while gels treated with TGF-ß1 and ≥10 µM SB-431542 showed no evidence of contraction (p < 0.0001). Upon removal of the compound, all gels contracted to control levels after 44 h in culture, necessitating sustained delivery for prolonged inhibition. To this end, SB-431542 was encapsulated in PLGA microspheres (SBMS) that had an average diameter of 87.5 ± 24 µm and a loading capacity of 4.3 µg SB-431542 per milligram of SBMS. Functional assessment of SBMS revealed sustained inhibition of TGF-ß1-induced gel contraction as well as hallmark features of myofibroblastic differentiation, including α-smooth muscle actin expression and connective tissue growth factor production. These results suggest that SB-431542 may be used to counter TGF-ß1-driven events in the fibrotic cascade in the knee cartilage. Impact statement Arthrofibrosis is the most prevalent comorbidity resulting from orthopedic procedures such as total knee arthroplasty that is characterized by excess deposition and accumulation of extracellular matrix. Despite its prevalence, treatments are generally palliative, and there is no effective prophylactic therapy. We report that the small molecule transforming growth factor beta-1 (TGF-ß1) receptor inhibitor, SB-431542, can inhibit the TGF-ß1-driven myofibroblastic differentiation of fibroblast-like synoviocytes. To provide sustained inhibition, we explored the use of SB-laden microspheres as a prophylactic therapy in a three-dimensional contraction model of fibrosis and propose that such therapies will have the potential to improve the standard of care for arthrofibrosis.

Sustained low-dose dexamethasone delivery via a PLGA microsphere-embedded agarose implant for enhanced osteochondral repair.

Articular cartilage defects are a common source of joint pain and dysfunction. We hypothesized that sustained low-dose dexamethasone (DEX) delivery via an acellular osteochondral implant would have a dual pro-anabolic and anti-catabolic effect, both supporting the functional integrity of adjacent graft and host tissue while also attenuating inflammation caused by iatrogenic injury. An acellular agarose hydrogel carrier with embedded DEX-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres (DLMS) was developed to provide sustained release for at least 99 days. The DLMS implant was first evaluated in an in vitro pro-inflammatory model of cartilage degradation. The implant was chondroprotective, as indicated by maintenance of Young's modulus (EY) (p = 0.92) and GAG content (p = 1.0) in the presence of interleukin-1ß insult. In a subsequent preliminary in vivo experiment, an osteochondral autograft transfer was performed using a pre-clinical canine model. DLMS implants were press-fit into the autograft donor site and compared to intra-articular DEX injection (INJ) or no DEX (CTL). Functional scores for DLMS animals returned to baseline (p = 0.39), whereas CTL and INJ remained significantly worse at 6 months (p < 0.05). DLMS knees were significantly more likely to have improved OARSI scores for proteoglycan, chondrocyte, and collagen pathology (p < 0.05). However, no significant improvements in synovial fluid cytokine content were observed. In conclusion, utilizing a targeted DLMS implant, we observed in vitro chondroprotection in the presence of IL-1-induced degradation and improved in vivo functional outcomes. These improved outcomes were correlated with superior histological scores but not necessarily a dampened inflammatory response, suggesting a primarily pro-anabolic effect. STATEMENT OF SIGNIFICANCE: Articular cartilage defects are a common source of joint pain and dysfunction. Effective treatment of these injuries may prevent the progression of osteoarthritis and reduce the need for total joint replacement. Dexamethasone, a potent glucocorticoid with concomitant anti-catabolic and pro-anabolic effects on cartilage, may serve as an adjuvant for a variety of repair strategies. Utilizing a dexamethasone-loaded osteochondral implant with controlled release characteristics, we demonstrated in vitro chondroprotection in the presence of IL-1-induced degradation and improved in vivo functional outcomes following osteochondral repair. These improved outcomes were correlated with superior histological cartilage scores and minimal-to-no comorbidity, which is a risk with high dose dexamethasone injections. Using this model of cartilage restoration, we have for the first time shown the application of targeted, low-dose dexamethasone for improved healing in a preclinical model of focal defect repair.

Cartilage Wear Particles Induce an Inflammatory Response Similar to Cytokines in Human Fibroblast-Like Synoviocytes.

The synovium plays a key role in the development of osteoarthritis, as evidenced by pathological changes to the tissue observed in both early and late stages of the disease. One such change is the attachment of cartilage wear particles to the synovial intima. While this phenomenon has been well observed clinically, little is known of the biological effects that such particles have on resident cells in the synovium. The present work investigates the hypothesis that cartilage wear particles elicit a pro-inflammatory response in diseased and healthy human fibroblast-like synoviocytes, like that induced by key cytokines in osteoarthritis. Fibroblast-like synoviocytes from 15 osteoarthritic human donors and a subset of three non-osteoarthritic donors were exposed to cartilage wear particles, interleukin-1α or tumor necrosis factor-α for 6 days and analyzed for proliferation, matrix production, and release of pro-inflammatory mediators and degradative enzymes. Wear particles significantly increased proliferation and release of nitric oxide, interleukin-6 and -8, and matrix metalloproteinase-9, -10, and -13 in osteoarthritic synoviocytes, mirroring the effects of both cytokines, with similar trends in non-osteoarthritic cells. These results suggest that cartilage wear particles are a relevant physical factor in the osteoarthritic environment, perpetuating the pro-inflammatory and pro-degradative cascade by modulating synoviocyte behavior at early and late stages of the disease. Future work points to therapeutic strategies for slowing disease progression that target cell-particle interactions. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1979-1987, 2019.

A Functional Tissue-Engineered Synovium Model to Study Osteoarthritis Progression and Treatment.

IMPACT STATEMENT: The synovium envelops the diarthrodial joint and plays a key regulatory role in defining the composition of the synovial fluid through filtration and biosynthesis of critical boundary lubricants. Synovium changes often precede cartilage damage in osteoarthritis. We describe a novel in vitro tissue engineered model, validated against native synovium explants, to investigate the structure-function of synovium through quantitative solute transport measures. Synovium was evaluated in the presence of a proinflammatory cytokine, interleukin-1, or the clinically relevant corticosteroid, dexamethasone. We anticipate that a better understanding of synovium transport would support efforts to develop more effective strategies aimed at restoring joint health.