Amy L. McNulty

Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage.

Significance Cartilage, a mechanically sensitive tissue that covers joints, is essential for vertebrate locomotion by sustaining skeletal mobility. Transduction of mechanical stimuli by cartilage cells, chondrocytes, leads to biochemical–metabolic responses. Such mechanotransduction can be beneficial for tissue maintenance when evoked by low-level mechanical stimuli, or can have health-adverse effects via cartilage-damaging high-strain mechanical stress. Thus, high-strain mechanotransduction by cartilage mechanotrauma is relevant for the pathogenesis of osteoarthritis. Molecular mechanisms of high-strain mechanotransduction of chondrocytes have been elusive. Here we identify Piezo1 and Piezo2 mechanosensitive ion channels in chondrocytes as transduction channels for high-strain mechanical stress. We verify their functional link to the cytoskeleton as important for their concerted function and offer a remedial strategy by application of a Piezo1/2 blocking peptide, GsMTx4, from tarantula venom. Diarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca2+ signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca2+ transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1- or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains.

Temporomandibular joint pain: a critical role for Trpv4 in the trigeminal ganglion.

&NA; Trpv4 is critical for sensitization of trigeminal ganglion sensory neurons that innervate the inflamed temporomandibular joint, as shown by using bite‐force measurements in the laboratory mouse to measure nocifensive behavior. &NA; Temporomandibular joint disorder (TMJD) is known for its mastication‐associated pain. TMJD is medically relevant because of its prevalence, severity, chronicity, the therapy‐refractoriness of its pain, and its largely elusive pathogenesis. Against this background, we sought to investigate the pathogenetic contributions of the calcium‐permeable TRPV4 ion channel, robustly expressed in the trigeminal ganglion sensory neurons, to TMJ inflammation and pain behavior. We demonstrate here that TRPV4 is critical for TMJ‐inflammation‐evoked pain behavior in mice and that trigeminal ganglion pronociceptive changes are TRPV4‐dependent. As a quantitative metric, bite force was recorded as evidence of masticatory sensitization, in keeping with human translational studies. In Trpv4−/− mice with TMJ inflammation, attenuation of bite force was significantly less than in wildtype (WT) mice. Similar effects were seen with systemic application of a specific TRPV4 inhibitor. TMJ inflammation and mandibular bony changes were apparent after injections of complete Freund adjuvant but were remarkably independent of the Trpv4 genotype. It was intriguing that, as a result of TMJ inflammation, WT mice exhibited significant upregulation of TRPV4 and phosphorylated extracellular‐signal–regulated kinase (ERK) in TMJ‐innervating trigeminal sensory neurons, which were absent in Trpv4−/− mice. Mice with genetically‐impaired MEK/ERK phosphorylation in neurons showed resistance to reduction of bite force similar to that of Trpv4−/− mice. Thus, TRPV4 is necessary for masticatory sensitization in TMJ inflammation and probably functions upstream of MEK/ERK phosphorylation in trigeminal ganglion sensory neurons in vivo. TRPV4 therefore represents a novel pronociceptive target in TMJ inflammation and should be considered a target of interest in human TMJD.

Synovial Fluid Concentrations and Relative Potency of Interleukin-1 alpha and beta in Cartilage and Meniscus Degradation

Cartilage degeneration with osteoarthritis (OA) is believed to involve the activities of interleukin‐1 (IL‐1), which exists as alpha and beta isoforms. The goal of this study was to measure the concentrations of both isoforms of IL‐1 in the synovial fluid of normal and spontaneously osteoarthritic porcine knees, and to test the hypothesis that physiologic concentrations of IL‐1α and IL‐1β exhibit different potencies in activating calcium signaling, the production of matrix metalloproteinases and nitric oxide, and the loss of proteoglycans and tissue mechanical properties in cartilage and meniscus. Median concentrations of IL‐1α were 0.043 ng/ml with mild OA and 0.288 ng/ml with moderate OA, whereas IL‐1β concentrations were 0.109 ng/ml with mild OA and 0.122 ng/ml with moderate OA. Both isoforms induced calcium signaling in chondrocytes and meniscal cells at all concentrations. Overall, cartilage and meniscus catabolism was significantly more sensitive to IL‐1α than IL‐1β at concentrations of 1 ng/ml or less, while few differences were observed between the two forms at 10 ng/ml. These data provide a range of physiologic IL‐1 concentrations that can serve as a framework for the comparison of various in vitro studies, as well as providing further insight for the development of anti‐cytokine therapies for OA.

The Mechanobiology of Articular Cartilage: Bearing the Burden of Osteoarthritis

Articular cartilage injuries and degenerative joint diseases are responsible for progressive pain and disability in millions of people worldwide, yet there is currently no treatment available to restore full joint functionality. As the tissue functions under mechanical load, an understanding of the physiologic or pathologic effects of biomechanical factors on cartilage physiology is of particular interest. Here, we highlight studies that have measured cartilage deformation at scales ranging from the macroscale to the microscale, as well as the responses of the resident cartilage cells, chondrocytes, to mechanical loading using in vitro and in vivo approaches. From these studies, it is clear that there exists a complex interplay among mechanical, inflammatory, and biochemical factors that can either support or inhibit cartilage matrix homeostasis under normal or pathologic conditions. Understanding these interactions is an important step toward developing tissue engineering approaches and therapeutic interventions for cartilage pathologies, such as osteoarthritis.

The Effects of Adipokines on Cartilage and Meniscus Catabolism

Obesity is one of the primary risk factors for osteoarthritis. Increased adiposity is associated not only with alterations in joint loading, but also with increased systemic and joint concentrations of adipose tissue-derived cytokines, or “adipokines,” that promote a state of chronic, low-grade inflammation that may act in concert with other cytokines in the joint to increase joint degeneration. However, the direct effect of adipokines, such as leptin, visfatin, and interleukin-6 (IL-6), on joint tissues, such as articular cartilage and meniscus, are not fully understood. In this study, we examined the hypothesis that these adipokines act synergistically with interleukin-1 (IL-1) to increase catabolism and the production of proinflammatory mediators in cartilage and meniscus. Explants of porcine cartilage and meniscus were treated with physiologically relevant concentrations of leptin, IL-6, or visfatin, alone or in combination with IL-1. Visfatin and IL-1 promoted the catabolic degradation of both cartilage and meniscus, as evidenced by increased metalloproteinase activity, nitric oxide production, and proteoglycan release. However, leptin or IL-6 at physiologic concentrations had no effect on the breakdown of these tissues. These findings suggest that the effects of obesity-induced osteoarthritis may not be through a direct effect of leptin or IL-6 on cartilaginous tissues, but support a potential role for increased visfatin levels in this regard. These data provide an important first step in understanding the role of adipokines in regulating cartilage and meniscus metabolism; however, these adipokines may have different effects in the context of the whole joint and must be evaluated further.

Dynamic Loading Enhances Integrative Meniscal Repair in the Presence of Interleukin-1

OBJECTIVE Meniscal tears are a common knee injury and increased levels of interleukin-1 (IL-1) have been measured in injured and degenerated joints. Studies have shown that IL-1 decreases the shear strength, cell accumulation, and tissue formation in meniscal repair interfaces. While mechanical stress and IL-1 modulate meniscal biosynthesis and degradation, the effects of dynamic loading on meniscal repair are unknown. The purpose of this study was to determine the effects of mechanical compression on meniscal repair under normal and inflammatory conditions. EXPERIMENTAL DESIGN Explants were harvested from porcine medial menisci. To simulate a full-thickness defect, a central core was removed and reinserted. Explants were loaded for 4h/day at 1 Hz and 0%-26% strain for 14 days in the presence of 0 or 100 pg/mL of IL-1. Media were assessed for matrix metalloproteinase (MMP) activity, aggrecanase activity, sulfated glycosaminoglycan (S-GAG) release, and nitric oxide (NO) production. After 14 days, biomechanical testing and histological analyses were performed. RESULTS IL-1 increased MMP activity, S-GAG release, and NO production, while decreasing the shear strength and tissue repair in the interface. Dynamic loading antagonized IL-1-mediated inhibition of repair at all strain amplitudes. Neither IL-1 treatment nor strain altered aggrecanase activity. Additionally, strain alone did not alter meniscal healing, except at the highest strain magnitude (26%), a level that enhanced the strength of repair. CONCLUSIONS Dynamic loading blocked the catabolic effects of IL-1 on meniscal repair, suggesting that joint loading through physical therapy may be beneficial in promoting healing of meniscal lesions under inflammatory conditions.

Integrative repair of the meniscus: Lessons from in vitro studies

Current therapies for meniscal injury seek to preserve and repair damaged tissue since loss of meniscal tissue is associated with degenerative changes in the joint, ultimately leading to osteoarthritis (OA). After a meniscal tear, the difficulty of integrating juxtaposed meniscal surfaces continues to be an obstacle. In order to determine the local factors that are necessary for successful tissue repair, previous studies have developed in vitro model systems that allow both biological and quantitative biomechanical measures of meniscus repair. Many studies have shown the importance of individual factors in meniscus metabolism, but there is a complex interplay among a variety of factors that influence meniscal healing, including inflammatory cytokines, growth factors, mechanical loading, and zonal differences in cell and tissue properties. In particular, the upregulation of inflammatory cytokines following joint injury appears to have significant catabolic influences on meniscal cell metabolic activity that must be overcome in order to promote repair. In the presence of inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor alpha (TNF-alpha), intrinsic meniscal repair in vitro is significantly inhibited. While anabolic growth factors, such as transforming growth factor-beta1 (TGF-beta1), enhance meniscal repair, they cannot completely overcome the IL-1-mediated inhibition of repair. The mechanisms by which these mediators influence meniscal repair, and their interactions with other factors in the microenvironment, such as mechanical loading, remain to be determined. Future studies must address these complex interactions during meniscal healing to ultimately enhance meniscal repair.

Cartilage viability and catabolism in the intact porcine knee following transarticular impact loading with and without articular fracture

Posttraumatic arthritis commonly develops following articular fracture. The objective of this study was to develop a closed joint model of transarticular impact with and without creation of an articular fracture that maintains the physiologic environment during loading. Fresh intact porcine knees were preloaded and impacted at 294 J via a drop track. Osteochondral cores were obtained from the medial and lateral aspects of the femoral condyles and tibial plateau. Chondrocyte viability was assessed at days 0, 3, and 5 postimpact in sham, impacted nonfractured, and impacted fractured joints. Total matrix metalloproteinase (MMP) activity, aggrecanase (ADAMTS‐4) activity, and sulfated glycosaminoglycan (S‐GAG) release were measured in culture media from days 3 and 5 posttrauma. No differences were observed in chondrocyte viability of impacted nonfractured joints (95.9 ± 6.9%) when compared to sham joints (93.8 ± 7.7%). In impacted fractured joints, viability of the fractured edge was 40.5 ± 27.6% and significantly lower than all other sites, including cartilage adjacent to the fractured edge (p < 0.001). MMP and aggrecanase activity and S‐GAG release were significantly increased in specimens from the fractured edge. This study showed that joint impact resulting in articular fracture significantly decreased chondrocyte viability, increased production of MMPs and aggrecanases, and enhanced S‐GAG release, whereas the same level of impact without fracture did not cause such changes.

Mechanobiology of the meniscus

The meniscus plays a critical biomechanical role in the knee, providing load support, joint stability, and congruity. Importantly, growing evidence indicates that the mechanobiologic response of meniscal cells plays a critical role in the physiologic, pathologic, and repair responses of the meniscus. Here we review experimental and theoretical studies that have begun to directly measure the biomechanical effects of joint loading on the meniscus under physiologic and pathologic conditions, showing that the menisci are exposed to high contact stresses, resulting in a complex and nonuniform stress-strain environment within the tissue. By combining microscale measurements of the mechanical properties of meniscal cells and their pericellular and extracellular matrix regions, theoretical and experimental models indicate that the cells in the meniscus are exposed to a complex and inhomogeneous environment of stress, strain, fluid pressure, fluid flow, and a variety of physicochemical factors. Studies across a range of culture systems from isolated cells to tissues have revealed that the biological response of meniscal cells is directly influenced by physical factors, such as tension, compression, and hydrostatic pressure. In addition, these studies have provided new insights into the mechanotransduction mechanisms by which physical signals are converted into metabolic or pro/anti-inflammatory responses. Taken together, these in vivo and in vitro studies show that mechanical factors play an important role in the health, degeneration, and regeneration of the meniscus. A more thorough understanding of the mechanobiologic responses of the meniscus will hopefully lead to therapeutic approaches to prevent degeneration and enhance repair of the meniscus.

In vivo cartilage strain increases following medial meniscal tear and correlates with synovial fluid matrix metalloproteinase activity

Meniscal tears are common injuries, and while partial meniscectomy is a frequent treatment option, general meniscus loss is a risk factor for the development of osteoarthritis. The goal of this study was to measure the in vivo tibiofemoral cartilage contact patterns in patients with meniscus tears in relation to biomarkers of cartilage catabolism in the synovial fluid of these joints. A combination of magnetic resonance imaging and biplanar fluoroscopy was used to determine the in vivo motion and cartilage contact mechanics of the knee. Subjects with isolated medial meniscus tears were analyzed while performing a quasi-static lunge, and the contralateral uninjured knee was used as a control. Synovial fluid was collected from the injured knee and matrix metalloproteinase (MMP) activity, sulfated glycosaminoglycan, cartilage oligomeric matrix protein, prostaglandin E2, and the collagen type II cleavage biomarker C2C were measured. Contact strain in the medial compartment increased significantly in the injured knees compared to contralateral control knees. In the lateral compartment, the contact strain in the injured knee was significantly increased only at the maximum flexion angle (105°). The average cartilage strain at maximum flexion positively correlated with total MMP activity in the synovial fluid. These findings show that meniscal injury leads to loss of normal joint function and increased strain of the articular cartilage, which correlated to elevated total MMP activity in the synovial fluid. The increased strain and total MMP activity may reflect, or potentially contribute to, the early development of osteoarthritis that is observed following meniscal injury.