Chondrocytes are enveloped within the pericellular matrix (PCM), a structurally intricate network primarily demarcated by the presence of collagen type VI microfibrils and perlecan, resembling a protective cocoon. The PCM serves pivotal functions in facilitating cell mechanoprotection and mechanotransduction. The progression of osteoarthritis (OA) is associated with alterations in the spatial arrangement of chondrocytes, transitioning from single strings to double strings, small clusters, and eventually coalescing into large clusters in advanced OA stages. Changes in cellular patters coincide with structural degradation of the PCM and loss of biomechanical properties. Here, we systematically studied matrix metalloproteinases (MMPs), their distribution, activity, and involvement in PCM destruction, utilizing chondrocyte arrangement as an OA biomarker. Cartilage specimens were obtained from 149 osteoarthritis (OA) patients, and selected based on the predominant spatial pattern of chondrocytes. Immunoassays were employed to screen for the presence of various MMPs (-1, -2, -3, -7, -8, -9, -10, -12, -13). Subsequently, the presence and activity of elevated MMPs were further investigated through immunolabeling, western blots and zymograms. Enzymatic assays were utilized to demonstrate the direct involvement of the targeted MMPs in the PCM destruction.Introduction
Methods
Within articular cartilage, chondrocytes reside within the pericellular matrix (PCM), collectively constituting the microanatomical entity known as a chondron. The PCM functions as a pivotal protective shield and mediator of biomechanical and biochemical cues. In the context of Osteoarthritis (OA), enzymatic degradation of the PCM is facilitated by matrix metalloproteinases (MMPs). This study delves into the functional implications of PCM structural integrity decline on the biomechanical properties of chondrons and impact on Ca2+ signaling dynamics. Chondrons isolated from human cartilage explants were incubated with activated MMP-2, -3, or -7. Structural degradation of the pericellular matrix (PCM) was assessed by immunolabelling (collagen type VI and perlecan, n=5). Biomechanical properties of chondrons (i.e. elastic modulus (EM)) were analyzed using atomic force microscopy (AFM). A fluorescent calcium indicator (Fluo-4-AM) was used to record and quantify the intracellular Ca2+ influx of chondrons subjected to single cell mechanical loading (500nN) with AFM (n=7).Introduction
Method
Piezo1 is a mechanosensitive Ca2+ ion channel that has been shown to transduce hyper-physiologic mechanical loads in chondrocytes. In osteoarthritic cartilage, Piezo1 expression was shown to be upregulated by interleukin-1 alpha (IL-1α) and resulted in altered calcium dynamics and actin cytoskeleton rarefication. Together these studies highlight the importance of Piezo1 channels during joint injury. However, the mechanism by which Piezo1 regulates chondrocyte physiology and mechanotransduction during homeostasis is still largely unknown. In this study, we investigate the impact of Piezo1 activation on nuclear mechanics and chromatin methylation state. Porcine chondrocytes (n=3-5 pigs) were treated with Yoda1, a Piezo1-specific agonist, for either 2, 5, 15 or 180 minutes. To characterize chromatin state, we monitored the abundance of a chromatin methylation marker (H3K9Me3) using immunofluorescence (IF). Atomic force microscopy (AFM, 25 nm cantilever) was employed to quantify the nuclear elastic modulus (NEM) of individual cell nuclei. To explore the interplay between cytoskeletal dynamics and nuclear mechanics, chondrocytes were treated with Latrunculin A (LatA), an actin polymerization inhibitor.Introduction
Methods
Osteoarthritis (OA) is the leading cause of pain and disability worldwide and is characterized by the degenerative changes of articular cartilage. Joint loading is required for cartilage maintenance; however, hyper-physiologic loading is a risk factor for OA. Mechanosensitive ion channels Piezo1 and Piezo2 synergistically transduce hyper-physiologic compression of chondrocytes, leading to chondrocyte death and onset of OA. This injury response is inhibited by Piezo channel loss of function, however the mechanistic role of Piezo channels Aggrecan-Cre Knockout of Chondrocyte-specific
Meniscal tears are common knee injuries that subsequently lead to degenerative arthritis, attributed to changes in stress distribution in the knee. In such cases there is need to protect the articular cartilage by repairing or replacing the menisci. While traditionally, meniscal replacement involves implantation of allografts, problems related to availability, size matching, cost and risk of disease transmission limit their use. Another optional treatment is that of biodegradable scaffolds which are based principally on tissue engineering concepts. The variability in body response to biodegradable implants and the quality of the tissue formed still pose a problem in this respect, under intense knee loading conditions. Moreover, biological solutions are mostly limited to younger patients <40 years old. Therefore, the goal of this study was, to develop a synthetic meniscal implant which can replace the injured meniscus, restore its function, and relieve pain. A composite, non-fixed self-centering discoid-shaped meniscus implant (NUsurafce®, AIC, Memphis, TN), composed of polycarbonate-urethane (PCU) and reinforced circumferentially with UHMWPE fibers is proposed (Fig. 1). The implant geometry was based on an extensive MRI study of over 100 knee scans [1]. The proposed structure aims to mimic the circumferential collagen reinforcement of the natural meniscus. Biomechanical evaluation of the implant was focused on in-vitro measurements of contact pressure under the implant in cadaver knees and computational finite element (FE) analyses [2,3]. Pressure distribution on the tibial plateau (under the meniscus implant) was measured by pressure sensitive films (Tekscan, MA) and quantified with respect to the natural meniscus. FE analyses were used to evaluate internal stress and strains, and to support the selection of optimal implant configuration. The last pre-clinical step was a large-animal (sheep) study in which the cartilage condition was evaluated microscopically over six months [4].Statement of Purpose
Methods
Meniscus replacement still represents an unsolved problem in orthopedics. Allograft meniscus implantation has been suggested to restore contact pressures following meniscectomy. However, graft availability, infection, and size matching still limit its use. A synthetic meniscal substitute could have significant advantages for meniscal replacement, as it could be available at the time of surgery in a substantial number of sizes and shapes to accommodate most patients. In the current study we present an optimization method for meniscal implant design and employ in the development of artificial polycarbonate-urethane (PCU) meniscus implant in an ovine model. The construction of the gross implant structure was based on 3D interpolation of MRI scans of the native sheep meniscus in-situ. PCU-based samples based on this design were produced for testing. 35 ovine knee joints were tested. An experimental evaluation of the implants’ biomechanical performances was conducted by measuring pressure distributions on the tibial plateau (TP) during loading. Subsequently, a pressure score of 0 to 100% was calculated. The score reflects on the magnitude of peak pressure and contact area coverage with respect to the natural meniscus. Implant design was reevaluated following changes to the initial implant configuration, e.g., modification of implant geometry, adding reinforcement material, and the applying of different fixation forces during implantation. The effect of these changes on pressure distribution was assessed by additional compression tests. The initial all-PCU implant showed limited ability to distribute pressure, The pressure score of 37% calculated for this case reflects on the small contact area (151mm2) subject to relatively high contact pressures (>
1.85MPa). The implant’s ability to distribute pressure improved significantly when circumferential reinforcement fibers were added. Applying a pretension force of 20N during fixation, improved pressure distribution and increased the contact area (273mm2). A small region of focal pressure concentration still existed in this case, but the pressure score increased markedly to 77%. Finally, it was found that optimal pressure distribution (87%) can be attained when a force of 30 to 50N is applied. In this configuration, peak pressures and coverage area (1.65MPa and 310mm2) were similar to those of the natural meniscus (1.61MPa and 373 mm2, respectively). We conclude that peripheral reinforcement of the implant (similar to the natural meniscus microstructure), in addition to pretension of 30 to 50N can significantly improve TP pressure distributions. The results are in agreement with other studies, reported on pressure distribution improvement due to reinforcement and/or pretension. We believe that the current device can be used in future as a practical solution for patients suffering from severe meniscal injury.
