Electromechanical and Polarization Microscopy Evaluation of Cartilage Repair and Degeneration

RÉSUMÉ L’arthrose est une condition physique prévalente pour laquelle aucun traitement permettant de modifier la maladie n’est disponible présentement. La recherche actuelle évalue des modèles pré-cliniques pour le développement et la vérification des approches thérapeutiques, ou de réparation, potentielles. Par la suite, des essais cliniques sont effectués sur des sujets humains sous conditions contrôlées pour évaluer l’efficacité des approches les plus prometteuses. Ces études représentent un effort considérable où le choix de méthodes sensibles et pertinentes pour l’évaluation du cartilage articulaire est très important pour leur succès. Les études pré-cliniques sur l’arthrose impliquent l’utilisation de modèles de blessures d’impact, où une blessure mécanique initiale est créée sur la surface du cartilage, laquelle peut initier les voies de dégradation qui culminent éventuellement en arthrose. L’étude des premiers événements de cette trajectoire est importante car les phases immédiates et aiguës après la blessure sont caractérisées par une augmentation de la réponse cellulaire et des activités de dégradation, ce qui représente une période très propice où les interventions thérapeutiques pour atténuer la progression de la maladie peuvent être les plus efficaces. Dans de telles études, une méthode d’évaluation suffisamment sensible doit être utilisée pour détecter les changements se produisant dans la matrice extracellulaire du cartilage. Dans les études cliniques, une évaluation directe de la qualité du tissu cartilagineux en réparation est réalisée histologiquement à l’aide de petites biopsies obtenues au cours d’une arthroscopie de réévaluation. Les systèmes actuels de notation des coupes histologiques évaluent un nombre de caractéristiques qui contribuent à la durabilité à long terme des tissus cartilagineux en réparation, mais omettent une catégorie spécifique qui permet l’évaluation structurée de l'organisation du collagène. Le réseau de fibrilles de collagène joue un rôle critique sur la capacité de chargement du cartilage et est donc possiblement indicateur de la durabilité de la réparation du cartilage à long terme. Concevoir un système de notation où différents niveaux----------ABSTRACT Osteoarthritis (OA) is a prevalent health condition for which no disease modifying strategies are presently available. Current research incorporates pre-clinical models for the development and testing of potential therapeutic or repair approaches, and subsequently, clinical trials are undertaken, where the most promising strategies are assessed in humans under controlled conditions. These are substantial endeavours requiring sensitive and appropriate evaluation methods for cartilage assessment to ensure their success. Pre-clinical studies of OA include the use of impact injury models, where an initial mechanical insult to the cartilage surfaces can initiate degradative pathways that eventually culminate in OA. Studying early time points in this trajectory are desirable because the immediate and acute phases after injury are characterized by elevated cellular response and degradative activity, representing a window of opportunity where therapeutic intervention to mitigate disease progression may be the most effective. In such studies a sensitive method of cartilage assessment is required to monitor the changes expected to occur in the cartilage extracellular matrix. In clinical trials, direct assessment of cartilage repair tissue quality is performed histologically using small biopsies obtained during a second-look arthroscopy. Current histological scoring systems evaluate a number of characteristics believed to contribute to the long term durability of repair tissues but lack a specific category where a structured evaluation of collagen organization is made. Collagen architecture plays a critical role in cartilage load bearing properties and is therefore likely indicative of the long term durability of cartilage repair tissues. Designing a score where different levels of collagen organization could be assessed using a method sensitive to collagen orientation is required to enhance the analysis of repair tissue quality

Increased Osteoblast GαS Promotes Ossification by Suppressing Cartilage and Enhancing Callus Mineralization During Fracture Repair in Mice

ABSTRACT GαS, the stimulatory G protein α‐subunit that raises intracellular cAMP levels by activating adenylyl cyclase, plays a vital role in bone development, maintenance, and remodeling. Previously, using transgenic mice overexpressing GαS in osteoblasts (GS‐Tg), we demonstrated the influence of osteoblast GαS level on osteogenesis, bone turnover, and skeletal responses to hyperparathyroidism. To further investigate whether alterations in GαS levels affect endochondral bone repair, a postnatal bone regenerative process that recapitulates embryonic bone development, we performed stabilized tibial osteotomy in male GS‐Tg mice at 8 weeks of age and examined the progression of fracture healing by micro‐CT, histomorphometry, and gene expression analysis over a 4‐week period. Bone fractures from GS‐Tg mice exhibited diminished cartilage formation at the time of peak soft callus formation at 1 week post‐fracture followed by significantly enhanced callus mineralization and new bone formation at 2 weeks post‐fracture. The opposing effects on chondrogenesis and osteogenesis were validated by downregulation of chondrogenic markers and upregulation of osteogenic markers. Histomorphometric analysis at times of increased bone formation (2 and 3 weeks post‐fracture) revealed excess fibroblast‐like cells on newly formed woven bone surfaces and elevated osteocyte density in GS‐Tg fractures. Coincident with enhanced callus mineralization and bone formation, GS‐Tg mice showed elevated active β‐catenin and Wntless proteins in osteoblasts at 2 weeks post‐fracture, further substantiated by increased mRNA encoding various canonical Wnts and Wnt target genes, suggesting elevated osteoblastic Wnt secretion and Wnt/β‐catenin signaling. The GS‐Tg bony callus at 4 weeks post‐fracture exhibited greater mineral density and decreased polar moment of inertia, resulting in improved material stiffness. These findings highlight that elevated GαS levels increase Wnt signaling, conferring an increased osteogenic differentiation potential at the expense of chondrogenic differentiation, resulting in improved mechanical integrity. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research

Non-invasive electroarthrography measures cartilage in live horses and correlates to direct measurements of cartilage streaming potentials in weight bearing regions of equine metacarpophalangeal joints

ABSTRACT: Objective To perform non-invasive Electroarthrography (EAG) on live horses and establish relationships between EAG and direct measurements of cartilage streaming potentials in weight bearing areas of the equine metacarpophalangeal joint. Design EAG was performed bilaterally on the metacarpophalangeal joints of live horses (n = 3). Separate experiments used metacarpophalangeal joint explants (n = 11) to measure EAG obtained during simulated loading followed by direct measurements of cartilage streaming potentials on joint surfaces using the Arthro-BST probe. Joints were assigned to relatively normal (n = 5) and mildly degraded (n = 6) groups based on histological scoring of Safranin-O/Fast Green stained sections. Results EAG, involving application of electrodes to skin surrounding the joint and repeated weight shifting, was well-tolerated in live horses. One pair of distal forelimbs were available for analogous ex vivo EAG testing and measurements were strongly correlated to in vivo EAG measurements obtained on the same joints (r = 0.804, p = 0.016, n = 8). Both indirect (EAG) and direct (Arthro-BST) measurements of cartilage streaming potentials distinguished between normal and mildly degraded cartilage with statistically significant differences at 5 of 6 and 4 of 6 electrodes during simulated standing and walking, respectively. Strong and moderate correlations for weight bearing regions on the dorsal phalanx and central metacarpus were detected during both standing and walking. At the metacarpus/sesamoid interface a moderate correlation occurred during walking. Conclusion Non-invasive EAG was used successfully in a clinical scenario and correlated to direct measurements of streaming potentials in weight bearing cartilage. These data support the potential of EAG to contribute to the diagnosis and treatment of degenerative joint diseases

Non-invasive Electroarthrography Measures Load-Induced Cartilage Streaming Potentials via Electrodes Placed on Skin Surrounding an Articular Joint

OBJECTIVE: We aimed to demonstrate that electroarthrography (EAG) measures streaming potentials originating in the cartilage extracellular matrix during load bearing through electrodes adhered to skin surrounding an articular joint. DESIGN: Equine metacarpophalangeal joints were subjected to simulated physiological loads while (1) replacing synovial fluid with immersion buffers of different electrolyte concentrations and (2) directly degrading cartilage with trypsin. RESULTS: An inverse relationship between ionic strength and EAG coefficient was detected. Compared to native synovial fluid, EAG coefficients increased (P < 0.05) for 5 of 6 electrodes immersed in 0.1X phosphate-buffered saline (PBS) (0.014 M NaCl), decreased (P < 0.05) for 4 of 6 electrodes in 1X PBS (0.14 M NaCl), and decreased (P < 0.05) for all 6 electrodes in 10X PBS (1.4 M NaCl). This relationship corresponds to similar studies where streaming potentials were directly measured on cartilage. EAG coefficients, obtained after trypsin degradation, were reduced (P < 0.05) in 6 of 8, and 7 of 8 electrodes, during simulated standing and walking, respectively. Trypsin degradation was confirmed by direct cartilage assessments. Streaming potentials, measured by directly contacting cartilage, indicated lower cartilage stiffness (P < 10(−5)). Unconfined compression data revealed reduced Em, representing proteoglycan matrix stiffness (P = 0.005), no change in Ef, representing collagen network stiffness (P = 0.15), and no change in permeability (P = 0.24). Trypsin depleted proteoglycan as observed by both dimethylmethylene blue assay (P = 0.0005) and safranin-O stained histological sections. CONCLUSION: These data show that non-invasive EAG detects streaming potentials produced by cartilage during joint compression and has potential to become a diagnostic tool capable of detecting early cartilage degeneration

Osteochondral Biopsy Analysis Demonstrates That BST-CarGel Treatment Improves Structural and Cellular Characteristics of Cartilage Repair Tissue Compared With Microfracture

OBJECTIVE: The efficacy and safety of BST-CarGel, a chitosan-based medical device for cartilage repair, was compared with microfracture alone at 1 year during a multicenter randomized controlled trial (RCT) in the knee. The quality of repair tissue of osteochondral biopsies collected from a subset of patients was compared using blinded histological assessments. METHODS: The international RCT evaluated repair tissue quantity and quality by 3-dimensional quantitative magnetic resonance imaging as co-primary endpoints at 12 months. At an average of 13 months posttreatment, 21/41 BST-CarGel and 17/39 microfracture patients underwent elective second look arthroscopies as a tertiary endpoint, during which ICRS (International Cartilage Repair Society) macroscopic scoring was carried out, and osteochondral biopsies were collected. Stained histological sections were evaluated by blinded readers using ICRS I and II histological scoring systems. Collagen organization was evaluated using a polarized light microscopy score. RESULTS: BST-CarGel treatment resulted in significantly better ICRS macroscopic scores (P = 0.0002) compared with microfracture alone, indicating better filling, integration, and tissue appearance. Histologically, BST-CarGel resulted in a significant improvement of structural parameters—Surface Architecture (P = 0.007) and Surface/Superficial Assessment (P = 0.042)—as well as cellular parameters—Cell Viability (P = 0.006) and Cell Distribution (P = 0.032). No histological parameters were significantly better for the microfracture group. BST-CarGel treatment also resulted in a more organized repair tissue with collagen stratification more similar to native hyaline cartilage, as measured by polarized light microscopy scoring (P = 0.0003). CONCLUSION: Multiple and independent analyses in this biopsy substudy demonstrated that BST-CarGel treatment results in improved structural and cellular characteristics of repair tissue at 1 year posttreatment compared with microfracture alone, supporting previously reported results by quantitative magnetic resonance imaging