Significance: Cartilage mineralization is imperative in various processes such as skeletal growth and fracture repair. However, this process may also be pathological, as in the case of the degenerative joint disease, osteoarthritis (OA). Using a posttraumatic OA model (PTOA), we find that cartilage-specificSirt1genetic nulls caused severe synovitis and mineralization of the lateral joint compartment, due to augmentedLef1gene expression. Conversely, cartilage-specificLef1nulls exhibited impaired synovitis and mineralization of the lateral joint, accompanied by a reduction of local pain. Consistently, transcriptomic profiles ofLef1-ablated chondrocytes exhibited enhanced anabolism, yet impaired pathways related to calcification and inflammation. Accordingly, cartilage mineralization of the lateral joint compartment relies on amplified inflammatory pathways, contributing to articular damage following PTOA.

Osteoarthritis (OA) is characterized by progressive, irreversible erosion of articular cartilage accompanied by severe pain and immobility. This study aimed to assess the effect and mechanism of action of HU308, a selective cannabinoid receptor type 2 (CB2) agonist, in preventing OA-related joint damage. To test the assumption that HU308 could prevent OA-related joint damage, Cnr2 null mice and wild type (WT) mice were aged to reach 20 months and analyzed for joint structural features. OA was induced in WT mice via a post-traumatic procedure or aging, followed by HU308 local (intra-articular) or systemic (intraperitoneal) administration, respectively. Additional analyses of time and dose courses for HU308 were carried out in human primary chondrocytes, analyzed by RNA sequencing, RT-PCR, chromatin immunoprecipitation, and immunoblotting. Our results showed that Cnr2 null mice exhibited enhanced age-related OA severity and synovitis compared to age-matched WT mice. Systemic administration of HU308 to 16-month-old mice improved pain sensitivity and maintained joint integrity, which was consistent with the intra-articular administration of HU308 in post-traumatic OA mice. When assessing human chondrocytes treated with HU308, we uncovered a dose- and time-related increase in ACAN and COL2A1 expression, which was preceded by increased SOX9 expression due to pCREB transcriptional activity. Finally, transcriptomic analysis of patient-derived human chondrocytes identified patient subpopulations exhibiting HU308-responsive trends as judged by enhanced SOX9 expression, accompanied by enriched gene networks related to carbohydrate metabolism. Collectively, the results showed that HU308 reduced trauma and age-induced OA via CB2-pCREB dependent activation of SOX9, contributing to augmented gene networks related to carbohydrate metabolism. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

In cranial flat bone fractures, spontaneous bone repair will occur only when the fracture ends are in close contact. However, in cases wherein bone discontinuity is extensive, surgical interventions are often required. To this end, autologous bone is harvested and surgically integrated into the site of fracture. Here we propose to use cartilage, as an alternative autologous source, to promote cranial fracture repair. The advantage of this approach is the potential reduction in donor site morbidity, likely due to the avascular and aneural nature of cartilage. As a first step we attempted to induce cartilage mineralization in vitro, using micromass primary chondrocyte cultures, incubated with BMP2 and/or WISP1, which were examined histologically following a 3-week culture period. Next, chondrocyte seeded collagen scaffolds were evaluated in vitro for expression profiles and ALP activity. Finally, chondrocyte-seeded collagen scaffolds were implanted in a Lewis rats 8 mm critical calvaria defect model, which was imaged via live CT for 12 weeks until sacrifice. End points were analyzed for microCT, histology, and serum levels of bone related markers. Micromass cultures exhibited an osseous inducing trend following WISP1 administration, which was maintained in chondrocyte seeded scaffolds. Accordingly, in vivo analysis was carried out to assess the impact of WISP1-pretreated chondrocytes (WCS) versus untreated chondrocytes (UCS) in calvaria defect model and compared to untreated control comprised of a defect-associated blood clot (BC) or empty collagen scaffold (CS) implant. Live CT and microCT exhibited higher mineralization volumes in critical defect implanted with UCS, with some structural improvements in WCS. Histological analysis exhibited higher anabolic bone formation in WCS and trabecular bone was detected in WCS and UCS groups. Chondrocytes implanted into critical cranial defect expedite the formation of native-like osseous tissue, especially after WISP1 priming in culture. Ultimately, these data support the use of autologous chondrocytes to repair critical maxillofacial defects.

Background: In a previous report, we have identified the cannabinoid receptor 2 (CB2) agonist HU308 to possess a beneficial effect in preventing age and trauma-induced osteoarthritis (OA) in mice. The effects of HU308 were largely related to the capacity of this compound to induce cartilage anabolism which was dependent on the CREB/SOX9 axis, and exhibited pro-survival and pro-proliferative hallmarks of articular cartilage following treatment. Here, we utilized the novel cannabinoid-fenchone CB2 agonists (1B, 1D), which were previously reported to render anti-inflammatory effects in a zymosan model. Methods: Initially, we assessed the selectivity of CB2 using a Gs-protein receptor cAMP potency assay, which was also validated for antagonistic effects dependent on the Gi-protein receptor cAMP pathway. Based on EC50 values, 1D was selected for a zymosan inflammatory pain model. Next, 1D was administered in two doses intra-articularly (IA), in a post-traumatic medial meniscal tear (MMT, Lewis rats) model, and compared to sham, vehicle, and a positive control consisting of fibroblast growth factor 18 (FGF18) administration. The histopathological assessment was carried out according to the Osteoarthritis Research Society International (OARSI) guidelines for rat models following 28 days post-MMT. Results: The G protein receptor assays confirmed that both 1B and 1D possess CB2 agonistic effects in cell lines and in chondrocytes. Co-administering a CB2 antagonists to 25 mg/kg 1D in a paw inflammatory pain model abolished 1D-related anti-swelling effect and partially abolishing its analgesic effects. Using an MMT model, the high dose (i.e., 24 µg) of 1D administered via IA route, exhibited reduced cartilage damage. Particularly, this dose of 1D exhibited a 30% improvement in cartilage degeneration (zonal/total tibial scores) and lesion depth ratios (44%), comparable to the FGF18 positive control. Synovitis scores remained unaffected and histopathologic evaluation of subchondral bone damage did not suggest that 1D treatment changed the load-bearing ability of the rats. Contrary to the anabolic effect of FGF18, synovial inflammation was observed and was accompanied by increased osteophyte size. Conclusion: The structural histopathological analysis supports a disease-modifying effect of IA-administered 1D compound without any deleterious effects on the joint structure.

Objective: Previous studies have shown that the cleavage of Sirt1 contributes to the development of osteoarthritis (OA). In fact, OA was effectively abrogated by the intra-articular (IA) administration of two compounds, one blocking Sirt1 cleavage (CA074me) and the other activating Sirt1 (SRT1720), using a post-traumatically induced model (PTOA) in young female mice. In this study, we attempted to understand if this local treatment is effective in preventing age-associated OA (AOA) progression and symptoms. Design: A group of 17-month-old female C57BL/6J mice were IA administered with CA074me and/or SRT1720 or their combination. Joint histopathological analysis and bone histomorphometry were carried out, with an assessment of knee mechanical hyperalgesia. A serum analysis for NT/CT Sirt1 was carried out along with immunohistochemistry for articular cartilage to detect p16^INK4A or γH2A.X. Similarly, meniscal cartilage was monitored for Lef1 and Col1a1 deposition. The data were compared for young female mice subjected to post-traumatic OA (PTOA). Results: Similar to PTOA, combination-treated AOA exhibited improved knee hyperalgesia, yet structural improvements were undetected, corresponding to unchanged NT/CT Sirt1 serum levels. Both AOA and PTOA exhibited unchanged staining for nuclear p16^INK4A or γH2A.X and lacked a correlation with OA severity. Contrarily to PTOA, the combination treatment with AOA did not exhibit a local reduction in the Lef1 and Col1 targets. Conclusions: When targeting Sirt1 cleavage, the PTOA and AOA models exhibited a similar pain response to the combination treatment; however, they displayed diverse structural outcomes for joint-related damage, related to Lef1-dependent signaling. Interestingly, nuclear p16^INK4A was unaffected in both models, regardless of the treatment’s effectiveness. Finally, these findings highlight the variations in the responses between two highly researched OA preclinical models, reflecting OA pathophysiology heterogeneity and variations in gender-related drug-response mechanisms.