ABSTRACT
OBJECTIVE: Osteoarthritis (OA) is a disease with sex-dependent prevalence and severity in both human and animal models. We sought to elucidate sex differences in synovitis, mechanical sensitization, structural damage, bone remodeling, and the synovial transcriptome in the anterior cruciate ligament rupture (ACLR) mouse model of post-traumatic OA (PTOA). DESIGN: Male and female 12-week-old C57/BL6J mice were randomized to Sham or noninvasive ACLR with harvests at 7d or 28d post-ACLR (n = 9 per sex in each group - Sham, 7d ACLR, 28d ACLR). Knee hyperalgesia, mechanical allodynia, and intra-articular matrix metalloproteinase (MMP) activity (via intravital imaging) were measured longitudinally. Trabecular and subchondral bone (SCB) remodeling and osteophyte formation were assessed by µCT. Histological scoring of PTOA, synovitis, and anti-MMP13 immunostaining were performed. NaV1.8-Cre;tdTomato mice were used to document localization and sprouting of nociceptors. Bulk RNA-seq of synovium in Sham, 7d, and 28d post-ACLR, and contralateral joints (n = 6 per group per sex) assessed injury-induced and sex-dependent gene expression. RESULTS: Male mice exhibited more severe joint damage at 7d and 28d and more severe synovitis at 28d, accompanied by 19% greater MMP activity, 8% lower knee hyperalgesia threshold, and 43% lower hindpaw withdrawal threshold in injured limbs compared to female injured limbs. Females had injury-induced catabolic responses in trabecular and SCB, whereas males exhibited 133% greater normalized osteophyte volume relative to females and sclerotic remodeling of trabecular and SCB. NaV1.8+ nociceptor sprouting in SCB and medial synovium was induced by injury and comparable between sexes. RNA-seq of synovium demonstrated similar injury-induced transcriptomic programs between the sexes at 7d, but only female mice exhibited a transcriptomic signature indicative of synovial inflammatory resolution by 28d, whereas males had persistent pro-inflammatory, pro-fibrotic, pro-neurogenic, and pro-angiogenic gene expression. CONCLUSION: Male mice exhibited more severe overall joint damage and pain behavior after ACLR, which was associated with persistent activation of synovial inflammatory, fibrotic, and neuroangiogenic processes, implicating persistent synovitis in driving sex differences in murine PTOA.
ABSTRACT
OBJECTIVES: Synovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/ß-catenin signalling, given its emerging importance in arthritis. METHODS: We subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain of function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types. RESULTS: We uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/ß-catenin signalling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages and chondrocytes. CONCLUSIONS: Synovial fibroblasts assume distinct functional identities during PTOA in mice, and Prg4hi lining fibroblasts secrete Rspo2 that may drive pathological joint crosstalk after injury.
