ObjectivesSynovium 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/β-catenin signalling, given its emerging importance in arthritis.MethodsWe 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.ResultsWe uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/β-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.ConclusionsSynovial fibroblasts assume distinct functional identities during PTOA in mice, and Prg4hi lining fibroblasts secrete Rspo2 that may drive pathological joint crosstalk after injury.

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Conventional tissue engineering approaches rely on scaffold-based delivery of exogenous proteins, genes, and/or cells to stimulate regeneration via growth factor signaling. However, scaffold-based approaches do not allow active control of dose, timing, or spatial localization of a delivered growth factor once the scaffold is implanted, yet these are all crucial parameters in promoting tissue regeneration. To address this limitation, we developed a stable cell line containing a heat-activated and rapamycin dependent gene expression system. In this study, we investigate how high intensity focused ultrasound (HIFU) can spatiotemporally control firefly luciferase (fLuc) transgene activity both in vitro and in vivo by the tightly controlled generation of hyperthermia. Cells were incorporated into composite scaffolds containing fibrin and hydroxyapatite particles, which yielded significant increases in acoustic attenuation and heating in response to HIFU compared to fibrin alone. Using 2.5 MHz HIFU, transgene activation was observed at acoustic intensities of 201 W/cm 2 and higher. Transgene activation was spatially patterned in the scaffolds by rastering HIFU at speeds up to 0.15 mm/s. In an in vivo study, a 67-fold increase in fLuc activity was