Spatiotemporal transcriptomic landscape of synovial joint repair – an in vivo murine multimodal model of osteochondral injury

Abstract Objective The repair response to focal osteochondral injuries frequently fails to truly restore native osteochondral tissue, predisposing the joint to the likelihood of progressive degeneration and post-traumatic osteoarthritis. The biological mechanisms governing the earliest stages of repair in these tissues remain poorly understood, limiting the development of effective regenerative therapies. We therefore aimed to define the early cellular and spatial organisation of repair in a reproducible murine osteochondral injury model by integrating single cell spatial transcriptomics across the whole joint with longitudinal structural imaging and histological analyses. Design A reproducible, non-critical osteochondral injury was created in the trochlear groove of female C57BL/6 mice. Structural repair was assessed using a multimodal approaching comprising quantitative histology, immunophenotyping, longitudinal magnetic resonance imaging (MRI) and micro-computed tomography (µCT), while whole-joint Xenium spatial transcriptomics at days 3 and 7 defined the cellular and molecular organisation of the early repair response. Results Spatial transcriptomics demonstrated that the first week after injury is characterised by the emergence of anatomically distinct immune, vascular and stromal microenvironments across the synovial joint. Resolution of the early inflammatory response was accompanied by regional organisation of repair-associated stromal populations by day 7 after injury. The synovium preferentially supported matrix-associated fibro-chondrocyte-like cells, whereas the osteochondral injury itself retained stress-responsive stromal states with comparatively limited representation of matrix-associated populations. These findings indicate that distinct anatomical niches within the joint are associated with transcriptionally distinct stromal cell phenotypes during early repair. Longitudinal MRI and µCT and histological analysis, demonstrated that these early spatial differences in cell phenotype were associated with progressive restoration of osteochondral architecture, with more effective regeneration of subchondral bone and limited restoration of native articular cartilage. Conclusions This study provides, to our knowledge, the first spatially resolved transcriptomic analysis of the early osteochondral repair response to injury across the whole synovial joint. Our findings demonstrate that the first week after injury establishes spatially organised immune, vascular and stromal cell microenvironments. Furthermore, these data suggest that incomplete cartilage repair may reflect an initial failure to establish and sustain matrix-associated stromal cellular states within the injury niche. These findings identify the early repair microenvironment as a critical determinant of tissue regeneration and provide a rationale for regenerative strategies that target repair with spatial and temporal precision.