SLIT3 promotes osteogenic differentiation in osteoarthritic subchondral bone

Dysregulated osteogenic activity in subchondral bone is closely associated with osteoarthritis (OA) progression. Slit guidance ligand 3 (SLIT3) has been implicated in bone metabolism and OA-associated angiogenesis; however, its cellular distribution in OA subchondral bone and its role in osteogenic differentiation remain incompletely understood. Here, we integrated microarray data from GSE51588 and single-cell RNA sequencing data from GSE196678 to characterize SLIT3 expression in OA subchondral bone and validated the findings in human tibial plateau subchondral bone samples. The role of SLIT3 in osteogenic differentiation was examined using siRNA-mediated Slit3 silencing and recombinant SLIT3 treatment in MC3T3-E1 cells, while its in vivo relevance was evaluated in a destabilization of the medial meniscus (DMM) model using global Slit3 -knockout mice. SLIT3 expression was elevated in OA subchondral bone, was enriched in osteoblast-lineage cell clusters, and showed a progressive increase along the inferred osteogenic differentiation trajectory. In vitro, Slit3 knockdown suppressed osteogenic differentiation, whereas recombinant SLIT3 enhanced osteogenic differentiation. In vivo, global Slit3 deficiency was associated with reduced osteogenic marker expression in subchondral bone, accompanied by attenuated cartilage degeneration in the DMM model. Collectively, these findings demonstrate that SLIT3 promotes osteogenic differentiation in vitro and support a role for SLIT3 in OA-associated osteogenic alterations in subchondral bone.