Osteoblast-derived CAR3 synergizing with collagen and bone sialoprotein enhances bone formation

Osteoblasts orchestrate the infiltration and crystallization of mineral precursors within collagen fibrils. Certain osteoblast-secreted mineralization-inducing proteins further stimulate bone formation. In this study, scRNA-seq analysis of murine skull and long bone revealed a striking expression pattern of carbonic anhydrase III (Car3) in osteoblasts. We uncovered a pivotal role for CAR3 in osteoblast lineage cells, revealing its critical function in skeletal development and homeostasis. Conditional ablation of Car3 in Prx1-lineage cells resulted in osteopenia and markedly impaired osteoblast activity, underscoring its functional role. Mechanistically, the primary transcription factor RUNX2 directly regulated Car3 expression, mediating its spatiotemporal expression during development. Notably, CAR3 promoted collagen intrafibrillar mineralization by forming a ternary complex with COL1A1 and bone sialoprotein (BSP), thereby facilitating mineral deposition. Furthermore, CAR3-functionalized scaffolds significantly improved bone repair and regeneration by promoting both matrix mineralization and recruitment of Prx1-lineage cells. These findings establish CAR3 as a critical coordinator of osteoblast differentiation and collagen interfibrillar mineralization, positioning it as a central mediator for maintaining skeletal integrity and enabling regeneration.