Mouse MFGE8^high Skeletal Stem Cells Drive Osteogenesis via Matrix Mechanics

The regeneration of large jawbone defects remains challenging as biological mechanisms that drive regional differences in mandibular osteogenesis are not well defined. To elucidate the intrinsic programs of skeletal stem cells (SSCs) that underlie these regional differences, we performed single-cell RNA sequencing of freshly isolated mouse mandibular cells. Single-cell RNA sequencing revealed site-dependent differences in immune cell composition between the mandibular body and ramus and uncovered a ramus-enriched SSC population characterized by high MFGE8 expression. Transcriptomic profiling further revealed that ramus-derived SSCs exhibit coordinated activation of extracellular matrix (ECM)-receptor interactions, focal adhesion, and ossification pathways. Compared with body SSCs, ramus SSCs exhibited superior osteogenic capacity both in vitro and in vivo, accompanied by enrichment of MFGE8⁺ SSCs. Consistent with these findings, MFGE8 knockdown impaired osteogenic differentiation in ramus SSCs, whereas recombinant MFGE8 enhanced osteogenic differentiation in body-derived SSCs. Mechanistically, MFGE8 modulation altered laminin expression and ECM viscoelastic behavior in SSCs, indicating that MFGE8 influences the mechanical properties of the local matrix environment. These effects require integrin-dependent signaling, as MFGE8 binds integrin β3 and promotes focal adhesion kinase (FAK) phosphorylation at the Y397 site. Moreover, inhibition of integrins or FAK abrogated MFGE8-induced osteogenic differentiation, demonstrating that integrin-FAK signaling is required for coupling MFGE8-associated matrix remodeling to osteogenic capacity. Together, these findings provide a mechanistic explanation for the enhanced osteogenic performance of ramus SSCs and identify MFGE8-mediated regulation of ECM biomechanics as a key role for improving jawbone regeneration.