Sex differences in osteoblast matrix maturation regulate osteoblast-endothelial interactions

Background Bone formation during development and repair is divergently modulated by osteoblast (OB)-derived vascular endothelial growth factor (VEGF) which drives the skeletal sexual dimorphism of the bone vasculature. While the extracellular matrix (ECM) provides both structural and instructive cues to developing vasculature, the contributions of the bone matrix to this skeletal vascular dimorphism in bone remains undefined at the cellular level. Methods Primary OBs were isolated from neonatal female and male C57BL/6 long bones and cultured under basal or osteogenic conditions. ECM composition was quantified by Raman spectroscopy. Primary murine bone marrow endothelial cells (BMECs) were seeded directly onto established OB layers and maintained in heterotypic cocultures to assess contact-mediated effects of OB ECM on BMEC survival and expansion. OB-conditioned media (CM) was used to evaluate soluble-factor contributions, with VEGF-A concentration quantified by ELISA. Results Raman spectroscopy, on individual OBs from monotypic cultures, revealed sexually dimorphic ECM signatures that were independent of cellular growth profiles. Female OB matrices were enriched with type I collagen-specific proline and hydroxyproline and octacalcium phosphate with enhanced collagen intra-strand stability consistent with a matrix-dominant signature. Male OB matrices exhibited relatively lower type I collagen content and higher carbonated apatite resulting in an elevated mineral-to-matrix ratio indicative of advanced mineral maturation. After 24-hours of heterotypic culture of BMECs with OBs, BMEC numbers were 1.39-fold higher when in contact with male OBs. CM treatment of BMECs did not recapitulate these effects despite higher VEGF-A release from male OBs. Conclusions Sex differences in OB ECM are linked to divergent, contact-dependent regulation of BMEC behaviour. These findings suggest that differences in matrix maturation stat contribute to the sex-specific regulation of the skeletal vascular niche. Elucidating the mechanisms that regulate sex-specific OB-ECM production may reveal new therapeutic targets for selectively modulating pathological skeletal angiogenesis in men and women. Summary Bone is a sexually dimorphic organ, with men and women differing in bone size, strength and risk of fracture. The skeletal vasculature is essential for bone growth and repair, with bone forming osteoblast (OB) cells influencing blood vessel development through the skeletal extracellular matrix (ECM). Although the interactions between OB and vascular cells are crucial for lifelong skeletal health, it is not yet known whether sex differences in bone structure between men and women arise from differences in OB activity, or whether this divergence is driven by sex differences in blood vessel growth. Here, we show that male and female mouse OBs deposit distinct ECMs that differentially influence vascular endothelial cell behaviour. Female OBs produce a collagen-rich matrix with low mineral content. In contrast, male OB matrices contain less collagen and more mineral while releasing elevated levels of blood vessel promoting VEGF-A than females. When placed directly onto these OBs, vascular cell growth was greater when in contact with male than female OBs. Notably, this sex-dependent effect requires direct contact between both cell types and was not reproduced by exposure to OB-derived substances alone. These findings identify a cellular mechanism by which sex differences in OB matrix composition influences vascular cell behaviour in bone. Understanding how OB-vascular interactions differ by sex may help explain variation in bone health, healing capacity and disease risk between men and women. Further, our approach may support the discovery of new therapeutic targets that support bone growth and repair while targeting abnormal blood vessel growth in a sex-specific manner. Highlights Primary OBs from male and female C57BL/6 mouse long bones synthesise compositionally distinct ECMs. Female OB matrices are type I collagen-rich and enriched with octacalcium phosphate, whereas male OB matrices contain less type I collagen and higher levels of carbonated apatite. Bone marrow-derived endothelial cell (BMEC) growth is enhanced in heterotypic cocultures with male, but not female, OBs after 24 hours. Male OBs release higher levels of the pro-angiogenic factor VEGF-A than female OBs. The sex-specific effects of the OB ECM on BMECs is contact-dependent and are not reproduced by treatment with OB-derived conditioned media.