Rapid bone formation and remodeling via vascular infiltration of a hydroxyapatite/collagen nanocomposite beneath the calvarial periosteum

The development of biomaterials that actively promote bone regeneration remains a major challenge in regenerative medicine and dentistry. This study investigated the time-dependent osteogenic potential of an organic-inorganic composite bone filler (hydroxyapatite/collagen bone-like nanocomposite, HAp/Col) using a rat calvarial model, assessed by immunohistochemistry and bone histomorphometry. By day 3, vascular endothelial growth factor receptor 2 (VEGFR2)-positive cells were detected on the HAp/Col surface, indicating early vascular invasion. By day 5, vascular infiltration had extended into the scaffold, where double staining with tartrate resistant acid phosphatase (TRAP)/ alkaline phosphatase (ALP) revealed osteoclast-mediated material resorption and osteoblast-mediated bone matrix deposition around vascular cavities. By day 7, bone remodeling within HAp/Col was markedly active, with clusters of osteoblasts producing new bone matrix. Outside the scaffold, osteoblasts adhered to its surface and elongated, enhancing bone formation through the space-forming effect of HAp/Col swelling. Furthermore, collagen triple helix repeat-containing protein 1 (CTHRC1) expression increased progressively from days 3 to 7, as confirmed by immunostaining and qRT-PCR, suggesting its role as a coupling factor between osteoclast activity and osteoblast differentiation. These findings suggest that HAp/Col is associated with cellular activities related to bone remodeling, including vascular invasion and osteoclast/osteoblast recruitment. HAp/Col demonstrates biocompatibility and in vivo tissue compatibility as a candidate biomaterial for medical and dental applications.