Bone Regeneration of Critical-Size Calvarial Defects in Rats Using Highly Pressed Nano-Apatite/Collagen Composites

Osteo-conductive bone substitute materials are required in dentistry. In this study, highly pressed nano-hydroxyapatite/collagen (P-nHAP/COL) composites were formed by a hydraulic press. Critical-size bone defects (Φ = 6 mm) were made in the cranial bones of 10-week-old Wistar rats, in which P-nHAP/COL and pressed collagen (P-COL) specimens were implanted. Defect-only samples (DEF) were also prepared. After the rats had been nourished for 3 days, 4 weeks, or 8 weeks, ossification of the cranial defects of the rats was evaluated by micro-computed tomography (micro-CT) (n = 6 each). Animals were sacrificed at 8 weeks, followed by histological examination. On micro-CT, the opacity of the defect significantly increased with time after P-nHAP/COL implantation (between 3 days and 8 weeks, p < 0.05) due to active bone regeneration. In contrast, with P-COL and DEF, the opacity increased only slightly with time after implantation, indicating sluggish bone regeneration. Histological inspections of the defect zone implanted with P-nHAP/COL indicated the adherence of multinucleated giant cells (osteoclasts) to the implant with phagocytosis and fragmentation of P-nHAP/COL, whereas active bone formation occurred nearby. Fluorescent double staining indicated dynamic bone-formation activities. P-nHAP/COL is strongly osteo-conductive and could serve as a useful novel bone substitute material for future dental implant treatments.