Effects of Hydroxyapatite Pretreated with Two Sodium Fluoride Concentrations on Early Bone Healing in Beagle Dogs

Hydroxyapatite (HA) is widely used as an osteoconductive bone substitute, and fluoride can influence bone-forming cells in a concentration-dependent manner. This study examined how pretreating resorbable HA with low or high concentrations of sodium fluoride (NaF) affects early mandibular bone healing in beagle dogs. Twelve beagle dogs were used in the project, including three in a pilot study and nine in the main experiment. At each observation time point (Days 4, 7, and 14), one dog was assigned to each of the three HA-based treatment conditions (HA, HA + 0.2% NaF, or HA + 4% NaF). Four experimental defects in each dog received the same assigned treatment, while four contralateral defects were left unfilled as controls. The four defects were considered within-animal subsamples rather than independent biological replicates, and all treatment comparisons were descriptive and exploratory. Before implantation, HA was exposed to the designated NaF solution for 3 min and then washed with 100 mL of distilled water. Healing was evaluated by H&E, Azan, Masson’s trichrome and toluidine blue staining, immunohistochemistry, and quantitative real-time RT-PCR. Histological examination showed progressive tissue and matrix organization around the implanted material, with more evident new bone and osteoblast-associated changes in the low-NaF group at Days 7 and 14. Runx2 immunoreactivity was more prominent in the early healing phase, and osteocalcin was more prominent in the later healing phase. Osteocalcin mRNA gradually increased until Day 14, whereas Runx2 mRNA was maximally expressed at Day 7. Both responses were strongest with low-NaF-pretreated HA. High-NaF-pretreated HA retained osteogenic activity but did not produce a response of the same magnitude. These exploratory findings suggest that NaF pretreatment concentration may influence early HA-associated bone healing, warranting confirmation in studies with independent animal-level replication.