Effect of Bovine Hydroxyapatite–Gelatin–Vitamin D Supplementation on Bone Regeneration in a Rabbit Femoral Defect Model

Bone defects resulting from fractures remain a major clinical challenge due to impaired bone continuity and limited regenerative capacity, particularly in critical-sized defects. Biomimetic supplementation combining osteoconductive scaffolds, organic matrices, and osteogenic regulators may enhance bone regeneration by accelerating osteogenic maturation. This study aimed to evaluate the effectiveness of a capsule formulation containing bovine hydroxyapatite (BHA), gelatin, and vitamin D in promoting bone regeneration in a rabbit femoral bone defect model using osteogenic markers Runx2, alkaline phosphatase (ALP), and osteocalcin (OCN). Fifteen healthy male New Zealand white rabbits were divided into three groups (n = 5): bone defect (control), commercialized calcium supplementation, and BHA–gelatin–vitamin D supplementation. A standardized femoral bone defect (4 mm diameter × 6 mm depth) was created surgically. Supplements were administered orally for 28 days. Femoral bone samples were collected and analyzed using immunohistochemistry for Runx2, ALP, and OCN expression. Immunoreactive scores (IRS) were assessed semi-quantitatively. Data were analyzed using the Kruskal–Wallis test (p < 0.05). The BHA–gelatin–vitamin D group showed significantly higher IRS values for Runx2, ALP, and OCN than the control group (p < 0.05). No significant differences were observed between the BHA-based formulation and commercial calcium supplementation, indicating comparable osteogenic efficacy. The combination of BHA, gelatin, and vitamin D effectively accelerates bone regeneration by enhancing osteogenic differentiation, matrix formation, and osteoblast maturation. This formulation shows potential as an alternative strategy for promoting bone healing with efficacy comparable to commercially available calcium supplements.