Accelerated bone defect closure after administration of nano bovine hydroxyapatite-calcium sulfate-gelatin scaffold in bone defect models

Context: Bone defects associated with fractures exceeding 2 cm in circumference present significant clinical challenges due to the high risk of nonunion or malunion. Scaffold implantation offers a non-toxic, effective strategy to support bone regeneration and restore structural integrity. Aims: To evaluate and compare the regenerative effects of bovine hydroxyapatite-calcium sulfate-gelatin (BHA-CS-gelatin) and nano-BHA-CS-gelatin scaffolds. Focus was placed on cellular morphology, including osteoblasts, osteocytes, and osteoclasts, cell migration, and the expression of osteogenic markers: collagen type I (COL1), Runx2, osteocalcin, and osteoprotegerin (OPG). Methods: In vivo experiments used New Zealand White rabbits with surgically induced bone defects treated with either BHA-CS-gelatin or nano-BHA-CS-gelatin scaffolds. Bone regeneration was assessed by histological, immunohistochemical, and ELISA analyses. Rabbits were sacrificed on days 7 and 28 for histology and immunohistochemistry evaluations. OPG levels were measured by ELISA on days 0, 1, 3, 5, 7, 14, and 28. Results: Histological analysis showed enhanced migration of osteoblasts, osteocytes, and osteoclasts in the nano-BHA-CS-gelatin group. Immunohistochemistry revealed significantly higher expression of COL1, Runx2, and osteocalcin in this group at day 28 (p<0.05). ELISA demonstrated a significant increase in OPG levels on day 14 compared with the conventional BHA-CS-gelatin scaffold group (p<0.05). Conclusions: Nano-BHA-CS-gelatin scaffolds significantly accelerate bone defect closure and enhance osteogenic marker expression more effectively than conventional scaffolds. These results support their preclinical potential application in bone repair and regenerative medicine.