Ovine bone graft enhances osteogenic commitment in human osteoblasts via selective suppression of miR-6797-5p: In silico and in vitro study

Xenogeneic grafts are widely employed in oral and maxillofacial rehabilitation, but their biological influence extends beyond osteoconduction. Central to osteoblast differentiation is the transcription factor RUNX2, which is tightly regulated by networks of microRNAs (miRNAs). While the osteogenic effects of bovine and porcine grafts have been well studied, little is known about whether ovine bone grafts can alter RUNX2-related post-transcriptional regulation. This study aimed to investigate whether ovine bone graft–derived soluble factors modulate RUNX2-associated miRNAs and thereby influence osteogenic commitment in human osteoblast-lineage cells. Hydroxyapatite was isolated from ovine femoral bone and used to prepare conditioned medium (OBG-CM). Human osteoblastic cells and mesenchymal stem cells were exposed to OBG-CM for 24 h. Candidate RUNX2-targeting miRNAs were first identified in silico using miRDB and STarMir, with overlapping predictions screened by qRT-PCR. Predicted binding was further analysed by TargetScan. Functional assays were carried out by transfecting cells with hsa-miR-6797-5p mimics and inhibitors, followed by assessment of RUNX2 mRNA and protein expression. Computational analysis highlighted several high-confidence RUNX2-targeting miRNAs, including hsa-miR-6797-5p. Exposure to OBG-CM selectively reduced the expression of hsa-miR-6797-5p, hsa-miR-889-5p and hsa-miR-122-5p, while other candidates showed minimal change. TargetScan identified a conserved 8-mer seed match for hsa-miR-6797-5p within the RUNX2 3′UTR. Transfection with the mimic suppressed RUNX2 expression at both transcript and protein levels, whereas inhibition produced the opposite effect. Ovine bone graft–derived factors can selectively remodel the miRNA landscape in osteoblast-lineage cells. These insights may guide the development of bioactive grafts optimised for post-transcriptional regulation.