Background: Fracture healing involves complex cellular and molecular interactions. While osteoprotegerin (OPG) facilitates bone repair by inhibiting osteoclast activity, its role in chondrocyte differentiation and cartilage formation remains unclear. This study examined the effects of OPG on bone and cartilage during fracture repair.

Methods: A murine femoral fracture model was established and treated with subcutaneous injections of OPG recombinant protein or OPG-neutralizing antibodies. Bone healing was assessed by micro-computed tomography (Micro-CT) and Safranin O staining to evaluate callus size and cartilage formation. Immunohistochemistry detected Ace-tubulin, γ-tubulin, Ki-67, and Collagen II expression. qRT-PCR assessed chondrogenic markers, including SRY-box transcription factor 9 (Sox9), Collagen II, Aggrecan, and Collagen X. OPG shRNA was transfected into chondrocytes to evaluate cell viability (CCK-8 assay), Ace-tubulin and γ-tubulin expression (immunofluorescence), and acidic mucopolysaccharides (Alcian blue staining). Western blot was performed to examine the effect of OPG on the Transforming Growth Factor-β (TGF-β) pathway.

Results: OPG expression was significantly elevated during fracture healing (p < 0.05). Administration of recombinant OPG increased fracture callus size and improved bone parameters, including bone volume/total volume (BV/TV) ratio, trabecular number (Tb.N), and trabecular thickness (Tb.Th), while reducing trabecular separation (Tb.Sp) (p < 0.05). Conversely, OPG blockade produced opposite effects (p < 0.05). OPG treatment also upregulated Ace-tubulin, γ-tubulin, Ki-67, Sox9, Collagen II, Aggrecan, and Collagen X (p < 0.05), whereas anti-OPG reduced their expression (p < 0.05). In vitro, OPG knockdown reduced chondrocyte viability, diminished Ace-tubulin and γ-tubulin expression, reduced acidic mucopolysaccharide accumulation, and downregulated chondrogenic markers (p < 0.05). Furthermore, OPG silencing suppressed TGF-β signaling by lowering TGF-β1, TGF-βRI, and Smad2/3 phosphorylation (p < 0.05).

Conclusions: OPG promotes chondrocyte differentiation and bone formation by regulating TGF-β signaling, highlighting its potential as a therapeutic target for enhancing fracture healing.