Comparative In Vitro Evaluation and Osteogenic Mechanisms of Representative Bone Graft Substitutes: Bioactive Glass, Beta-Tricalcium Phosphate, and Deproteinized Bovine Bone

Objectives: Autologous bone grafting remains the gold standard for maxillofacial reconstruction but is limited by tissue scarcity and donor-site morbidity. Consequently, substitutes like bioactive glass (BG), beta-tricalcium phosphate (β-TCP), and deproteinized bovine bone (DBB) are widely used. However, comprehensive mechanistic comparisons among them remain scarce. Materials and Methods: We systematically evaluated these substitutes under standardized in vitro conditions to compare their physicochemical transformations, degradation profiles, biological performances, and underlying osteogenic molecular pathways. Results: In simulated body fluid, BG underwent rapid hydroxyapatite mineralization, whereas the highly porous DBB and dense β-TCP remained structurally inert. Degradation assays revealed BG exhibited the fastest mass loss and ion release, β-TCP showed intermediate degradation, and DBB maintained high in vitro structural stability. Biologically, all materials showed favorable cytocompatibility and comparable angiogenic potential. However, BG demonstrated significant antibacterial activity (E. coli, S. aureus) and a strong potential to enhance osteogenic differentiation, significantly upregulating the protein-level expression of RUNX2 and OCN, alongside the transcriptional upregulation of Bmp2, Runx2, and Ocn. Transcriptomic profiling and pharmacological validation suggest that the enhanced osteogenic performance of BG might be associated with specific regulatory pathways, supporting the hypothesis that the suppression of NF-κB-mediated inflammation and the activation of the ECM-Integrin-FAK mechanotransduction axis play potential roles. Conclusions: BG offers high bioactivity and notable potential to enhance osteogenic differentiation in vitro but degrades rapidly. DBB ensures structural durability without intrinsic osteoinductivity, and β-TCP provides a balanced, intermediate profile. These in vitro mechanistic insights provide a theoretical foundation for future in vivo evaluations and designing next-generation bone scaffolds.