Effects of HCl concentration and immersion time on physicochemical properties, BMP-2 detection, degradation, and osteoblast response of human demineralized tooth matrix

Objective: Human demineralized tooth matrix (hDTM) is a promising tooth-derived graft material; however, demineralization protocols vary widely and may alter matrix structure, degradation behavior, and growth factor detectability. This study investigates the combined effects of hydrochloric acid (HCl) concentration (0.5 vs 1.0M) and immersion time (10 vs 20 min) on the physicochemical characteristics, Bone Morphogenetic Protein-2 (BMP-2) detection, degradation behavior and osteoblast response of hDTM.Methods: Caries-free permanent teeth (particle size 500–1000 µm) were demineralized using four HCl protocols: 0.5M/10 min, 0.5M/20 min, 1M/10 min, and 1M/20 min. Morphology, crystallinity, and surface features were characterized by scanning electron microscopy (SEM), X-ray diffraction, X-ray fluorescence, and Brunauer-Emmett-Teller analysis. Total protein and BMP-2 were quantified using Bradford and enzyme-linked immunosorbent assay (ELISA) assays. In-vitro degradation was monitored for 60 days in simulated body fluid. MC3T3-E1 osteoblast adhesion and proliferation were evaluated using the SEM and PrestoBlue® assay. Two-way analysis of variance was performed to assess main and interaction effects of concentration and time.Results: Both HCl concentration and immersion time significantly influenced hDTM properties. Lower concentration enlarged dentinal tubules and collagen exposure, whereas higher concentration produced surface microcracking and reduced collagen exposure. Higher HCl concentration increased degradation (1M/20 min: 23.76 ± 3.23% vs. 0.5M/20 min: 9.02 ± 0.63%, p < 0.01), while longer immersion increased ELISA-detected BMP-2 levels (1M/20 min: 11.8 ± 1.9 ng/g; 0.5M/20 min: 5.3 ± 1.6 ng/g vs. control: 0.1 ng/g, p < 0.001). Significant independent and interactive effects of HCl concentration and immersion time were observed (p < 0.001). Among the test conditions, a 0.5M/20 min protocol demonstrated balanced collagen preservation, controlled degradation, and favorable osteoblast proliferation.Conclusion: HCl concentration and immersion time independently and interactively modulate hDTM properties. Within the test conditions, 0.5M HCl for 20 minutes provides a practical solution and balances collagen preservation, controlled degradation, and osteoblast proliferation, supporting its use as a feasible approach for fabricating tooth-derived bone graft materials.