In vitro biomimetic development of artificial dental calculus: a proof of concept study

BACKGROUND: To develop an in vitro model to generate dental calculus through a standardized process of biofilm mineralization, and compare it with natural human dental calculus . METHODS: The proposed in vitro model consisted of two staged procedures: first biofilm formation and then its mineralization. After a 72-hour pre-culture, biofilms of Streptococcus mutans were formed on a glass dish in artificial saliva with 1% sucrose for 3 h. Then the calcification process consisted of incubating the sample in supersaturated mineral solution for 21 h. These steps were repeated for 5 days. The samples were then divided into 3 groups based on the different processes; (i) mineralization only, (ii) biofilm formation only, (iii) biofilm formation and mineralization. Obtained samples were compared morphologically with human supragingival calculus using scanning electron microscopy, quantitative light-induced fluorescence, and energy-dispersive X-ray spectroscopy. Confocal laser scanning microscopy was used to assess bacterial distribution and viability, and X-ray diffraction analysis was performed to evaluate the crystallinity of the mineral deposits. A solubility test was also conducted to assess structural stability. RESULTS: The artificially developed calculus exhibited similar characteristics to human dental calculus including a layered structure with bacterial embedded in a porous mineral matrix, and comparable ratio of inorganic components. No weight loss was observed before or after the solubility test. CONCLUSION: Artificial calculus synthesized through this novel biofilm mineralization model demonstrated structural and chemical characteristics consistent with early-stage human dental calculus.