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

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 .

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.

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.

Artificial calculus synthesized through this novel biofilm mineralization model demonstrated structural and chemical characteristics consistent with early-stage human dental calculus.