Dental microcosm biofilm formation

Dental microcosm biofilms were formed using the model described in detail by Lee et al.¹⁹ Ethical approval for collecting human saliva was granted by the Ethics Committee of Yonsei Dental Hospital, Korea (IRB No. 02-2015-0051). The study was performed according to the guidelines of the Declaration of Helsinki. Saliva samples were collected from healthy adult donors who had not performed oral hygiene within 24 h, and had not taken antibiotics or exhibited any cariogenic activity or periodontal disease within the last three months. Informed written consent was obtained from the donors. The collected saliva samples were filtered through sterilized glass wool (Duksan Pure Chemicals Co., Ltd., Ansan, Korea) and then diluted in sterile glycerol (Duksan Pure Chemicals Co., Ltd., Ansan, Korea) to prepare stock solutions with a final concentration of 30%. The stock solutions were stored at − 80 °C, and the same batch of frozen saliva was used as an inoculum for each set of experiments. The stock saliva (1.5 ml) was inoculated with each of the specimens in a 24-well cell plate and incubated at 37 °C and 10% CO₂ for 4 h. The specimens were transferred to new wells with a basal medium mucin growth medium mixed with 0.3% sucrose and incubated in an anaerobic environment (80% N₂, 10% CO₂, and 10% H₂) at 37 °C for 7 days to form microcosm biofilms.