The association of vitamin D and severe early childhood caries: a case-control study / Manal Mohamed Mansour Almoudi

Objectives: This research consists of in vivo and in vitro studies. The in vivo study aims to study the association between serum and saliva vitamin D and severe early childhood caries (S-ECC) in a group of young Malaysian children. The other objective was to determine if salivary LL37 and mutans streptococci levels are related to S-ECC. The in vitro study aims to evaluate the antimicrobial effects of the cholecalciferol vitamin D3 against Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus). Methods: The in vivo study included the recruitment of 120 healthy children; 93 with S-ECC and 27 were caries-free (CF). Clinical examinations were performed, blood and saliva samples were collected, and parents completed a questionnaire about family demographic characteristics, child’s oral health and nutritional habits. The ELISA Kit was used to determine vitamin D and salivary LL37 levels. Mutans streptococci bacteria were isolated and identified using a modified SB-20 culture medium. In the in vitro study, the antimicrobial effects of vitamin D3 were evaluated against S. mutans and S. sobrinus using the agar disc diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of vitamin D3 were determined using a microdilution method. Scanning electron microscope (SEM) was used to evaluate the morphological changes of bacterial cells following exposure to vitamin D3. Data were analysed with descriptive statistics, bivariate, Spearman’s rank correlation and multiple regression analysis. A p-value ≤ 0.05 was considered statistically significant. Results: S-ECC children had lower median serum and saliva vitamin D levels compared to CF children; however, these differences were not significant. Vitamin D levels in serum were significantly higher than in saliva, with a positive moderate correlation between them. S-ECC children had significantly lower median salivary LL37 levels and exhibited a higher count of S. mutans and S. sobrinus compared to CF children, with an inverse weak correlation between salivary LL37 levels and caries experience. Regression analysis disclosed that mothers with higher education levels were 92.9 % less likely to have children with dental caries and children that had both S. mutans and S. sobrinus were almost 12 times more likely to have S-ECC compared to children with no bacteria. In vitro results demonstrated that S. sobrinus was more sensitive to vitamin D3 compared to S. mutans bacteria. The MIC values of vitamin D3 against S. sobrinus and S. mutans were 60 μg/mL and 250 μg/mL respectively whereas the MBC values were 120 μg/mL and 500 μg/mL, respectively. Moreover, significant changes in the bacterial morphology were observed in treated bacterial cells with vitamin D3 as compared to the untreated control bacteria using SEM. Conclusion: The association between serum and saliva vitamin D and dental caries in young children was inconclusive. S-ECC children exhibited lower serum and saliva vitamin D levels, lower salivary LL37 levels and higher S. mutans and S. sobrinus counts compared to CF children. Mother’s education level and the presence of both S. mutans and S. sobrinus were the main factors that predicted dental caries experience in this age group. Also, this study supported the protective role of salivary LL37 against dental caries. Vitamin D3 showed excellent antimicrobial effects against S. mutans and S. sobrinus in vitro. Further studies are warranted to investigate the definite relation between vitamin D levels and dental caries, and the use of saliva vitamin D as a non-invasive alternative method over blood samples, and to elucidate the mechanism of vitamin D3 on S. mutans and S. sobrinus