Effects of calcium-containing solutions on fluorescence-detectable plaque area and enamel mineral volume: an in vitro and exploratory clinical study

Abstract Background Calcium-containing biomimetic materials have been investigated as adjunctive approaches for managing early enamel demineralization; however, clinical evidence regarding calcium-containing oral sprays remains limited. This study evaluated the effects of calcium-based solutions on fluorescence-detectable plaque-associated area on tooth surfaces through an in vitro experiment and an exploratory randomized clinical trial. Methods Extracted teeth with visible enamel carious lesions were immersed for 6 h in ultrapure water, artificial saliva, calcium solution, or artificial saliva plus calcium solution. Threshold-segmented enamel mineralized volume and fluorescence-detectable plaque-associated area were assessed using micro-computed tomography and quantitative light-induced fluorescence (QLF). In an exploratory randomized clinical trial, fifteen children were randomly assigned to placebo, a calcium-containing spray (Formula 1; 0.3% calcium lactate, 0.01% dicalcium phosphate, and 0.01% mesoporous bioactive glass), or the same formulation supplemented with 225 ppm fluoride (Formula 2) for two months. Fluorescence-detectable plaque-associated area and salivary calcium concentration were evaluated. Results In vitro, threshold-segmented enamel mineralized volume showed numerically higher post-treatment values in the artificial saliva and calcium-containing solution groups, while QLF-detected plaque-associated fluorescent area decreased across groups. Changes in mineralized volume were significantly negatively correlated with changes in fluorescence-detectable plaque-associated area (Spearman’s ρ = −0.771, P < 0.0001). Clinically , salivary calcium increased in both calcium-containing spray groups, although within-group changes did not reach statistical significance (both P = 0.0625). The calcium-plus-fluoride comparator demonstrated a significantly greater increase in Δsalivary calcium than placebo ( P < 0.05). Median fluorescence-detectable plaque-associated area decreased from 5.47% to 2.33% in Formula 1 and from 8.63% to 3.06% in Formula 2, whereas the placebo group showed minimal change (2.42% to 2.76%). Exploratory ANCOVA adjusted for baseline QLF, age, and sex supported a significant overall treatment effect ( P = 0.009). Conclusions Both calcium-containing oral sprays were associated with greater reductions in the primary clinical outcome, fluorescence-detectable plaque-associated area, compared with placebo. No statistically significant difference in the primary outcome was observed between Formula 1 and Formula 2. Complementary in vitro findings showed numerically higher post-treatment mineralized volume values and a significant inverse association between ΔMV and ΔQLF-detected fluorescent area under controlled conditions. Together, these exploratory findings support further investigation of calcium-containing oral sprays in larger, adequately powered clinical trials. Clinical significance Calcium-containing sprays may have potential as an adjunctive home-care approach for reducing fluorescence-detectable plaque-associated signals in children with early enamel demineralization. Clinical trial registration NCT07269730, retrospectively registered on November 25, 2025. URL https//clinicaltrials.gov/study/NCT07269730.