Caries Control by CAPE Toothpaste: In Vitro, In Vivo, and In Situ Analysis

Dental caries is closely associated with microbiome dysbiosis. Incorporating antimicrobial agents can enhance the efficacy of fluoride toothpaste. Our previous studies showed that caffeic acid phenethyl ester (CAPE), derived from propolis, effectively inhibited cariogenic bacteria. To formulate a novel CAPE-containing fluoridated toothpaste and establish a multistage evaluation system assessing its caries-controlling efficacy. The CAPE toothpaste's physicochemical properties were characterized. Its in vitro antimicrobial activity against <i>Streptococcus mutans</i> was examined using quantitative suspension and checkerboard microdilution assays. In vivo anticaries efficacy and biosafety were evaluated in a rat caries model (<i>n</i> = 9/group) comparing 5 groups: untreated control (group NC), base toothpaste without CAPE (group B, 600 ppm F^-), CAPE-fluoride toothpaste (group C, 0.16 mg/mL CAPE + 600 ppm F^-), fluoride control (group F, DARLIE^®, 600 ppm F^-), and propolis nonfluoride control (group P, Red Seal^®). Caries severity was scored using the Keyes method. Finally, in situ enamel repair (<i>n</i> = 24 enamel blocks) and plaque microbiome modulation (<i>n</i> = 6 samples) were assessed in a 7-d clinical study (ChiCTR2400089643) with 4 groups (groups NC, C, F, and P). The novel formulation showed stable physicochemical properties. Group C reduced <i>S. mutans</i> by 1.5-log10 within 3 min (<i>P</i> = 0.0062 vs NC), and CAPE plus fluoride exhibited additive antibacterial properties (fractional inhibitory concentration index = 0.75). In rats, group C showed the lowest smooth-surface score (21.0 ± 1.7, <i>P</i> < 0.005 vs. all) and reduced sulcal lesion severity (slight dentinal lesions: <i>P</i> = 0.0181 vs. NC and <i>P</i> = 0.0318 vs. F). In situ, group C achieved 27.58% surface microhardness recovery (<i>P</i> < 0.01 vs. all), with significant reductions in mineral loss and lesion depth (<i>P</i> < 0.001 vs. all). Microbiome analysis revealed a preserved microbial diversity, increased <i>Streptococcus oralis</i>, and reduced cariogenic populations. The novel CAPE-containing fluoridated toothpaste effectively inhibited the onset and development of caries. Wider-ranging and longer-term clinical investigations are still needed.