Evaluation of the preventive effects of fluoride varnish, CPP-ACP, nHAp, and self-assembling peptide P11-4 on enamel demineralization around and under clear aligner attachments: An in vitro study

This study aimed to evaluate and compare the effects of fluoride varnish (FV), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), nano-hydroxyapatite (nHAp), and self-assembling peptide (SAP) P11-4 on the prevention of enamel demineralization around and under orthodontic clear aligner attachments.

Ninety-six freshly extracted human premolar teeth were randomly assigned to six groups (n=16): FV, CPP-ACP, nHAp, SAP, positive control (CO+), and negative control (CO-). Attachments were bonded to the buccal surfaces of the teeth in the treatment groups. The positive control group received attachments without additional treatment. The negative control group received no attachment and was immersed only in deionized water. The remineralizing agents were then applied to the buccal surfaces of the teeth. All groups, except for the negative control group, were subjected to a pH-cycling process for 15 days. Each tooth was sectioned buccolingually; one half was utilized for cross-sectional microhardness (CSMH) testing and the other half for energy-dispersive X-ray spectroscopy (EDX) analysis. Both tests were conducted at the occlusal, middle, and gingival areas of the attachment. Enamel CSMH and EDX calcium/phosphorus (Ca/P) ratio were assessed at multiple depths from the external enamel surface in each area. Statistical analysis was performed using a three-way mixed-design ANOVA and Games-Howell post hoc tests.

The group effect was significant for both CSMH (F(5,90)=123.8, P<0.001, partial η²=0.87) and the Ca/P ratio (F(5,90)=30.0, P<0.001, partial η²=0.63). SAP showed the highest values among the remineralizing agents (CSMH, 189.8, 95% confidence intervals [CI], 186.6-193.0; Ca/P, 2.13, 95% CI, 2.10-2.16). For CSMH, SAP values were significantly higher than those for nHAp and the positive control (p ≤ 0.002). Differences among SAP, FV, and CPP-ACP were not significant once within-tooth clustering was accounted for. For the Ca/P ratio, SAP values were significantly higher than those for FV, CPP-ACP, nHAp, and the positive control (P≤0.03). All agents showed significantly higher values than the positive control (P<0.02). CSMH and the Ca/P ratio increased with depth (P<0.001), and gingival values were lower than occlusal and middle values (P<0.001).

All tested remineralizing agents demonstrated variable preventive effects against enamel demineralization around and under clear aligner attachments when compared with the positive control group. SAP showed the most favourable results among all treatment groups.