In vitro effect of amorphous calcium phosphate paste applied for extended periods of time on enamel remineralization

Abstract Dental applications based on the unique characteristics of amorphous calcium phosphate stabilized by casein phosphopeptides (CPP-ACP) have been proposed, as well as the improvement of its properties. Objectives: The objective of this study was to determine the ability of topically applied CPP-ACP from a commercial product to remineralize subsurface lesions when applied for extended periods of time (3 h and 8 h). Material and Methods: Artificially induced carious lesions were produced in 50 bovine enamel blocks previously selected by surface hardness. After treatments with gel without F and CPP-ACP applied for 1 minute (Placebo); 2% NaF neutral gel applied for 1 minute (Fluoride 1 min); CPP-ACP applied for 3 min (ACP 3 min); and CPP-ACP applied for 3 h (ACP 3 h) and for 8 h (ACP 8 h), the enamel blocks were submitted to the remineralization pH-cycling. Surface hardness and synchrotron micro-tomography were used to determine the percentage of surface hardness recovery (%SHR) and to calculate mineral concentration (gHAp.cm−3), respectively. The data were submitted to ANOVA followed by the Student-Newman-Keuls test (p<0.05). Results: Fluoride gel presented higher %SHR followed by ACP 3 min (p<0.001). No difference (p = 0.148) was found for Placebo, ACP 3 h and ACP 8 h groups for %SHR. Fluoride gel showed greater mineral concentration (p<0.001) when compared with the other groups. ACP 3 min demonstrated a significant difference (p<0.001) from ACP 3 h and ACP 8 h. The ACP 3 h and 8 h presented a subsurface lesion with development of laminations in all blocks. Conclusion: In this in vitro study the use of CPP-ACP for extended periods of time did not produce an additive effect in the remineralization process.


Introduction
Dental applications based on the unique characteristics of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) have been proposed, as well as the improvement of its properties and associations with similar products that have antidemineralizing and remineralizing potential. The developed systems use a special milk-derived protein (casein phosphopeptides; CPP) to stabilize the calcium phosphate ions from amorphous calcium phosphate (ACP) 4 . The CPP-ACP may then act as a calcium phosphate reservoir, buffering the free calcium and phosphate ion activities, thereby helping to maintain a state of supersaturation with respect to tooth mineral depressing enamel demineralization and enhancing remineralization 12 .
CPP-ACP has been incorporated into various products in order to exert a topical effect. These products include commercially available sugar-free chewing gum, mints and topical gels 12 . The indications for use are during bleaching, following professional for caries active patients, and those suffering from erosion or xerostomia. In view of its broad methods of application for dental care, the CPP-ACP can be used safely from infants through the elderly 12 .
The application of the pastes containing CPP-ACP as a home-care product is commercially available.
CPP-ACP has been used as re-mineralizing agent in caries active patients, dentin sensitivity, post bleaching sensitivity, and for tooth erosion 12,20 . For these patients, the extended topical application of CPP-ACP may bring a greater reduction in the severity of these dental problems. Since the CPP-ACP formulation is non-toxic 12 it could be swallowed instead of spat out, allowing the overnight use in a tray by the patient, mainly after dental bleaching. Nevertheless, there is no published research using CPP-ACP with different approaches such as a longer treatment time 15 .
Therefore, further research is required to provide a clinical applications.
supported clinical recommendation for its use, the aim of this in vitro study was to determine the ability of a commercial product containing CPP-ACP to remineralize subsurface lesions when applied for extended periods of time (3 h and 8 h). The null hypothesis was that the product with CPP-ACP applied for 3 minutes would present the same ability to produce remineralization when applied for 3 h or 8 h.

Experimental design
Enamel blocks (4x4 mm) were obtained from bovine incisor teeth that were stored in 2% formaldehyde solution with a pH of 7.0 for 30 days 7 . The enamel surface of the blocks was then

Results
The Fluoride 1 min group presented the highest mean value for SH 2 and %SHR (Table 1) (Table 1). There was a statistically 3 min and the ACP 3 h and ACP 8 h groups (Table   between SH   that the longer the CPP-ACP stays in the mouth, the more effective it will be releasing calcium and phosphate continuously for the 3 hours. As there is the necessity of more research using CPP-ACP with  Clinically, the patient would be instructed to apply the paste using a tray for 3 or 8 hours (overnight), mainly after dental bleaching.
Application of CPP-ACP for 3 minutes (ACP 3 min) was capable of improving enamel remineralization (Table 1,Table 2 and Figure 1), reducing mineral loss (IML and IML) and lesion depth (μm). As described in the literature, the CPP-ACP has the ability to adhere to enamel and supersaturate the environment with free calcium and phosphate ion activities 3,11,17 . Both the outer part and the inner part of the lesion showed a more homogeneous remineralization ratio ( Table   2) (Table 1 and Figure 1).
in the outer part of the lesion (zone A, Table 2 and Figure 1a) mainly due its strong deposition into the outer zones of the enamel lesion 13,14 . In addition, the presence of fluoride promoted higher ionic activity to HF 0 and greater degree of saturation with on remineralization 11 products in the outer part of enamel resulted in higher surface hardness (Table 1) and mineral content (zone A, Table 2) than the products precipitated by CPP-ACP.  Figure 1a, 1b and 1c).
The prolonged contact with the enamel leads to a   Additional studies should be performed using MI Paste