The development of caries involves a dynamic biological process where acids produced by bacterial glycolysis of dietary carbohydrates cause demineralization of dental hard tissues. Noncavitated white spot lesions (WSLs) are indicative of early stage of dental caries. These lesions are considered reversible if detected early. ¹ Topical application of fluoride-releasing materials has been used over three decades to combat dental caries. ² , ³ They are considered important adjuvants in clinical practice, particularly when the patient's level of cooperation is low. ⁴ The major mechanism of fluoride ions in preventing enamel demineralization is by promoting the formation of fluorapatite in enamel in the presence of calcium and phosphate ions produced by plaque bacterial organic acids. ⁵ , ⁶ Fluoride ions can also drive the remineralization of previously demineralized enamel if enough salivary or plaque calcium and phosphate ions are available when the fluoride is applied. ⁷ Therefore, on topical application of fluoride ions, the availability of calcium and phosphate ions can be the limiting factor for net enamel remineralization to occur. ⁷ To overcome the limited bioavailability of calcium and phosphate ions, calcium phosphate-based remineralization systems such as amorphous calcium phosphate (ACP), calcium sodium phosphosilicate bioactive glass, and casein phosphopeptide-stabilized ACP were developed. The remineralizing potential of casein phosphopeptide-ACP (CPP-ACP) in cariogenic studies on animals, was demonstrated by researchers in 1995, and only in 2009 was it used for the treatment of WSLs. ⁸ CPP-ACP nanocomplexes is a technology based on ACP stabilized by casein phosphopeptides (CPP). ⁷ CPPs stabilize high concentrations of calcium and phosphate ions, together with fluoride ions, at the tooth surface by binding to pellicle and plaque. These ions are freely bioavailable to diffuse down concentration gradients into enamel subsurface lesions, thereby effectively promoting remineralization in vivo. ⁷

Varnishes allow for the delivery of high concentrations of fluoride in small amounts. ⁹ The effectiveness, the relative safety, high fluoride uptake, and the ease of application, offer significant advantages over other professionally applied topical fluoride treatments such as fluoride gels, foams, and mouthrinses. ⁹ CPP-ACPF varnish is unique in that it uses Recaldent (CPP-ACP) technology incorporated with 5% sodium fluoride. The inclusion of CPP-ACP in MI varnish® (GC America Inc., Alsip, IL, USA) has been proven to inhibit enamel demineralization to a much greater extent than fluoride varnishes without CPP-ACP. ¹⁰ , ¹¹ Literature search revealed three systematic reviews on CPP-ACP, with one focusing on effectiveness of MI paste and the other two reviewing effect of all formulations of CPP-ACP in human randomized clinical trials. ¹² , ¹³ , ¹⁴ Review of in vitro studies comparing remineralizing potential of CPP-ACPF varnish with other fluoride varnishes has not been done. Hence, this systematic review aimed to evaluate the efficacy of CPP-ACPF varnish in remineralizing WSLs in vitro on human teeth.

[TAG:2]Materials and Methods [/TAG:2]The Cochrane Handbook for Systematic Reviews of Interventions ¹⁵ and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement guidelines were followed for review. ¹⁶ The Population, Intervention, Comparison, Outcome, and Study design method as applicable is presented in Table 1. The protocol was registered at the International Prospective Register of Systematic Reviews (PROSPERO) (CRD42021237900).{Table 1}

Search strategy for article identification

An extensive electronic search for in vitro clinical trials via three databases, namely Medline (via PubMed), The Cochrane Controlled Clinical Trials Register, and Google Scholar, till January 29, 2021, was done. The outcomes of the search and Medical Subject Headings are summarized in Table 2. Hand searching was performed for relevant journals and the reference lists of all eligible studies for additional relevant studies. No restrictions on the language or date of publication were applied during the search. Reference articles were retrieved and exported to the Mendeley Desktop 1.13.3 software (Elsevier. Mendeley Ltd, London, United Kingdom). ¹⁷ The authors were not blinded to country or journal names.{Table 2}

Selection of studies

The review included in vitro studies on human teeth from 2010 to January 2021. The studies assessing depth of WSL, microhardness due to artificial demineralization or remineralization and calcium, phosphate ion release, and mineral loss/gain were considered for review. Case reports, abstracts, editorials, review articles, non-English articles, animal studies, and studies testing formulations of CPP-ACP, such as sugar-free gums, lozenges, fluoridated gels, mouth rinse, paste, and antibacterial gels, were excluded from review.

Data extraction

One author searched the studies, screened the titles and abstracts of each study based on the criteria, and extracted data. Two authors independently rechecked the full text of the screened studies. Any disagreement between the two authors, was resolved by a third reviewer. Data collected for each study included information pertaining to year of publication, authorship, sample size, study characteristics like intervention, comparison, follow-up period, examination methods, and results.

Assessment of risk of bias

A customized risk assessment tool was prepared using Office of Health Assessment and Translation risk-of-bias tool ¹⁸ and checklist for reporting in vitro studies guidelines. ¹⁹ The domains evaluated are listed in Figure 1. All included studies were assessed independently by two review authors who were not blinded to identifying details of articles. Each domain was classified as having a low, high, or unclear risk of bias. The Cochrane risk of bias assessment tool was used to generate the risk of bias summary graph. ²⁰ Figure 1

Cochrane risk of bias of the included studies (a) graph, (b) summary

Study selection and description

Through the literature search, 913 studies were identified, including 822 duplicates. Seventy-four articles were identified after excluding duplications. A total of 17 articles evaluating the efficacy of MI paste were excluded after reading abstract. Full-text articles were retrieved for 57 relevant studies. Thirty-nine articles were excluded after full-text reading. Finally, 18 studies which met the inclusion criteria were considered Figure 2. Review evaluated three different outcomes: lesion depth, surface microhardness, and mineral loss/gain. Studies assessing the remineralization effect of CPP-ACP varnish over a period of 7 days in comparison to other fluoride varnish were pooled. Results of four studies measuring surface microhardness were synthesized using forest plot. Figure 2

Preferred Reporting Items for Systematic Reviews and Meta-Analyses flow diagram of review methodology

This systematic review and meta-analysis underline the significant remineralizing effect of CPP-ACPF varnish compared to fluoride varnishes. Majority of in vitro studies ¹¹ , ³³ , ³⁶ , ³⁹ have shown pronounced remineralizing effect of CPP-ACPF varnishes compared with other fluoride varnishes. However, few in vitro ³⁴ , ³⁸ studies have shown contradictory results. Meta-analysis showed no significant difference between CPP-ACPF varnish and other fluoride varnishes in improving the surface microhardness after remineralization during 7-day period.

Fluoride varnishes are more effective compared to other forms of delivery because of long contact periods resulting in high fluoride uptake and the formation of CaF ₂deposits that act as fluoride reservoirs. ⁴⁰ The CPP-ACP has been shown to interact with fluoride ions to produce an additive anticariogenic effect through the formation of CPP-stabilized amorphous calcium fluoride phosphate (ACFP) phase. ⁴¹ , ⁴² , ⁴³ , ⁴⁴ The in situ study by Reynolds ⁴⁵ showed that CPP-ACP plus fluoride formulation not only increased fluoride incorporation into plaque but also in subsurface enamel and substantially increased remineralization of subsurface lesions of enamel compared with fluoride alone. An in vitro study by Duraisamy et al. reported that demineralization inhibitory potential on the additive use of F − varnish + CPP-ACP was superior to fluoride varnish or CPP-ACP applied alone on the enamel of young permanent teeth. ⁴⁶ The presence of the CPP-ACFP nanocomplexes in MI varnish has shown to be superior to fluoride alone in inhibiting enamel demineralization and promoting remineralization in a number of in situ and in vivo randomized controlled clinical trials. ⁸ , ⁴⁵ , ⁴⁷ , ⁴⁸ , ⁴⁹

An in vitro study by Thakkar et al. found that CPP-ACPF varnish had significant demineralization inhibitory property compared to CPP-ACP paste and CPP-ACPF paste plus. Remineralizing effect did not differ significantly compared to both groups. ³⁷ A previous study, which evaluated remineralizing efficiency of CPP-ACPF varnish and paste groups, showed increased values of calcium, phosphate contents, higher percent surface microhardness recovery, decrease in lesion depth, and mineral loss compared to paste groups. ²⁸

In the studies considered for review, fluoride varnishes contained a variety of combinations, which accounted for substantial clinical heterogeneity. CPP-ACPF varnish showed significantly higher demineralization inhibitory effect and greater amount of fluoride release as compared to fluor protector for a period of 1 month. ¹⁰ An in vitro study compared calcium phosphate-containing varnishes such as enamel pro, Clinpro, and CPP-ACPF varnish with other fluoride varnishes. The results showed that only CPP-ACPF varnish significantly inhibited demineralization than fluoride alone varnishes such as Duraphat and Profluorid. ¹¹ Furthermore, CPP-ACPF varnish provided 130% greater inhibition when compared with the Duraphat fluoride-alone control. ¹¹ The presence of the CPP-ACFP nanocomplexes and combination with fluoride would explain the superior ability of CPP-ACP varnish in inhibiting demineralization.

Remineralization potential on artificial enamel caries-like lesions in permanent teeth was significantly better with CPP-ACPF varnish compared to 5% sodium fluoride varnish ³³ , ³⁶ and Clinpro varnish. ³³ However, CPP-ACPF varnish did not achieve better remineralization of artificial enamel carious lesions when compared with Duraphat varnish. ³⁴ , ³⁸ In a clinical trial by Obradović et al., CPP-ACPF varnish showed better remineralization of initial smooth surface caries lesions of primary teeth compared to conventional varnish with fluoride. ⁵⁰ A systematic review and meta-analysis on randomized controlled trials by Tao et al. concluded that fluorides combined with CPP-ACP treatment produce significantly better remineralization of occlusal early caries lesions. For lesions on smooth surfaces, the meta-analysis showed no significant difference between using fluorides with CPP-ACP and using fluorides alone. ¹³ The CPP-ACFP are amorphous electroneutral nanocomplexes and their hydrodynamic radius allows rapid diffusion out of the varnish and is reported to enter the porosities of an enamel subsurface lesion through intraprismatic spaces favoring remineralization. ⁵¹

Most varnishes in the market contain 5% NaF with an alcohol- and resin-based solution. The evaporation of the alcohol makes the system fast drying, and the resin functions to allow the fluoride to adhere to the teeth. ⁵² The most common resin additive is colophony, a resin derived from pine tree sap. ⁵² The main complaint about varnish, “stickiness,” and a noticeable film on the teeth is due to the carrier. The resin base in fluoride varnishes allows them to stick to tooth surfaces and stay for up to 24 h whereby fluoride is gradually released from the varnish and is taken up by the tooth enamel and dentin. ⁵² The strongly bound fluoride, incorporated onto the surface of the crystals of apatite, can reduce the solubility of the tooth mineral and hence inhibit demineralization due to acids generated by plaque bacteria. ⁵³ Varnishes are preferred over other topical forms since they are user friendly and requires less handling and application time, When compared to gels, they are less likely to be ingested by young children. Varnishes are a successful strategy of prevention of caries in individuals with special requirements, such as those with developmental disabilities, because they stick to the tooth surface for longer lengths of time and requires less patient compliance. ⁵⁴ A randomized trial by Huang et al. assessed the effectiveness of MI Paste Plus and PreviDent fluoride varnish for treatment of WSLs. The results showed no significant difference between two groups compared to a standard oral hygiene and toothpaste. This result can be attributed to the presentation of material in the form of varnish and not in toothpaste form. ⁴ , ⁵⁵

A Cochrane review in 2016 suggested the superiority of resin-based fissure sealants over fluoride varnish applications for preventing occlusal caries in permanent molars, but the quality of evidence was low. ⁵⁶ Fluoride varnish was shown to be less expensive than fissure sealant in a randomized clinical trial that used mobile dental clinics to target children with high caries risk. However, caries prevention was not significantly different when either strategy was used twice a year. ⁵⁷ A cost-effectiveness analysis based on a clinical decision tree found the application of fluoride varnish as a cost-effective community strategy to prevent early childhood caries among rural children in nonfluoridated areas. ⁵⁸ Cost-effectiveness of varnish against sealant was assessed over a period of 4 years in a school-based setting. Varnish appeared to be more cost-effective than sealants as the latter required expertise in application. ⁵⁹ The application of varnish requires very little infrastructure, and it can be more readily applied in nontraditional settings (dental chair with illumination and fluid evacuation to maintain a dry field). ⁵⁹ Even health-care providers with minimal training can also apply varnish. ⁵⁹

In vitro studies make possible the inexpensive and rapid yet sensitive assessment of any new inventions in a highly controlled environment representing a key component of product activity confirmation. However, major limitation is their inability to simulate the complex biological processes involved in caries. And, also the oral conditions that prevail in the mouth. ⁶⁰ In in-situ and in -vitro investigations, quantitative measurements of mineral loss and lesion depth are regarded as standard primary outcomes for evaluating re and demineralization. ⁶⁰ Surface microhardness is indirect technique which complements direct measures of mineral gain and loss. ⁶⁰ There were very few studies which assessed mineral loss/gain in the present review. ¹¹ , ³⁴ , ³⁵ , ³⁶

Lesion depth defines the magnitude of penetration and damage from acid caused by varnishes. Transverse microradiography and polarized light microscopy are desirable methods ⁶¹ for direct assessment of lesion depth which was followed in only a few studies. Some studies had a small sample size. There was heterogeneity in the method of outcome assessment for lesion depth and mineral loss/gain, which limited meta-analysis for these parameters. Future research would benefit from a uniform method of assessment for mineral loss/gain and lesion depth using these products. Being cognizant about heterogeneity in reporting of methodology and evaluation, including the duration of follow-up and assessment, the evidence should be considered with caution. Future studies should be planned overcoming these limitations and dental association bodies should frame guidelines for reporting of efficacy of caries remineralizing agents in scientific studies which will help in review of articles and framing of clinical practice guidelines.

CPP-ACPF varnish appears to be equally effective as other fluoride varnishes in remineralizing WSLs, but quality of evidence is low.

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Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.