Evaluation of the efficacy of casein phosphopeptide-amorphous calcium phosphate on remineralization of white spot lesions in vivo and in vitro: A systematic review and meta-analysis


 Abstract
Background: This systematic review with meta-analyses sought to answer whether casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) provided a remineralizing benefit superior to that of nonintervention or placebo.
Methods: The Cochrane databases, PubMed, EmBase, and Ovid up to May, 2019, were scanned, with no restrictions. Study information extraction and methodological quality assessments were accomplished independently by two reviewers. The “Criteria for judging risk of bias in the ‘Risk of bias’ assessment tool” was used for methodological quality assessment. The continuous data was analyzed by mean difference (MD) or standardized mean difference (SMD) with a 95% confidence interval (CI). Review Manager 5.3 was used for statistical analysis. Outcome variables include quantitative light-induced fluorescence in vivo, average surface roughness and surface microhardness in vitro.
Results: There were significant differences in the quantitative light-induced fluorescence (SMD = −0.43, 95% CI: [−0.79, −0.07], P = 0.02), average surface roughness (SMD = −8.21, 95% CI: [−10.37, −6.04], P < 0.01), Vickers microhardness (SMD = 1.19, 95% CI: [0.72, 1.66], P < 0.01), and Knoop microhardness (SMD = 3.52, 95% CI: [2.68, 4.36], P < 0.01) between the CPP-ACP and control groups or baseline.
Conclusion: Within the limitations of this meta-analysis, CPP-ACP exhibited excellent remineralization effects evaluated in vivo and in vitro, indicating outstanding restoration of form, aesthetics, and function in treating white spot lesions.

remineralization, initiated by acid-producing bacteria in the micro-environment. White spot lesions (WSLs), characterized as primitive enamel surface and subsurface demineralization without cavitation [1], are developed by dental plaque accumulation, commonly owning to inadequate oral hygiene [2][3][4]. WSLs are commonly clinically characterized by a chalky, opaque appearance located in pits, fissures, or smooth surfaces on the teeth. As the demineralization process progresses, the intact dental surface ultimately collapses and cavitates [5]. The traditional treatment approach for carious teeth involved caries excavation and restoration, which is frequently invasive [6,7].
However, several decades of research have culminated in "minimally invasive" approaches, emphasizing prevention rather than conventional surgical techniques.
Minimally invasive dentistry utilizes programs that restore form, function, and aesthetics with minimal removal of sound tooth tissue [8]. Research indicates that demineralization can be arrested or reversed with the help of remineralization agents in WSLs or noncavitated carious lesions [9]. Therefore, enhancing the remineralization of WSLs may be a relatively less invasive treatment of the disease [10,11]. In conclusion, it is of great significance to explore novel agents and strategies to enhance the remineralization process.
In order to conserve tooth tissues, fluoride has been widely recommended as a remineralization agent for preventing WSLs. Despite the cariostatic effects of high concentrations of topical fluoride, its treatment capacity does have certain limitations.
Because topical fluoride solutions cannot infiltrate the lesion, they do not eliminate its opaque whitish aspect [12]. Moreover, the cariostatic effects of fluoride are insufficient to manage patients with high caries risk [13] and the careless handling of fluoride may lead to adverse effects such as fluorosis [14]. To maximize the clinical significance of remineralization, a series of preventive agents containing non-fluoridated products has been developed to promote enamel remineralization. lists of included studies. The medical subject headings (MeSH) words and free text words were included during the search. "Casein phosphopeptide-amorphous calcium phosphate nanocomplex," "CPP-ACP," "GC tooth mousse," "Recaldent," "milk derivate," "casein derivate," "dental caries," "enamel demineralization," "white spot lesion," "remineralisation," "RCT," "Randomized Controlled Trials," "Controlled Clinical Trials," "Equivalence Trial," and "Pragmatic Clinical Trial" were used in combination with other strategies.
Based on the titles and abstracts, initial screening of the retrieved studies was carried out. After the removal of the duplicated and obviously irrelevant studies, full texts of potential interests were reassessed and only those meeting inclusion criteria were included. This work was accomplished by two reviewers (X.L. and X.M.), independently.
When any disagreement occurred, a third reviewer (F.X.) was consulted and a decision arrived at by consensus after the issues solved.

Inclusion criteria
Study inclusive criteria: (1) randomized controlled trials, retrospective and prospective studies, which were placebo-controlled or blank-controlled and had a parallel-group design.
(2) Participants for in vivo study including patients with early enamel carious had to be randomized to test or control groups. Participants for in vitro study using extracted human teeth had to utilize teeth free of any enamel defects, microcracks, or caries. Quality assessment and data extraction The Cochrane Collaboration methodology was used to assess the risk of bias of every retrieved study included. The assessment tool included random sequence generation, allocation concealment, blinding of assessment, incomplete outcome data, selective reporting, and other possible sources of bias so as to appraise the methodological quality of included studies. Bias in every study was classified as "low risk of bias," "high risk of

Statistical analysis
The Cochrane Handbook for Systematic Reviews of Interventions was used to conduct the statistical analysis [19]. This meta-analysis of randomized control trials (RCTs) was performed to evaluate the effectiveness of CPP-ACP for WSL treatment. The data type for the outcome measurement was mainly continuous data. To avoid errors caused by different measuring instruments, the SMD was used instead of MD with a 95% CI to generalize the effectiveness of treatment in each report. P-values were used to test the heterogeneity across studies. For P < 0.05, the data is considered significantly heterogeneous. In the meantime, the degree of inconsistency of the statistical analysis was assessed by I 2 [20]. The new quantity I 2 has the range 0% to 100%; the values 25%, 50%, and 75% represent low, moderate, and high heterogeneity, respectively [21]. If all the included studies showed good homogeneity, the fixed effects model was used. When the clinical and methodological heterogeneity was high or P < 0.05, we used the random effects models to combine the studies [21]. RevMan statistical software version 5.3 (The Nordic Cochrane Centre, Copenhagen, Denmark) was used to conduct the statistical analyses. If there were 10 or fewer studies, publication bias was not assessed, because more than 10 studies are required to check funnel-plot asymmetry [19]. Sensitivity analysis was performed by the leave-one-out approach in this review. The analysis was carried out using STATA version 14.1 (StataCorp, College Station, Texas, USA).

Results
Results of the search measurement was performed to detect changes in fluorescence loss (ΔF). As for the in vitro study, the total number of teeth in the CPP-ACP group ranged from 6 to 15, the total number of teeth in the control group ranged from 10 to 15 and the laboratory samples came from incisors, canines, premolars, and molars. The effects of remineralization of artificial dental caries were investigated through average surface roughness and surface microhardness (SMH) which were observed by atomic force microscopy (AFM) and measured by nanoindentation respectivel.

Assessment of methodological quality
Results of the assessment of methodological quality are shown in Fig. 2A and 2B. The judgements about each risk of bias item for each included study are presented in Fig. 2A. Fig. 2B illustrates our judgements about each risk of bias item, presented as percentages across all included studies. All of the items in one study were judged as "low risk of bias [22]." All included studies had low risks of bias in selective reporting. However, one study was judged as "high risk of bias" in incomplete outcome data, on account of the loss of follow-up data [23]. Because the proportion of high risk of bias was so small, it would not seriously weaken confidence in the results. Only one study had low risk of bias in blinding of outcome assessment and the rest had unclear risks of bias. Overall, the included studies in vitro had unclear risks of random-sequence generation and allocation concealment [24][25][26][27][28][29][30][31][32].

Average surface roughness from in vitro experimentation
All three studies provided average surface roughness data and were included in the analysis [15, 27,28]. When the data from the three studies were pooled, no significant heterogeneity was found (Chi 2 = 2.77, df = 2, P = 0.25, I 2 = 28%). Meta-analysis demonstrated a significant difference between the two groups of CPP-ACP versus the control, based on the average surface roughness measurement (SMD = −8.21, 95% CI: [−10.37, −6.04], P < 0.00001) (Fig. 3B).

Sensitivity analysis and publication bias
The leave-one-out approach was used to assess the sensitive ity of meta-analysis. When individual studies are eliminated in turn, all results are consistent with the meta-analysis results using all studies and the directions of the polled estimates of SMH do not vary considerably (Fig. 4). This means that the meta-analysis had good reliability and stability.
Publication bias was not evaluated for these results because the detection of funnel plot asymmetry requires more than 10 studies.

Discussion
The use of the proposed minimally invasive technique can not only induce recovery of the natural tooth appearance but also promote enamel remineralization in depth, so it may be considered a potential alternative to conventional operative treatment. The proposed minimally invasive technique mainly utilizes a combined approach of microabrasion and enamel remineralization 33. Fluoride therapy has long been considered as the base non- performed. The SMH test offers a relatively simple, rapid, and non-destructive approach in demineralization and remineralization studies 40. Different description units of SMH (Vickers hardness and Knoop hardness) could all induce heterogeneity within one study when comparing outcomes; therefore, we conducted subgroup analysis according to different testing methods, such that the heterogeneity reduced I2 from 77% to 16% and 0%, respectively (Fig. 3C). This indicated that the different testing methods were the main factors inducing heterogeneity, so the subgroup analysis permitted comparison of the outcomes. After CPP-ACP treatment and remineralization, the mean SMH values increased significantly compared to those of the control group, whether measured with a Vickers microhardness tester or Knoop hardness tester. This result can be attributed to the mineralization induction of CPP-ACP: after the localization of ACP at the enamel surface, free calcium and phosphate ions were buffered, thereby helping to maintain a state of supersaturation with respect to tooth minerals, depressing enamel demineralization, and promoting remineralization 34, 41. Some potential limitations of the study should be addressed. Our meta-analysis, including average surface roughness and surface microhardness, was based on in vitro environments, which was not reproduced in vivo; therefore, many limitations were unavoidable. For instance, the effects of salivary enzymes, proteins, pellicle, dental plaque, and additional fluoride sources on demineralization and remineralization cycles in the oral environment were not included 42. However, from another perspective, considering the good control of interfering factors for in vitro studies, the results may be more stable and convincing. Furthermore, there is considerable risk of lowering the quality of the evidence in surface microhardness analysis. The CPP-ACP group included both "GC Tooth Mousse Plus" and "MI Paste Plus," which contains a small amount of fluoride, as expounded in Table 2

Conclusions
Based on this study's results analyzing in vitro and in vivo data, CPP-ACP exhibited excellent remineralization of WSLs compared to the other groups or baseline, with greater percentages of WSL regression, lower enamel surface roughness, and the highest surface microhardness recovery. This indicates that CPP-ACP can dramatically restore form, aesthetics, and function. Therefore, CPP-ACP seems effective for the remediation of WSLs. Explanations: "n.r" = "not reported," "qd" = "once daily," "bid" = "twice daily," s Mean (standard deviation).     Outcome of sensitivity analysis of SMH in vitro.