Analysis of Dental Enamel Remineralization: A Systematic Review of Technique Comparisons

The demineralization process conditions the structure of the enamel and begins with a superficial decalcification procedure that makes the enamel surface porous and gives it a chalky appearance. White spot lesions (WSLs) are the first clinical sign that can be appreciated before caries evolves into cavitated lesions. The years of research have led to the testing of several remineralization techniques. This study’s objective is to investigate and assess the various methods for remineralizing enamel. The dental enamel remineralization techniques have been evaluated. A literature search on PubMed, Scopus, and Web of Science was performed. After screening, identification, and eligibility processes 17 papers were selected for the qualitative analysis. This systematic review identified several materials that, whether used singly or in combination, can be effective in the process of remineralizing enamel. All methods have a potential for remineralization when they come into contact with tooth enamel surfaces that have early-stage caries (white spot lesions). From the studies conducted in the test, all of the substances used to which fluoride has been added contribute to remineralization. It is believed that by developing and researching new remineralization techniques, this process might develop even more successfully.


Introduction
The process of demineralization is a gradual process that leads to the gradual dissolution of hydroxyapatite crystals in enamel, resulting in the loss of tooth tissue. This process will be discussed extensively below, along with its causes. Demineralization of enamel and dentin appears to be an increasingly common problem, despite technological development in oral hygiene and awareness campaigns. The main consequences related to it are increased dentin sensitivity and the development of dental caries [1][2][3].
The enamel protein matrix is produced by ameloblasts and later mineralized through calcium phosphate crystals [6,7]. Enamel is an acellular tissue in that ameloblasts die soon after producing it [8].
Its highly mineralized structure makes it on the one hand very resistant, and on the other hand susceptible to the phenomenon of demineralization [2,6]. Among the most prominent clinical signs preceding the onset of caries are WSLs, which are the first clinical sign of dental demineralization. On objective examination, they appear as opaque, chalky surfaces as a result of increased enamel porosity [26,27]. If caught early, WSLs result in reversible lesions. If, on the contrary, the acidic environment is not eliminated or reduced, WSLs can evolve into cavitated lesions ( Figure 2) [18,28,29]. Loss of mineralized structure beneath an apparently intact layer characterizes carious lesions. There is an increase in porosity within the lesion itself, which gives rise to the chalky appearance of white spots [10].
WSLs can be classified into active lesions if they have a chalky, rough appearance and inactive lesions if they have a shiny, smooth appearance [30].
Very often, WSLs can be visible both clinically and radiographically even after the use of remineralization agents [31]. This happens because the deepest part of the lesion has the lowest potential for remineralization and continues to diffuse light differently. Therefore, even if the white spot becomes hard, smooth, and shiny (and therefore inactive), some internal opacity is likely to remain [32].
To try minimize the occurrence of carious lesions, it is essential to implement effective prevention protocols.
Regenerative dentistry is a revolutionary idea that pushes mainstream dentistry to accelerate dental research and integrate scientific discoveries into innovative treatments in the future. The approach is founded on an understanding of the underlying mechanisms of tooth development, as well as the biological processes of healing and repair, resulting in a solid understanding of principles that could be applied in maximizing the inherent healing potential of dental tissues or regenerating (engineering) the damaged tissue enamel.
Remineralization from an external source allows for the deposition of new minerals within the tooth structure [33]. The altered mineral has a greater uptake of locally applied fluoride, which allows for more effective remineralization of non-cavitated lesions. This mechanism can be better understood by looking at the structure of hydroxyapatite ( Figure 3) [34][35][36]. During remineralization, fluoride is classically incorporated by replacing or displacing hydroxyl ions.
As a result of fluoride incorporation, Ca-F covalent bonds and/or OH-F hydrogen bonds are formed, and the unit cell volume of the apatite is reduced, increasing the hardness and acid resistance (chemical durability) of dental enamel (Figure 3b) [38][39][40][41].
Another important function of fluoride ions is to act as antibacterial agents and as inhibitors of the bacterial enzyme enolase, resulting in reduced acid production by bacteria [42].
The purpose of this systematic review of the scientific literature is to compare the various remineralizing agents most widely used in clinical practice, emphasizing the advantages and disadvantages of each. What differentiates it from other literature reviews is that it has included and analyzed materials that have been most widely used in the past 5 years included in the PRISMA.

Protocol and Registration
The current systematic review was carried out in compliance with the standards of the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) and the International Prospective Register of Systematic Review (PROSPERO) guidelines (ID 404428).

Search Processing
Pubmed, Scopus, and Web of Science were searched to find papers that matched our topic, dating from 1 January 2018 up to 14 February 2023. The search method was developed by combining phrases that suited the goal of our review, which deals with the analysis of the advantages and disadvantages of white spot lesion remineralization techniques.

Inclusion Criteria
The articles were selected using the following inclusion criteria: (1) studies only on human subjects; (2) open access studies; (3) clinical trials and randomized controlled trials; and (4) English language.

Data Processing
Two reviewers (R.M. and M.D.S.) searched the database to extrapolate the studies and assessed their quality independently, according to selection criteria. The selected articles were downloaded in Zotero (version 6.0.15). Any discrepancies between the two authors were resolved by consulting a senior reviewer (F.I.).

Results
From the search of the records on the Scopus, PubMed, and Web of Science databases, 771 articles were found, of which 142 duplicates of these records were excluded from the search before the screening.
Hence, 629 items were reviewed and 607 items were excluded, because they were excluded from our materials and methods. Twenty-two reports were sought for retrieval and 4 reports were not retrieved. Eighteen reports were assessed for eligibility and 1 report was excluded because it was off-topic. A final number of 17 studies were included in the review for qualitative analysis ( Figure 4) ( Table 1).

Discussion
WSLs are the first clinical sign that can be appreciated before caries evolve into cavitated lesions. Individuals most susceptible to the occurrence of WSLs include patients undergoing orthodontic treatment. These patients tend to accumulate more plaque as a result of the presence of orthodontic accessories (brackets, elastic, and metal binding) that make oral hygiene maneuvers difficult. In both orthodontic and non-orthodontic patients, the best prevention is proper home oral hygiene instruction and motivation. This, however, is very often not enough, and therefore remineralizing agents are introduced to prevent or treat the occurrence of white spot lesions. Various remineralizing agents that can complement oral hygiene include fluoride applications (in the form of gels, varnishes, or creams) either in the office or at home. Another option may be the application of remineralizing agents independent of patient compliance, including glass ionomer composites and dental sealants [27].

Casein-Phosphopeptide-Based Studies
The most widely used and well-known products in clinical practice for WSL remineralization include both fluoride-based products and technologies based on caseinphosphopeptide-stabilized amorphous calcium phosphate complexes (CPP-CP) [30].
CPP-ACP is a casein bioactive substance that can regulate supersaturated calcium and phosphorus ions in the oral environment. When the pH of the oral cavity falls, phosphorus ions stabilize the pH, while calcium ions induce the remineralization process [60].
Among the main capabilities of CPP-ACP is to increase the permeability of fluoride ions. Therefore, the ability of fluoride ions to reach deeper lesions allows for better remineralizing effects to be achieved [61].
There are few studies in the literature investigating the efficacy of CPP-ACP topical cream used as a toothpaste compared to traditional fluoride toothpaste. However, the study by Al-Batayneh et al. found that the combination of GC Tooth Mousse and toothpaste with 500 ppm fluoride provided no greater benefit than the isolated use of the two agents [43].
The use of MI Paste Plus (casein phosphopeptide-amorphous calcium fluoride phosphatebased agent, CPP-ACFP) also made no real improvement in WSLs in post-orthodontic patients after 1 year of use [53].
Hamdi et al. concluded that an application of CPP-ACP and tricalcium silicate (TCS)based agents twice daily achieves satisfactory remineralizing effects [52].
In the current study with these agents, the area of WSLs significantly decreased in the MI Paste Plus (65%) and Remin Pro (60%) groups over the course of the experiment, whereas there was no distinguishable improvement in the WSL area in the control group between the T2-T3 and T3-T4 time points (7% reduction over the study period). This finding suggests that MI Paste Plus and Remin Pro can successfully minimize the degree of WSLs throughout a three-month treatment period by remineralizing enamel caries.
It is thought that MI Paste Plus can keep the surface of the enamel saturated with calcium and phosphate. Moreover, MI Paste Plus's fluoride component enhances the remineralizing capability of CPP-ACP by working in concert with it [48,55,56,62,63].
Rechmann et al. used the MIV and MIPP agents to assess CPP-ACP's impact on WSL reversal and prevention during fixed appliance orthodontic therapy. The prescribed treatments were the use of an OTC toothpaste with 1100 ppm fluoride twice a day, daily use of MIPP, and quarterly application of MIV, and they found an improvement in the remineralization of both agents, but much better with MIPP. WSLs do not appear to be reduced appreciably by daily MIPP or quarterly MIV treatments during fixed orthodontic treatment [54].
It is possible to state how it is much more difficult to control WSLs in patients who wear orthodontic appliances than in those who do not. The potential of remineralizing agents can truly be evaluated after the removal of orthodontic appliances, which will have a long-term beneficial effect on the structure and acidity of bacterial plaque. This happens precisely because of the presence of orthodontic accessories (such as ligatures, braces, or elastics) that prevent the proper application of the remineralizing agent, which compromises its effectiveness, as can be seen in the study by Rechmann et al. [54].
Reviewing the results of the above studies, it can be stated that calcium-phosphatebased remineralization technology has proven to be a promising adjunctive treatment to fluoride therapy in the management of early carious lesions. This is possible because CPP-ACP-based remineralization technology allows high concentrations of calcium and phosphate ions to be stabilized by CPP and fluoride ions. In addition, since CPP-ACP can bind to the biofilm, it allows the pH of the plaque itself to be buffered by exerting the remineralizing effect.

Tricalcium Phosphate Studies
Another viable alternative to using fluoride is tricalcium phosphate (TCP) contained within ClinPro toothpaste. This agent allows for a greater penetration of fluoride ions. In particular, ClinPro 5000 (with 1.1% Sodium Fluoride) has been shown to achieve better effects than ClinPro Tooth Crème with 0.21% sodium fluoride. The use of ClinPro Tooth Crème with 0.21% sodium fluoride was more effective than ClinPro 5000, precisely because of the higher amount of sodium fluoride content [45].
The use of ClinPro Tooth Crème (0.21% NaF toothpaste with 950 ppm fluoro and f-TCP) achieves greater remineralizing results than MI Varnish with RECALDENT (CPP-ACP); while the main advantage of the latter is that of independent compliance [47].
Comparing patients treated with ClinPro Tooth Crème with a control group, AlFeel et al. demonstrated its remineralizing potential in treating WSLs. This split-mouth design is not an appropriate design for leachable materials, as described by Alfeel, 2021. If you apply Clinpro tooth creme on one side of the mouth, saliva, and the tongue can carry it to the other side of the mouth, so the result is biased and of low strength. However, despite the use of Clinpro Tooth Crème, no improvements in the aesthetics of WSLs themselves were found [50]. In contrast, ClinPro Tooth Crème used as a toothpaste may have greater efficacy, probably related to the action of brushing. The latter might give the remineralizing agent a chance to penetrate deeper. It is important to remember, however, that the brushing maneuver turns out to be highly dependent on the manual skill and cooperation of the patient. For this reason, varnishes always have a higher efficacy when compared to the former.

Self-Assembling Peptide 11-4 Studies
The introduction of the self-assembling peptide p11-4 (SAP11-4) enabled de novo regeneration of carious enamel, as it acts as a biomimetic. It can form new hydroxyapatite in the deeper early carious lesions, just as amelogenin supports the formation of the enamel itself [64].
Notably, the new hydroxyapatite crystals generated by SAP11-4 do not have a prismatic structure, but exhibit a "fan-like" structure, as they are arranged tangentially to the matrix fibers [65].
Broseler et al. compared the efficacy between fluoride varnish (control group) and SAP11-4 (test group) in treating early carious lesions. After one year of follow-up, a significant superiority of the results obtained with SAP11-4 over fluoride varnishes was found [56].
Even in comparison with TCPF varnish, SAP11-4 peptide demonstrated greater remineralizing potential due to its ability to self-assemble into three-dimensional fibrillar scaffolds [49].
One of the main advantages of SAP11-4 is precisely its biomimetic structure, which has enabled it to achieve better results than using fluoride or tricalcium phosphate varnishes that are more easily removed from tooth surfaces.

Nano-Hydroxyapatite Studies
Among the novel materials capable of remineralizing tooth enamel are nano-materials. Nano-hydroxyapatite, in particular, has distinct properties, such as increased solubility, improved biocompatibility, and greater surface energy [46].
Toothpastes composed of nano-hydroxyapatite make the remineralization process possible by releasing hydroxyapatite particles at the level of enamel prisms and dentinal tubules. Because of this ability, toothpaste containing nano-hydroxyapatite is used for the treatment of WSLs [66,67].
The first study conducted in vivo confirmed the excellent performance of nanohydroxyapatite-containing toothpaste. With a 6-month follow-up, toothpaste containing nano-hydroxyapatite reported better results than toothpaste containing only fluoride in remineralizing WSLs [44].
Nano-hydroxyapatite exerts a significantly greater remineralizing impact on early enamel lesions than the commonly used fluorides [68].

Ozone Therapy Studies
Another agent with high remineralizing capabilities is ozone [69,70]. Ozone has a high bactericidal function that is exploited to treat initial caries [71]. In addition, ozone, by dilating the dentinal tubules, increases the perfusion of remineralizing agents [72]. Despite the important remineralizing abilities of ozone, its combination with a hydroxyapatite gel has been observed to produce better effects. Ozone application alone allowed for the remineralization process to take place only in the outermost portion of the enamel [46].
As compared to nano-hydroxyapatite or ozone treatment alone, the combination of both approaches delivers the best results. To obtain the impact of nonrestorative caries therapy, the treatment techniques should be sustained for a long period.

Fluoride Agent Studies
Fluoride and xylitol varnishes can both mineralize non-cavitated lesions, as seen by the lesions in each group regressing after application [73]. To avoid NCLs and promote remineralization during orthodontic treatment, xylitol varnishes appear to be an alternative to fluoride varnishes. After application, fluoride varnishes created a thin layer of firm varnish that adhered to the enamel surface. However, due to the complex oral environment and movement caused by the buccal muscle, tongue, mastication, saliva wash, and oral hygiene practices, a fluoride varnish is likely to be removed quickly [55,74].
Although varnishes may be easier to apply and reapply, both MI Varnish and ProSeal sealant offer comparable levels of protection while undergoing orthodontic treatments. White spot carious lesions should be treated with CPP-ACFP varnish, which is strongly advised [58,59].
One advantage is its easy application, while a disadvantage is the action of muscles and saliva that can easily remove it from the tooth surface.

Sealant Agent Studies
Finally, the last approach involves the use of hydrophilic resin sealant and glass ionomer sealant materials. In contrast to glass ionomer sealants, resin sealants are very sensitive to a moist environment, which can compromise their stability over time [75]. Although glass ionomer sealants have a hydrophilic nature and allow high fluorine release, they have poor retention over time. Alsabek et al. demonstrated the superiority of resinbased sealants (particularly EmbraceTM WetBondTM) over glass ionomer sealants with a 6-month follow-up [57].
A particular advantage of these sealants is that their effectiveness does not depend on patient cooperation, and therefore the results obtained following their application can be considered more reproducible and reliable.

Conclusions
After reviewing the studies, we can conclude that CPP-ACP-based remineralizing agents remain the gold standard in clinical practice, especially when combined with fluoride-containing agents. Nevertheless, from the reviewed studies, excellent results were also obtained in treating WSLs with ozone therapy, SAP11-4, nano-hydroxyapatite, and sealants. Given the unevenness and heterogeneity of the analyzed studies, it is not possible to establish the superiority of one remineralizing agent over another.