Mineralizing agents to manage early carious lesions. Part I: mode of action

Chemistry of remineralization: mode of action

The natural formation of enamel (amelogenesis) is a complex biomineralization process involving cellular activity.¹⁰ Proteins self-organize to regulate the crystallization of hydroxyapatite in an ordered manner, that is, the extracellular protein matrix continuously forms when enamel crystals grow in length and the enamel thickens, followed by the transition and maturation stage when the protein matrix degrades as crystals grow in width and is eventually removed upon the completion of enamel formation. Therefore, mature enamel is acellular, and once demineralized, any repair cannot occur through autonomous biomineralization, but through extrinsic remineralization processes. Remineralization (perhaps better termed mineral deposition) can be described as the relatively disordered deposition of apatitic minerals onto/within the demineralized tissue. Efforts in investigating these remineralization processes have resulted in several commercialized agents available for clinical use. Depending on the mechanism, these can be categorized into the following groups:^8,11

Fluoride-based systems

Fluoride is often considered the ‘gold standard’ in enamel remineralization. The mechanisms by which fluoride facilitates enamel mineralization have been well documented. In an aqueous environment saturated with mineral ions and with a pH >5.5, fluoride is absorbed into the demineralized enamel. The electronegative ions act as nucleation sites, attracting calcium ions owing to the ionic charge.¹² It is suggested that a set of reactions, including ion exchange, chemisorption and hydrolysation can occur when fluoride reacts with hydroxyapatite (HA), which eventually results in transformation of HA to fluorapatite (FA) or fluoridated hydroxyapatite (FHA).¹³ FA and FHA have lower solubility in oral fluids at the critical pH for carious lesion formation (pH 5.5) thus increasing resistance to demineralization.¹⁴

Fluoride delivers significant remineralization potential at a low concentration (sub-ppm level),^13,15,16 but with a significant positive dose-response, with increased mineral gain with an increasing fluoride concentration.¹⁷ However, an in vitro study noted that the fluoride dose-response may also be dependent on the severity of the lesion, that is, the earlier the lesion, the better the dose-response, with possible reasons being that, in more demineralized lesions, the developed surface zone blocks the influx of mineral ions due to hypermineralization and/or the lack of soluble mineral ions leaves more acid-resistant minerals in the lesion.¹⁸

The clinical mode of action of fluoride in remineralization/mineral deposition is more sophisticated because biological factors may affect its efficacy. Fluoride can diffuse into the natural plaque biofilm, which may act as a reservoir to maintain fluoride concentration in the oral fluids close to the tooth surface.¹⁹ When the biofilm is immature, in vitro studies have revealed that fluoride failed to promote remineralization significantly, implying that elevated levels of fluoride in plaque and saliva exert a significant impact.^20,21 The level of fluoride in plaque is associated with many factors, including plaque disruption rate and pH.²² In addition, calcium fluoride (CaF₂) may form alongside FA and FHA, and be deposited onto the enamel surface. CaF₂ is readily soluble in acidic pH, when it dissolves and releases both calcium and fluoride into oral fluids, therefore acting as an alternative reservoir of calcium.²³ However, high-dose fluoride leads to excessive formation of CaF₂, which may in turn clog the lesion surface, resulting in reduced remineralization capacity.²⁴

Non-fluoride-based systems

Non-fluoridated mineralization agents can be further subcategorized into pH modifiers and calcium-phosphate systems (Table 1).²⁵

Biomimetic systems

Biomimetic approaches have gained increasing interest in recent years. Self-assembling peptides are an analogue to enamel matrix proteins, which could guide oriented apatite growth. P11-4 peptides, for example, are tailored for subsurface remineralization. Some studies indicate that P11-4 can diffuse into the carious lesion body, self-assemble into 3D hierarchical structures under highly ionic concentrations to provide nucleation sites for mineral ions, therefore favouring remineralization.^42,43 Although laboratory studies have found conflicting results regarding its efficacy in remineralizing incipient carious lesions,^44,45 recent clinical trials showed that P11-4 induced significantly greater remineralization than other agents including f-TCP and fluoride.^46,47 This material has been successfully launched as Curodont by Credentis for dental professionals.

Histological characteristics for mineralization

Incipient carious lesions (white spot lesions, WSLs) present clinically as a white, chalky and roughened enamel surface owing to the light scattering that occurs because of the difference in refractive index between the air/water in the porosities within the lesion and mineral, which intensifies with air drying. This poses an aesthetic and functional problem.⁴⁸ A histological diagram is shown in Figure 1. A recent in vitro pH-cycling study monitored the colour change in artificial WSLs using a spectroradiometer and attributed the greatest reversal of the white appearance observed the 5000ppm fluoride group to remineralization, which was evidenced by scanning electron microscopy (SEM) and electron microprobe analysis.⁴⁹ However, despite this remineralization, the white spot remained clinically evident after fluoride application. Similarly, CPP-ACP was found to show a significantly greater colour reversal compared to artificial saliva in vitro.⁵⁰ Further examinations suggested that such colour change was associated with mineral deposition filling the microporosities at the lesion surface. However, another in situ study compared the aesthetic characteristics of artificial WSLs remineralized by 5000ppm fluoride, bioglass and CPP-ACP and found no significant difference among all materials tested.⁵¹

Carious lesions are unique because, histologically, they consist of a well-mineralized surface zone in which the porosity is 1–2%, overlying the body of the lesion, which accounts for 25–50% porosity (Figure 1).⁵² Remineralization will affect these histological zones and, consequently, induce a series of changes.

The remineralization capabilities in the surface zone have been evidenced by studies employing various remineralization agents. For example, in an in vitro study, Milly et al demonstrated that bioglass, either pure or modified with polyacrylic acids, could precipitate apatitic crystals and assist the growth of the existing enamel prisms, therefore filling the superficial interprismatic spaces.⁵³ Some reports predict that the recrystallization mode depends on the calcium level of the solution. A low calcium concentration favours crystallization on existing damaged crystals, while higher concentrations may support nucleation and new crystal formation.⁵⁴ Other agents possess similar surface remineralization efficacy, including fluoride, CPP-ACP, f-TCP and n-HA.^53,55–57 The mineral gain in the surface zone can be measured as the recovery of surface microhardness resulting from the micro-structure reinforcement by the newly formed crystals. A recent in vitro pH-cycling study by Wang et al, comparing bioglass, CPP-ACP and fluoride on surface remineralization, suggested that treatment with these agents yielded significantly greater surface hardness recovery than in controls, which correlated well with morphological observations by SEM.⁵⁸ Caution is required because techniques, such as SEM and microhardness, are surface characterizations only. The Knoop microhardness indenter, for example, produces indentations with limited penetration to approximately 1.5 μm.⁵⁹ Thus, the interpretation of these results must be made with care when extrapolating to the clinical situation.

The subsurface body of the incipient carious lesion accounts for its bulk, and mineral infill in this part is crucial to successful long-term lesion repair. Some pH-cycling studies showed that by increasing the concentration, fluoride could diffuse through the depth of the lesion and initiate a deeper remineralization.⁶⁰ Fluoride tablets (4350ppm) exhibited extensive remineralization from the base of lesions, resulting in significant reduction in lesion depth, when compared to 1450ppm fluoride. Polarized light microscopy also showed an increased translucent zone after high-dose fluoride application, representing mineral infill in the inner part of the lesion. However, a recent in situ study suggested the opposite. Micro-radiographic observations by Amaechi et al indicated that HA induced a more homogeneous remineralization throughout the lesion depth than 500ppm fluoride, where remineralization occurred mostly in the surface zone.⁶¹ Nevertheless, both studies exhibited clear lamination after treatment, implying that the diffusion pathways in the lesion surface were blocked by the superficial mineral deposition. This is in agreement with previous studies.⁶²

Similarly, CPP-ACP is claimed to possess subsurface remineralization capabilities.⁶³ Longitudinal microhardness and microradiography suggested that CPP-ACP could favour more subsurface mineral regain than other agents, including calcium silicate and fluoride.^57,64 However, it was noted that the lesion surface remineralized by CPP-ACP exhibited comparably weaker mechanical properties. The conjecture was that newly precipitated mineral by CPP-ACP was less acid resistant, hence the pathways for ion diffusion remained open. A comparative study, on the other hand, suggested that CPP-ACP and fluoride were not superior to HA in remineralizing the subsurface lesion. All mineral gain followed an outside-inwards direction and predominately took place in/on the lesion surface.⁶⁵ These contradictory findings may arise from the disparities in study design, including concentration of the agent, study duration, type of study, etc.

Challenges for remineralization

Deep mineral gain within the body of the carious lesion remains the challenge for remineralization strategies. The porosity of the lesion surface zone is relatively small and can be more easily occluded when topical agents react and readily deposit on the surface, thus hindering ion penetration into the lesion depth. The lesion surface is arrested at the sacrifice of the lesion body in which the caries process and subsurface damage may still continue. The salivary pellicle, a tenacious semi-permeable organic layer on the enamel surface, retards transportation of ionic matter across the enamel.⁶⁶In vitro studies have demonstrated that this pellicle could inhibit remineralization on artificially formed WSLs.⁶⁷ It is, therefore, the delivery of calcium and phosphate ions into the depths of a lesion that controls remineralization. Figure 2 demonstrates the influence of mineral ions (concentration and exposure time) on the remineralization of incipient enamel lesions.

Some agents, such as self-assembling peptides and CPP-ACP, are claimed to possess the potential for subsurface remineralization. The mechanisms were discussed earlier. Although CPP-ACP has been shown to boost subsurface remineralization, a recent study found that extended application time with CPP-ACP failed to produce an additive remineralization effect.⁶⁴ Pre-conditioning the lesion surface to create better conditions for diffusion may provide an alternative route. Self-assembling peptides require conditioning through use of oral prophylaxis to remove biofilms and existing mineral deposits, which may prevent peptide monomers from penetrating.⁶⁸ Meanwhile, acid etching the lesion surface could also be considered to improve diffusion. Similar efforts have also been reported for other agents. For example, subsurface remineralization using a bioglass slurry was enhanced by surface pre-conditioning with air abrasion using bioglass particles modified by polyacrylic acids.⁶⁹ Mineral depositions were observed at an approximately 40–μm depth. Similarly, subsurface remineralization, to some degree, was enhanced by surface pre-conditioning with chitosan before bioglass application.⁷⁰ A dense layer consisting of newly deposited minerals was found to cover the lesion surface after the remineralization regimen. However, will this level of subsurface remineralization be effective long term? Perhaps these surface pre-conditioning techniques may require continuous/multiple applications by oral healthcare professionals, making home-based treatment difficult. Thus, designing an effective method for mineral-ion penetration is paramount for new effective remineralization strategies.

The speed of ion release is another challenge, especially for toothpastes and mouthwash carriers. Toothpastes cannot release active ingredients until dispensed, and having interacted with water or saliva. Other factors, such as toothbrush head geometry and filament size, saliva quality and secretion rate, are further complexities to consider.⁷¹ Mouth rinse has better ion-release potential. However, retention of such mineralizing agents remains an issue. The active phase of these agents is transient after brushing or rinsing, because of the diluting effect caused by the abrasive challenges by tongue, cheek movement, and/or pH change.^72,73

The future of biomimetic remineralization

In recent years, attention has been drawn to the biomineralization strategy for carious lesion repair, which aims to incorporate enamel proteins, such as amelogenin, to induce remineralization. Tooth enamel is the hardest tissue in the human body, and this unique characteristic is reportedly due to its delicate seven-level hierarchical structure.⁷⁴ Current commercialized remineralization agents can achieve mineral gain in the lesion, but to the best of the authors' knowledge, seldom replicate the hierarchical structure of enamel during remineralization. Amelogenins constitute 90% of matrix proteins involved in the process of enamel growth and the self-assembly ability is thought to be pivotal in forming the complicated hierarchical structure of enamel.⁷⁵ Molecular mechanisms include selective binding to specific crystal facets to regulate orientated crystal growth, and reaction with ACP to form intermediate pre-nucleation clusters that transform into organized crystals in subsequent stages.⁷⁵ Remineralization efficacy of amelogenin and its derivatives has been investigated. A hydrogel composed of chitosan-amelogenin, rP172, was found to biomimetically form mineral depositions with enamel-like crystals on both the surface and the subsurface of acid-etched and artificial carious lesions after pH-cycling for 7 days.^76,77 Leucine-rich amelogenin peptide, an amelogenin derivative, has also been found to reduce lesion depth and facilitate biomimetic reconstruction of enamel.⁷⁸ Meanwhile, there are certain concerns regarding amelogenin strategy, including the extraction and storage of amelogenins and duration required for the treatment.¹¹ However, clinical evidence is sparse, hence the requirement for more research to prove clinical efficacy.

Conclusions

Remineralization of early carious enamel lesions has seen promising progress in recent decades. Fluoride, calcium phosphate and biomimetic approaches, and their combinations, could successfully repair the surface and, to some degree, the subsurface zones of such incipient carious lesions. The lack of sufficient remineralization in the lesion body remains the greatest challenge for all commercialized mineralizing agents. New biomineralization strategies employing amelogenin and its derivatives may provide an alternative route, which have the potential to restore the hierarchical structure of tooth enamel. Extensive clinical investigations are necessary to prove the clinical benefits for these remineralization agents.