Antimicrobial, remineralization, and infiltration: advanced strategies for interrupting dental caries

The synthetic agents

Currently, commonly utilized synthetic agents include fluoride, chloride, and heavy metal agents. Fluoride can effectively inhibit acid production by S. mutans through direct targeting of enolase enzyme to impede the glycolytic pathway (Figure 4A) [33], while simultaneously enhancing enamel resistance against acidic solutions through the formation of fluorapatite [34]. Abundant evidence-based resources of high quality are readily available to support the utilization of fluoride toothpaste in caries prevention [35]. Well-controlled clinical trials consistently demonstrate significant reductions in caries incidence, up to 30 %, with the use of fluoride toothpastes such as Cheerio gel, Colgate sensitive, Close up deep action, Senquel-F, and Sensodent-KF [34]. The utilization of commercially available fluoride-containing varnish and toothpaste has become widespread [36]. Though the use of fluoride additives for the societal improvement and personal consumption has been prevalent, their effectiveness needs to be evaluated in light of the varying levels of fluoridated water consumed across different regions. Consequently, recommended dosage regimens vary regionally [36]. It should be noted that fluoride intake is a critical factor in the development of fluorosis. Therefore, it is important to remain cognizant of its potential negative effects. It is well-established that exposure to fluoride during post-secretory or early maturation period of enamel development increases the risk of fluorosis [36]. Reducing the fluoride content in water supplies is a viable strategy to mitigate the risk of dental fluorosis while maintaining nearly optimal caries prevention. Several countries have already implemented this measure. For example, in 2007–2008, Ireland and Canada reduced the target fluoride concentration from 0.9/1.0 to 0.7 ppm F. Currently, the United States Public Health Service recommends 0.7 ppm as the optimal fluoride concentration, which is at the lower end of the previous range of 0.7–1.2 ppm F [36]. The levels of fluorosis and dental caries will be continuously monitored as the ultimate test of this strategy.

Figure 4:

Antimicrobial mechanisms of chemical agents. (A) Mechanisms underlying the antimicrobial effects of fluoride and potential sites implicated in fluoride resistance (image source: Liao et al. [33] with permission). (B) Schematics illustrating the CPC-mont antibacterial technology (image source: Matsuo et al. [39] with permission). (C) The antibacterial mechanism of metal nanoparticles (image source: Chen et al. [42] with permission).

Chlorhexidine, a widely used antimicrobial agent against S. mutans, carries a positive charge in solution, enabling it to bind to the bacterial cell wall and subsequently destroy it [37]. Cetylpyridinium chloride (CPC) disrupts microbial cell membranes by perturbing their electrical equilibrium [38]. Given its inhibitory impact on S. mutans, CPC is frequently integrated as an antimicrobial agent in mouthwashes, toothpastes, resins, and adhesives to effectively reduce the incidence of dental caries [39]. In order to extend the release duration of CPC, Matsuo et al. incorporated CPC into montmorillonite (CPC-Mont) for controlled release, thereby significantly enhancing its antibacterial efficacy (Figure 4B) [39]. However, the cytotoxicity of CPC requires careful consideration. Research has shown that CPC dilutions exhibit cytotoxic effects on L929 cells [40], which is attributed to its mitochondrial inhibitory activity, specifically through the inhibition of mitochondrial oxygen consumption and ATP synthesis in vitro. Additionally, mouthwashes containing CPC have been found to be cytotoxic to neonatal melanocytes [41]. Therefore, it is crucial to carefully regulate the dosage of CPC to mitigate potential cytotoxic effects.

The inhibitory effects of silver nanoparticles, zinc nanoparticles, and gold nanoparticles on S. mutans have been extensively investigated (Figure 4C) [42], with silver nanoparticles being the subject of the most comprehensive studies. The antibacterial activity of silver compounds is attributed to their ability to disintegrate the cell membrane, penetrate into the interior of bacterial cells, and disrupt intracellular organelles. Additionally, silver compounds exhibit an additional mechanism that enhances their efficacy by inhibiting bacterial DNA replication capability and inactivating bacterial enzymes [43]. The incorporation of silver nanoparticles into adhesive systems and composite resins has demonstrated significant efficacy in the prevention of secondary caries [44], 45]. However, it is imperative to further evaluate their biosafety implications. Compared to pure metal nanoparticles, metal oxide nanoparticles demonstrate lower cytotoxicity to the human body [46] and are more cost-effective [47]. Although their precise antimicrobial mechanisms remain under debate, several distinctive mechanisms have been proposed. These include the generation of reactive oxygen species, the release of metal ions, internalization of particles into bacterial cells, and direct mechanical disruption of bacterial cell walls and/or membranes [48]. Zinc oxide nanoparticles can be incorporated into conventional glass without altering its fundamental mechanical properties [49], thereby imparting antimicrobial characteristics beneficial for dental restorations. However, it has been observed that the incorporation of Zinc oxide nanoparticles into composite resins affects their chemical and mechanical properties [50], thus limiting their clinical applications. In addition, copper oxide nanoparticles are often added to adhesives to prevent the development of early carious white spot lesions [51]. Given the variations in antimicrobial activity, biocompatibility, and dispersibility among different nanoparticles, the combination of various metals and metal oxides holds the potential to produce a wide array of antimicrobial materials with enhanced properties.

Antimicrobial peptides (AMPs)

Antimicrobial peptides (AMPs) represent a widely distributed class of peptides found in living organisms, which serve as a defense mechanism against foreign microorganisms and certain mutated cells. As a primary line of defense in innate immunity, antibacterial peptides exhibit broad-spectrum antimicrobial activity and a lower propensity for drug resistance compared to fluoride and chlorhexidine [52], 53]. The bactericidal mechanism of AMPs involves their positively charged properties and their interaction with the negatively charged surface of microorganisms. These peptides exhibit antibacterial activity by displacing lipids and integrating themselves into the outer leaflet of the lipid bilayer in the cytoplasmic membrane (Figure 5A) [54]. The mechanisms of ‘barrel-stave’, ‘carpet’, or ‘toroidal-pore’ facilitate the adherence and insertion of AMPs into membrane bilayers (Figure 5B) [55]. Additionally, some AMPs inhibit biofilm formation by competing with bacteria for adherence [56].

Figure 5:

Schematic representation of the bactericidal mechanism of AMPs. (A) Disruption of the cell membrane by AMPs (image source: Luo et al. [54] with permission). (B) The ‘barrel-stave’ (a), ‘carpet’. and (b), ‘toroidal-pore’ models of membrane disruption by AMPs (image source: Hafeez et al. [55] with permission).

The initial AMPs identified in mammals are known as defensins, which can be categorized into two subfamilies in humans: α-defensin (HNP) and β-defensin (hBD). The expression of hBDs (hBD-1, hBD-2, and hBD-3) has been observed in dental pulp and odontoblasts [56], 57]. Defensins hBD-2 and hBD-3 demonstrated activity against various oral pathogens, particularly the cariogenic organism S. mutans [58]. Moreover, LL37, α-defensins (HNP 1–3), and Histatin-5 all exhibited inhibitory effects against S. mutans [59], [60], [61]. In order to facilitate the application of AMPs in caries management, researchers are continuously studying synthetic analogs or fragments of natural AMPs derived from humans or other species. Due to the relatively short amino acid sequences of some AMPs, chemical synthesis offers a convenient approach to producing peptides that are structurally and functionally identical to their natural counterparts. The most commonly employed method is solid-phase peptide synthesis [62]. The core principle of solid-phase peptide synthesis involves anchoring the carboxyl terminus of an amino acid to a polystyrene resin support through chemical linkers. The amino terminus is then exposed to facilitate peptide chain elongation. The incoming amino acids are protected at their amino terminus and side chain groups, with only the carboxyl terminus activated for the coupling reaction. This activated carboxyl terminus reacts with the exposed amino terminus on the resin. By sequentially removing the amino protective group, adding an amino acid, and repeating this deprotection-coupling cycle, the desired peptide chain is synthesized iteratively [63].

The stability and inhibitory activity of AMPs against oral bacteria can be improved through amino acid substitutions, introduction of hydrophobic groups, and shortening of amino acid residues based on the structural characteristics of different AMPs [64], 65]. The AMP pleurocidin, derived and engineered from the epidermal mucus of Pleuronectes americanus exhibits potent bactericidal effects against S. mutans [66]. The derived peptide AT-7 from Lactobacillus, the de novo designed amphipathic α-helices peptide GH12, and the novel gallic acid (GA)-grafted AMP cathelicidin LL37 have all shown favorable inhibitory effects on S. mutans [67], [68], [69]. The recent advancements in designing AMPs for caries prevention have primarily focused on specifically targeting S. mutans, with the aim of restoring a healthy microbial community.

Xiang et al. employed virtual screening of lipopeptide libraries to develop C10-KKWW, which effectively inhibits and disrupts S. mutans biofilms by specifically targeting the l-ascorbate-specific PtxA component of its phosphoenolpyruvate sugar phosphotransferase system, as confirmed through software simulations [70]. SspB (390-T400K-402), a peptide derived from the adhesin SspB, exhibits high affinity for salivary glycoprotein-340 peptide SRCRP2, competitively inhibiting the adhesion of S. mutans to saliva and impeding biofilm formation [71]. Another targeting strategy involves pH-activated AMPs, which exhibit potent antimicrobial activity exclusively in acidic environments while remaining ineffective under neutral physiological conditions [72]. The dual-sensitive AMP, pHly-1, engineered by Zhang et al., undergoes a conformational transition from coil to helix upon interaction with bacterial membranes within the acidic cariogenic biofilm microenvironment, effectively eradicating cariogenic bacteria [73]. Conversely, under normal physiological conditions, pHly-1 adopts a β-sheet conformation and self-assembles into nanofibers, resulting in minimal cytotoxicity towards oral microbes. Jiang et al. engineered LH12 to incorporate histidine-rich sequences, thus enhancing its antimicrobial efficacy in acidic environments [74]. Protonation of LH12 in the acidified cariogenic microenvironment results in increased cationicity and improved interactions with bacterial membranes, enhancing the competitiveness of commensal bacteria under acidic conditions.

The limited half-life of AMPs presents a significant obstacle as therapeutic agents, impeding their clinical utility. Moreover, the economic challenges associated with AMP development should not be disregarded. Hence, ongoing efforts in caries prevention and treatment are focused on identifying or synthesizing medications that offer optimal safety profiles, consistent effectiveness, and economic viability for human use.

Natural medicines

The diverse range of chemical constituents, extensive origins, potent pharmacological activity, and negligible toxicity make natural medicines a pivotal reservoir of efficacious therapeutic agents that have been harnessed and explored for over three millennia [75]. Therefore, screening natural compounds for effective agents against S. mutans is a promising approach towards developing anti-caries drugs.

Phenolic compounds derived from Galla chinensis, Magnolia officinalis, and green tea have been extensively utilized in studies focused on dental caries prevention and control [76], [77], [78]. Phenolic compounds are secondary metabolites characterized by the presence of multiple phenolic hydroxyl groups, readily combining to form polyphenols or phenolic acids such as catechins and tannins. Their antibacterial effect primarily involves inducing the denaturation of pellicle proteins and bacterial cell lysis (Figure 6A) [79]. Additionally, polyphenols contribute to addressing periodontitis, halitosis, and oral cancer (Figure 6B) [80], making them key components in toothpaste formulations, mouthwash solutions, and chewing gum products (Figure 6C) [80]. Furthermore, phenolic compounds possess antioxidant and anti-inflammatory properties, rendering them essential constituents of numerous traditional Chinese medicines that exert significant pharmacological effects [81].

Figure 6:

The antimicrobial mechanisms and applications of polyphenols. (A) Antimicrobial mechanisms of polyphenols (image source: Flemming et al. [79] with permission). (B) Polyphenols in dental caries, periodontal diseases, halitosis, and oral cancers [80]. (C) Current approaches and prospects of polyphenols-based oral health (image source: Guo et al. [80] with permission).

The therapeutic potential of honokiol has been utilized for generations in the traditional herbal treatments of China, Japan, and Korea [82]. In caries research, it has been demonstrated that the high permeability of honokiol exerts bactericidal effects on S. mutans biofilms [83]. Ren et al. investigated the specific mechanism underlying their resistance against S. mutans and discovered that honokiol, a Gtf inhibitor, effectively impeded biofilm formation and EPS accumulation. Furthermore, it exhibited inhibitory effects on the down-regulation of the lactate dehydrogenase gene in S. mutans, resulting in reduced lactic acid production and tooth demineralization [82].

Galla chinensis extract (GCE), obtained from leaf galls induced by parasitic aphids, primarily comprises hydrolyzable tannins such as gallotannin and GA [84]. The growth-inhibiting and metabolic effects of GCE on dental caries pathogens, along with its capacity to promote enamel remineralization and prevent demineralization, have been substantiated by extensive in vitro and in vivo experimentation [85], 86]. These advantageous properties can be attributed to GCE’s interference with bacterial physiological processes and suppression of Gtfs activity [87]. However, the inefficacy of GCE against diverse biofilms under alkaline conditions underscores the optimal pH for applying GCE solutions as an anticariogenic agent to be 5.5 [88]. Nonetheless, it is important to note that a lower pH level may potentially exacerbate enamel demineralization, thus limiting the application of GCE due to this property.

The global consumption of tea is extensive, leading to variations in its polyphenol composition and content as a result of diverse processing methods. Notably, green tea contains active constituents such as catechins including gallocatechin and epigallocatechin-3-gallate (EGCG) [89]. The inhibitory effect of EGCG on the growth and viability of S. mutans was observed only within the concentration range of mg/mL, while its ability to inhibit in vitro biofilm formation occurred at a minimum inhibitory concentration level of 15.6 μg/mL [90]. These findings suggest that one mechanism underlying the antimicrobial activity of EGCG contributes to the inhibition of biofilm formation, which was further confirmed by Real-time PCR analysis, which demonstrated its ability to suppress Gtfs genes [91]. Therefore, though the polyphenol concentration in the oral cavity cannot be consistently maintained at a high level following green tea consumption, it remains sufficient to effectively inhibit biofilm formation. This finding has been confirmed by randomized clinical studies [92]. Overall, these findings underscore the potential of phenolic compounds, particularly those derived from natural sources, as promising agents for preventing and controlling dental caries. Their multifaceted properties make them valuable candidates for oral care products and traditional medicine formulations.

In summary, effective management of plaque biofilms is crucial for preventing dental caries. Strategies aimed at disrupting biofilm formation and targeting cariogenic bacteria constitute the fundamental approach in the prevention and control of caries. However, solely relying on antibacterial treatment while overlooking the repair of the defective tissue is insufficient for preserving the integrity and functionality of the tooth. In the subsequent section, we delve into the research progress on remineralization, with demineralization as a pivotal clinical manifestation of caries.

Calcium phosphate system

The presence of mineral ions is essential for the progression of mineralization, and the inclusion of additional Ca²⁺ and PO₄ ³⁻ proves advantageous in expediting the process of mineralization under conditions of insufficient salivary secretion, while also enhancing fluoride-mediated remineralization [109]. The rationale for employing nano-hydroxyapatite (NHAP) in achieving remineralization is substantiated by the predominant constituents of enamel minerals. NHAP demonstrates a structure and chemical formula comparable to that of enamel crystals, while its nanoscale dimensions confer enhanced bioactivity and heightened surface energy, facilitating robust binding with the enamel surface [110]. The incorporation of NHAP into toothpaste formulations is a common practice for the prevention of dental caries [111]. Moreover, the utilization of this toothpaste after orthodontic treatment has been demonstrated to effectively alleviate the roughness and sensitivity associated with post-debonding tooth surfaces [112]. While the mineralization process described adheres to the classical theory of crystal formation through ion deposition, it fails to consider the involvement of other elements in enamel development [113] and is no longer regarded as the prevailing paradigm in mineralization theory.

The uniqueness of ACP in remineralization has been elucidated in the preceding chapter. In addition, ACP demonstrates a significantly higher release of Ca²⁺ and PO₄ ³⁻ compared to other calcium phosphate systems, including HAP, due to its exceptional solubility as the most soluble calcium phosphate phase [114]. However, ACP is thermodynamically unstable and susceptible to transformation into more stable crystalline phases such as octacalcium phosphate and HAP. Therefore, prior stabilization is necessary before application, which is commonly achieved through pairing with various stabilizers and includes casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). Casein, the primary phosphoprotein found in milk, predominantly functions as a calcium phosphate-stabilized micellar complex. The excellent biocompatibility of CPP-ACP makes it a commonly used principal active ingredient in toothpastes for the management of caries and desensitization purposes. This also exemplifies the direct ACP delivery technology. It exerts a long-lasting remineralizing effect on early carious lesions [115], 116]. The clinical applications of CPP-ACP, however, are limited due to allergic reactions in certain populations, thus hindering further advancements in its usage.

ACP mineralized systems, which are typically in liquid form, pose challenges for clinical application due to their state. One proposed solution is integrating ACP particles into various dental resins, a concept introduced in the early 20th century [117]. However the mechanical properties of ACP composites are relatively inferior, with their flexural strength being approximately half that of unfilled resins, rendering them unsuitable for use as dental filling materials [118]. Xu et al. developed nanoscale amorphous calcium phosphate (NACP) composites, which exhibited comparable release of Ca²⁺ and PO₄ ³⁻ to previous CaP composites at a 20 % NACP content, while demonstrating at least twice the strength of previous ACP composites [119]. This superior performance can be attributed to the higher specific surface area of NACP compared to ACP, enabling enhanced release of Ca²⁺ and PO₄ ³⁻ even at low filler levels, as well as providing additional space for glass fillers to achieve exceptional mechanical properties. The composite, containing 40 % NACP, is capable of rapidly elevating the pH level of its surroundings from 4.0 to 5.7 in 10 min, effectively mitigating mineral degradation caused by acidity [120], 121]. What’s more, to achieve long-term remineralization, Zhang et al. developed a rechargeable NACP composite that incorporates pyromellitic glycerol dimethacrylate capable of chelating with external Ca²⁺, which can facilitate both the charging and re-release of mineral ions [122]. However, the practical clinical applications should consider the pertinent biosafety concerns.

Amelogenin and its analogs

Amelogenin constitutes more than 90 % of the enamel matrix and is believed to play a crucial role in regulating crystal growth and directing alignment during the process of enamel biomineralization [101]. Amelogenin protein is characterized by its relatively hydrophobic nature and consists of three distinct structural domains: a tyrosine-rich N-terminal domain with pronounced hydrophobicity that facilitates intermolecular interactions, a C-terminal domain rich in hydrophilic residues capable of establishing multiple charged interactions with the mineral surface, and a central region abundant in hydrophobic proline [123], 124]. Amelogenin demonstrates conformational versatility in diverse environments and upon interaction with various molecules. In the presence of Ca²⁺ and PO₄ ³⁻, amelogenin undergoes self-assembly into nanospheres, exhibiting a propensity for further extension into nanochains and nanofibers [125]. This hierarchical assembly confers advantages in stabilizing ACP and regulating crystal arrangement. Numerous studies have shown that recombinant human amelogenin possesses the capability to effectively synthesize fibrous apatite crystals from calcium phosphate saturated solutions. This phenomenon can be attributed to the contrasting charges exhibited by amelogenin and apatite [126]. In addition, the combination of amelogenin and fluoride can induce the formation of oriented bundles in needle-like fluorapatite [127].

Given the challenges associated with synthesizing full-length amelogenin and the potential immunogenicity of non-functional amelogenin sequences, recent research has been dedicated to developing various analogues of amelogenin for mineralization reproduction. Therefore, a comprehensive understanding of the specific structure and physiological function of amelogenin is imperative. The conservation level is high for both the hydrophilic C-terminus and hydrophobic N-terminus of amelogenin. However, unlike the recombinant full-length amelogenin, amelogenin with a truncated mutation in its hydrophilic C-terminal region showed an incapability to self-assemble into nanochains or induce parallel bundle formation of apatite [128]. Subsequently, upon deletion of peptide 14P2 from the N-terminus region, it was observed that the formation of nanoribbons was impeded even in the presence of calcium and phosphorus ions [129]. Therefore, the N-terminal domain plays a pivotal role in facilitating the self-assembly of amelogenin into amyloid-like aggregates through β-sheet stacking and providing a structural template for the growth of HAP crystals. Conversely, the C-terminal domain is responsible for promoting parallel alignment among these crystals [130].

In recent years, the technique of selective splicing has been utilized to synthesize truncated peptide fragments derived from major functional segments of amelogenin, such as tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide (LRAP) [127], 131]. Notably, LRAP demonstrates the capacity to substitute for the full-length amelogenin, thereby inducing the transformation of disordered calcium phosphates into organized crystals and facilitating the formation of enamel-like structures [132], 133]. However, extracting and purifying amelogenin and non-amelogenin, natural proteins presents challenges due to their susceptibility to denaturation and the risk of potential contamination [134].

In order to overcome the aforementioned limitation, Fang et al. devised an innovative approach where they genetically modified LRAP by incorporating a 3-tyrosine peptide domain at its N-terminus, resulting in a novel polypeptide called mLARP [135]. This fusion protein combines the advantageous features of both TRAP and LARP. Additionally, mLARP was successfully combined with non-amelogenin proteins to create enamel matrix proteins that could significantly enhance the biomineralization process. The outcomes of mineralization experiments clearly demonstrated that the regenerated enamel displayed prismatic and interprismatic structures similar to those observed in natural enamel, thus offering a promising strategy for promoting HAP crystal growth. Mukherjee et al. engineered peptides P26 and P32 by manipulating and fusing distinct functional domains within amelogenin [136]. Both peptides demonstrated the ability to generate HAP layers in situ with comparable crystal morphology and mechanical properties. However, P32 induced larger structural dimensions of peptide assemblies and crystal size in vitro compared to P26, which could be attributed to its two additional polyproline repeat motifs. This suggests that an increased number of polyproline repeat motifs could significantly alter the size of HAP crystals. Additionally, P26 and P32 exhibitrf significant potential in facilitating dentin remineralization [137]. Ding et al. successfully intercepted Gln-Pro-X, a highly conserved sequence in amelogenin, and developed QP5 peptide consisting of five repeats of Gln-Pro-X along with the C-terminus [138]. QP5 demonstrates robust affinity towards HAP, which is crucial for promoting enamel remineralization (Figure 8A) [124]. It effectively stimulates the formation of well-organized bundles of HAP and enhances the efficiency of fluoride-induced remineralization in its presence. By co-loading QP5 with bioactive glass within a hydrogel matrix, Ca²⁺ and PO₄ ³⁻ are supplied to QP5 by the bioactive glass, resulting in the formation of a remineralized layer that can exhibite excellent resistance against both erosion and abrasion [139]. However, the synthesis of peptides is consistently costly irrespective of their length. Hence, there exists a necessity to discover a straightforward and cost-effective approach to imitate the function of amelogenin.

Figure 8:

Schematic demonstration of (A) amelogenin-derived peptide QP5 for promoting enamel biomimetic remineralization (image source: Hu et al. [124] with permission); (B) amelogenin and the PTL/C-AMG matrix to mediate the transition from ACP to HAP on enamel for in situ remineralization (image source: Wang et al. [130] with permission).

It has been observed that lysozyme (lyso), when catalyzed by a reducing agent that disrupts disulfide bonds, can undergo a phase transition and generate phase-transforming lysozyme (PTL) with numerous β-sheet structures [140]. PTL effectively emulates the amyloid-like structure present in the N-terminal end of amelogenin. Based on this, Wang et al. combined PTL with C-terminal synthetic peptides (C-AMG matrix), resulting in engineered materials capable of replicating both in vivo and in vitro growth of ordered HAP crystals (Figure 8B) [130]. Moreover, the aligned structure of these materials closely resembles natural enamel.

In conclusion, the strategy of emulating the function and structure of amelogenin for enamel tissue regeneration from a developmental promising data. However, the steep cost associated with peptides may pose a significant constraint and hinder their broader application in research.

Polymer system

In recent years, extensive research has been conducted to design and develop polymers with protein-like functionalities aimed at preventing and treating early enamel caries. These polymers aim to achieve enamel remineralization by mimicking the stabilizing effect of amelogenin proteins on ACP in vivo. Polyacrylic acid (PAA), a superabsorbent polymer known for its non-cytotoxic and biocompatible nature, demonstrates carboxylic acid activity [141]. Qi et al. found a positive correlation between the concentration of PAA and its ability to inhibit the aggregation of ACP prenucleation clusters at the initial crystallization stage [142]. Conversely, reducing PAA concentration increases the kinetics of crystallization from ACP to nanoapatite. Liu et al. introduced a biomimetic mineralization strategy using polyphosphate, where sodium trimetaphosphate (STMP) was used alongside PAA to stabilize ACP [143]. The anionic surfactant STMP phosphorylates PAA, resulting in the production of highly phosphorylated PAA that effectively remineralizes artificial carious lesions, particularly in areas lacking seed crystallites. Furthermore, polymers such as polyaspartic acid, polyglutamic acid, polyacrylamide hydrochloride, polylysine, and carboxymethyl chitosan exhibit a close resemblance to the mineralization mechanism of PAA [144], [145], [146]. These polymers possess the ability to facilitate biomimetic restoration of enamel by stabilizing ACP and promoting its transformation into crystalline structures.

The utilization of the carboxyl terminus of branched polymers has demonstrated promising outcomes in attracting Ca²⁺ ions, however, the mineral content of remineralized layers typically remains suboptimal due to the limited availability of carboxyl groups [147]. For effective biomineralization control, ACP stabilizers, akin to NCPs, should have a well-defined steric structure. Poly (amidoamines) (PAMAMs) are notable examples, characterized by their high branching, multiple reactive terminal groups, specific dimensions, forms, and internal cavities. PAMAMs function as effective nucleation templates, chelating and binding Ca²⁺ and PO₄ ³⁻ electrostatically, securing nano-sized ACP particles, and enhancing remineralization [148], 149]. The size and shape of HAP can be modulated in a concentration-dependent manner by modifying the surface groups of PAMAMs [150]. HAP nanorods induced by carboxylic-terminated PAMAM (PAMAM-COOH) and hydroxyl-terminated PAMAM (PAMAM-OH) exhibit distinct shapes and lengths, with PAMAM-COOH resulting in more elongated rods compared to PAMAM-OH due to variations in nucleation site localization [151]. Fan et al. conducted a comparative study on the in vitro remineralization capacities of PAMAM-OH, PAMAM-COOH, and amine-terminated PAMAM (PAMAM-NH₂) [152]. The results revealed that among them PAMAM-NH₂ exhibited the highest remineralization capacity while PAMAM-OH demonstrated the lowest capacity. This difference can be attributed to their distinctive surface charges. Precisely, enamel surfaces are known for their negative charge; however, PAMAM-NH₂ carries a positive charge, PAMAM-COOH exhibits a negative charge, while PAMAM-OH remains electrically neutral. As a result of electrostatic attraction, the binding affinity between PAMAM-NH₂ and the enamel surface is significantly enhanced. Therefore, it is crucial to enhance the affinity between remineralized material and enamel in order to facilitate the formation of a mineralized layer. To accomplish this, Wu et al. incorporated alendronate (ALN) into PAMAM-COOH, which facilitated ligand exchange with the HAP surface and significantly improved the structure and mechanical properties of the newly generated enamel layer (Figure 9A) [153]. In view of the abundance of phosphate groups in amelogenin that stabilize ACP, Chen et al. synthesized PAMAM-PO₃H₂, a phosphate-terminated PAMAM derivative, by incorporating phosphate groups into PAMAM (Figure 9B) [154]. The strong affinity between the phosphate group and Ca²⁺ facilitated the formation of an 11.23 μm-thick enamel remineralization layer, whereas PAMAM-COOH only achieved a thickness of 6.02 μm. Moreover, in vivo experiments demonstrated promising remineralization effects of PAMAM-PO₃H₂.

Figure 9:

Schematic demonstration of (A) the specific adsorption of ALN–PAMAM–COOH on the surface of tooth enamel and the subsequent in situ remineralization of HAP (image source: Wu et al. [153] with permission); (B) the adsorption of PAMAM-PO₃H₂ on the surface of tooth enamel and the subsequent in situ remineralization of HAP (image source: Chen et al. [154] with permission); and (C) amyloid-like matrix of PTL fibrils (mimicking the templating protein), CMC/ACP (mimicking the non-templating protein) and NaClO (mimicking the proteinase) accomplish the three “key events” of biomineralization, de novo remineralization enamel in vitro (image source: Ye et al. [155] with permission).

Previous studies have predominantly focused on stabilizing ACP or utilizing amelogenin and its analogues to interact with mineral ions, aiming to achieve enamel-like crystal formation. However, these approaches still fall short in accurately replicating the columnar structure of natural enamel and fully restoring its mechanical properties to normal levels. Moreover, it is imperative not to overlook the nucleation of non-amelogenins and the degradation of amelogenin by proteinases [100]. In the enamel remineralization system developed by Ye et al., PTL functioned as a mimic of amelogenin, while carboxymethyl chitosan and hypochlorite served as imitations of non-amelogenins and proteinases, respectively (Figure 9C) [155]. Through synchronous self-assembly and mineralization, the regenerated mineral crystals exhibites an oriented structure and mechanical properties akin to natural enamel. Therefore, it is imperative to further investigate and focus on restoring the entire in vitro biomineralization process for achieving rapid and effective repair of demineralized enamel.

Peptides

The absence of microbial resistance development in AMPs is a crucial characteristic that allows for the fusion of AMPs with remineralizing peptides, creating bifunctional peptides for both antimicrobial and remineralization purposes. Wang et al. successfully linked TD7, a functional peptide located at the C-terminal end of amelogenin, to the N-terminal end of GH12 (α-helical AMPs), which resulted in the formation of a bifunctional peptide called TDH19 [179]. To enhance its positive charge, glutamic acid and aspartic acid residues in TDH19 were substituted with glutamine and asparagine respectively, leading to the creation of TNH19. Furthermore, by replacing the asparagine residue in TNH19 with valine, TVH19 was generated to increase hydrophobicity. The comparison of the bacterial and remineralization capacities of the three bifunctional peptides revealed that TVH19 exhibited superior antimicrobial activity and induced the formation of optimal crystal morphology. These findings suggest that charge and global hydrophobicity are crucial structural parameters than can influence the efficacy of amphiphilic α-helical AMPs [180], with positive charge promoting peptide interaction with microbial surfaces, while global hydrophobicity facilitates effective insertion into biofilms. These results provide novel insights for designing subsequent bifunctional peptides.

Zhou et al. chose to synthesize the bifunctional peptide Sp-H5 by incorporating histone 5 (H5), a salivary antimicrobial component, and grafting phosphoserine (Sp) onto its N-terminal end [181]. Importantly, in addition to its broad range of antibacterial and antifungal activities, H5 plays a vital role as a constituent of the acquired enamel pellicle and actively inhibits enamel demineralization [182]. The presence of Sp allows for binding to HAP and facilitates the attraction of free Ca²⁺ for nucleation and initiation of mineralization, which are commonly investigated in studies related to tooth and bone regeneration. The application of Sp-H5 on the tooth surface has been demonstrated to generate a novel bioactive tooth surface with antibiofouling and mineralizing properties. Through the presence of positively charged amino acid residues, Sp-H5 adheres to the enamel surface and attracts Ca²⁺ via negatively charged residues, thus initiating the process of enamel surface mineralization. Simultaneously, Sp-H5 exhibits bactericidal effects against planktonic S. mutans and protects enamel from acidic environments through electrostatic repulsion. Moreover, its user-friendly application allows for tight sealing of demineralized surfaces, effectively promoting in situ caries self-healing. However, subsequent investigations revealed that the long-term antimicrobial and remineralization capabilities of Sp-H5 were insufficient, which promoted further enhancements [183]. The SpSp (DPS) structural domain was incorporated to enhance the Ca²⁺ chelating capacity and subsequently grafted onto the C-terminus of P-113, which is the smallest antimicrobial fragment in H5. This resulted in the construction of P-113-DPS (Figure 10) [183]. Notably, P-113-DPS demonstrated superior adsorption ability, antibiofouling properties, demineralization inhibition, remineralization promotion, stability, and remineralization compared to Sp-H5. The incorporation of DPS not only facilitated increased attraction to Ca²⁺, but also induced rapid nucleation and growth of hexagonal prismatic crystals. Furthermore, by localizing on bacterial surfaces and cross-linking with bacterial membrane phospholipids, P-113-DPS is effectively destructive against S. mutans. These findings highlight the potential of P-113-DPS in managing dental caries.

Figure 10:

Schematic diagram of multiple functions of P-113-DPS in this study. (1) P-113-DPS adheres to the enamel surface via positively charged amino acids attracted to negatively charged PO₄ ³⁻of HA. (2) P-113-DPS kills planktonic Streptococcus mutans. (3) Coating of P-113-DPS on enamel inhibits the adhesion of S. mutans. (4) Coating of P-113-DPS on enamel protects the enamel surface against demineralization. (5) Coating of P-113-DPS on enamel increases the thickness of the regenerated crystal layer. Ser13-p and Ser14-p are in the red circles. Carboxyl is in the green circle. Green dotted lines show the coordinate bond; the pink dotted lines show the hydrogen bond (image source: Zhou et al. [183] with permission).

In addition to enamel caries, bifunctional peptides can also be valuable in the management of dentin caries. Niu et al. developed a bifunctional peptide called GA-KR12 [184], where GA serves as the functional group grafted onto the KR12 peptide derived from LL-37, which is the sole human cathelicidin variant. The antimicrobial efficacy of GA requires no further elaboration for its phenolic compound nature and pyrogallol moiety has been demonstrated to induce and expedite mineralization [185]. Treatment with GA-KR12 resulted in remineralization of the extrafibrillar portion of dentin and inhibition of S. mutans biofilm growth in artificial dentine caries models. However, intrafibrillar mineralization was not achieved possibly due to hinderance by a size exclusion mechanism. Overall, while enamel caries arrest and dentin caries arrest processes differ, they both require remineralization capacity and antimicrobial efficacy.

Nano-coatings

The effective sealing of pits and fissures on tooth surfaces is vital for preventing dental caries [186]. However, long-term microleakage may still occur due to inherent disparities between sealers and enamel composition. Henceforth, an enduring solution for dental caries necessitates sealers with remineralization capabilities alongside antimicrobial properties. PTL’s diverse reactive groups including carboxyls, hydroxyls, and amines enable strong adhesion to various substrate surfaces. Capitalizing on this feature, Yang et al., enhanced PTL’s hydrophilicity by grafting poly (ethylene glycol) (PEG) onto lyso while simultaneously reducing disulfide bonds through reductive means (Figure 11A) [187]. Consequently, forming lyso-PEG oligomeric nanoparticles measuring only 30 nm in aqueous solutions, which is one-10th the size compared to traditional resin-based sealants, facilitates deep penetration into pit-fissure structures. When a solid interface is present, lyso-PEG aggregates to form a seamless nano-film coating that acts as an adhesive surface for attracting Ca²⁺ and PO₄ ³⁻ from saliva. This promotes epitaxial growth of enamel without any marginal gaps between the newly mineralized layer and natural enamel. Consequently, this process successfully restores the mechanical properties of enamel while simultaneously inhibiting bacterial adhesion through cell wall perturbation, thus showing great potential for caries prevention.

Figure 11:

Nano-coatings based on IDC concept. (A) The scheme illustrates the rapid formation of lyso-PEG oligomer nanoparticle-based nanofilm coatings in the deep zone of pits and fissures, followed by ion adsorption from saliva and spontaneous remineralization toward the formation of enamel-like HAP structures in pits and fissures to achieve in situ biomimetic sealing (image source: Yang et al. [187] with permission). (B) The PAsp-PEG compound interacts with calcium ions on the acid-etched enamel surface, effectively chelating free calcium and phosphate ions in the solution to enhance mineralization, and forming a brush-like barrier on the enamel surface to inhibit bacterial adhesion (image source: Hou et al. [188] with permission). (C) Schematic illustration of PEG-PAsp-ALN block polymers to mediate enamel remineralization and resist adhesion of bacteria and proteins (image source: Chu et al. [189] with permission).

In recent years, hydrophilic and antifouling PEG have gained increasing prominence in the field of biomedical applications. PEG acts as a brush-like barrier to effectively inhibit the nonspecific adsorption of bacteria, making it widely recognized as the “gold standard” for antibiofouling polymers [188]. Hou et al. utilized polyaspartic acid (PAsp) to mimic amelogenin’s ability to induce biomineralization, capitalizing on the role of aspartic acid found in amelogenin in facilitating binding with HAP (Figure 11B) [189]. By combining PEG with PAsp, a multifunctional material possessing both antifouling and remineralization properties can be achieved. The PAsp-PEG coating effectively prevents bacterial adhesion on dental surfaces through hydration and steric hindrance effects, without inducing an inflammatory or immune response due to the absence of viable and non-viable bacteria. However, the limited availability of carboxyl groups in the coatings and their weak binding affinity to Ca²⁺ constrain their potential for remineralization. To address this issue, Chu et al. utilized ALN sodium for copolymer conjugating to optimize the material and provide an abundance of PO₄ ³⁻ groups (Figure 11C) [190]. The experimental results confirmed that compared to PAsp-PEG, PEG-PAsp-ALN demonstrated enhanced absorption capacity on the enamel surface, improved stability, and induced a thicker mineral layer, thereby achieving superior effects in caries prevention and control. Furthermore, taking inspiration from the intricate process of enamel formation and recognizing the significance of organic-inorganic interactions in mineral assembly, Wong et al. utilized a layer-by-layer deposition technique to precisely manipulate the crystallization process and facilitate the self-assembly of crystals [191]. Through the combination of graphene oxide, alginate, and chitosan, they successfully synthesized a macroscopic bioactive material that closely emulates enamel in terms of its chemical composition, mechanical properties, and crystal structure. The exceptional antibacterial adhesion property and outstanding biocompatibility demonstrated by this material offer innovative perspectives for synthesizing an enamel remineralization layer.

In addition to addressing the requirements for antimicrobial activity and remineralization, Xu et al. have also taken into consideration the aspect of anti-inflammation, as long-term plaque accumulation and demineralization can potentially trigger dentin hypersensitivity and gingivitis [192]. Turkish gall extract (TGE) has gained attention due to its multifaceted properties including anti-inflammatory, antimicrobial, and astringent effects [193]. Furthermore, it has been observed that TGE is capable of forming a thin film and generating precipitates on the tooth surface when exposed to artificial saliva. Subsequent in vivo and in vitro experiments have provided evidence for the potential of TGE in facilitating in situ self-healing [192]. The polyphenols in TGE possess multiple hydroxyl groups, demonstrating a strong affinity for the tooth surface. Upon interaction with TGE, a coating is formed on the tooth surface, resulting in the formation of a robust TGE-HAP complex that exhibits significant affinity towards Ca²⁺. Moreover, it exerts concentration-dependent inhibition on the growth of S. mutans. The anti-inflammatory potential of TGE can also be attributed to its polyphenolic constituents that effectively mitigate inflammation by inhibiting the formation of hydrogen peroxide and superoxide anion O₂. Following 7 days of treatment with TGE rinsing, all gingivitis indicators in rats showed significant improvement, leading to an effective reduction in inflammatory factors IL-6 and IL-1β content and achieving a comprehensive anti-caries effect.

Using ACP as a mineral source

Direct delivery of ACP technology presents an effective and straightforward approach in restorative dentistry. However, due to its thermodynamic instability, ACP tends to convert into an apatite phase prior to use, necessitating combination with other constituents to maintain its efficacy and enhance its performance. The combination of EGCG and ACP by Dai et al. involved the utilization of EGCG’s phenolic hydroxyl group to stabilize Ca²⁺ and inhibit their binding to phosphate ions, thereby effectively stabilizing ACP [194]. However, this chelation process requires a partial depletion of phenolic hydroxyl groups in EGCG, which may slightly compromise its antimicrobial activity. Furthermore, in a cariogenic acidic environment, the release of ACP from EGCG-ACP exceeded 80 % within 12 h, thereby raising concerns regarding its long-term remineralization efficacy. To enhance the availability of carboxyl groups and optimize antimicrobial properties, He et al. developed poly (carboxybetaine acrylamide) (PCBAA), a polyzwitterion utilized for the stabilization of ACP (Figure 12A) [195]. The inspiration for PCBAA is derived from carboxybetaine polymers, which are characterized by a high abundance of quaternary amino groups [196]. These cationic groups exhibit potent bactericidal properties in acidic environments and possess exceptional resistance to nonspecific protein adsorption, bacterial adherence, and biofilm formation due to electrostatically induced hydration. Upon the introduction of calcium ions into the PCBAA solution, they engage in electrostatic interactions with carboxyl groups. Following this, PO₄ ³⁻ adsorb onto and establish complexes with both quaternary ammonium groups and Ca²⁺. This action reduces the saturation of mineral ions in solution, thereby enhancing the potential barrier for ACP crystallization and facilitating enamel remineralization. Additionally, the PCBAA/ACP composite possesses a smaller particle size, allowing for deeper penetration into dentin tubules and effective interfibrillar remineralization. Consequently, PCBAA/ACP exhibits significant potential in preventing and treating both dentin caries and enamel caries.

Figure 12:

IDC materials with additional minerals. (A) Schematic illustration of the PCBAA/ACP nanocomposite with dual antibiofilm and remineralization functions. (a) The zwitterionic PCBAA polymer has both positive quaternary ammonium groups (−R₃N⁺) and negative carboxyl groups (−COO⁻). When Ca²⁺ is added into the PCBAA solution, it preferentially binds to the negatively charged carboxyl groups on the PCBAA side chains via electrostatic attraction. After the addition of PO₄ ³⁻ ions, some of the anions adsorb to the positively charged quaternary ammonium groups, and the others are attracted by Ca²⁺. (b) Evaluation of the effect of the PCBAA/ACP nanocomposite on inhibiting cariogenic bacterial adhesion and biofilm formation on the enamel surface and promoting enamel remineralization and dentin tubules occlusion (image source: He et al. [195] with permission). (B) Scheme for fluorinated urchin-like hydroxyapatite-filled light-curing dental resin composites to protect dentin and deep pulp by inhibiting bacterial adhesion and growth in dentin caries restorations (image source: Zhang et al. [197] with permission).

Using CaP as a mineral source

It is widely acknowledged that the primary goal of dentin remineralization lies in achieving intrafibrillar mineralization, as this specific type of mineralization governs the mechanical properties of dentin [197]. Yuan et al. developed an antimicrobial nanocomplex utilizing quaternary ammonium salt as the antimicrobial agent, anionic polymer γ-polyglutamic acid as the template for mineralization, and CaP as a source of minerals [198]. After a three-day treatment with the nanocomposites, microcrystals were observed within the fibrils, exhibiting a structure similar to that of native fibrils. Moreover, dentin caries was effectively interrupted while the mechanical properties were restored excellently. Due to the high proportion of organic matter and limited residual crystal seeds, dentin remineralization often poses a significant challenge. Extensive research has been conducted on caries interrupting strategies that not only facilitate dentin remineralization but also possess antimicrobial properties [199]–203], effectively arresting cavity progression while managing dentin hypersensitivity and preventing chronic pulpitis (Figure 12B).

In recent years, enhanced management strategies implemented at specific sites and in a controlled manner have emerged as a prominent topic of discussion in the field of oral health management. Ingeniously designed responsive materials can effectively identify the caries microenvironment, enabling targeted intervention to prevent caries development. This innovative approach demonstrates significant potential for enhancing the safety and efficacy of antimicrobial anti-caries materials. Li et al. developed a magnesium organic framework with a unique structure that exhibits collapse in an acidic environment associated with caries, enabling the controlled release of GA and Mg²⁺ upon loading [204]. Moreover, polydopamine was incorporated and coated onto the surface to achieve pH and photothermal dual responsiveness, while CaP was added as a mineral source to enhance remineralization ability. This responsive material effectively responds to low pH induced by cariogenic bacteria, eradicating bacterial biofilms at multiple levels including elemental composition, genetic factors, and adhesion through photothermal effects. Additionally, the regulated release of mineral ions from CaP promotes orderly formation of new HAP crystals, preventing demineralization progression. However, a common challenge faced by acid-responsive materials is their potential for incorrect response when exposed to acidic foods [205], thus necessitating more appropriate responsive designs.

No additional mineral sources

While the ability of PAMAM to control HAP crystallization is well established, its remineralization capacity should not be the sole focus. Jia et al. characterized the surface morphology of their remineralized layers and observed that compared to the NaF group, PAMAM-COOH and PAMAM-NH₂ induced smoother surfaces with tighter HAP stacking, effectively reducing adhesion of S. mutans [206]. To enhance antimicrobial efficacy, Tao et al. loaded honokiol into the hydrophobic interval cavity of PAMAM-COOH to achieve prolonged antimicrobial activity in honokiol-loaded PAMAM (PAMH) [207]. Honokiol, a naturally derived antibacterial agent from plants, demonstrates excellent biological safety [208]. It effectively enhances bacterial membrane permeability and inhibits the activity of glucose transferases, thereby exerting the ability to against S. mutans. Additionally, it has been observed that the carboxyl group and amino group of PAMAM exhibit distinct dissociation states under different pH conditions [207]. As a result, honokiol is completely released from the cavity of PAMAM within 96 h at pH 7.0, while it requires 300 h for complete release at pH 5.5. This sustained release effect in acidic conditions promotes the long-term antibacterial efficacy of honokiol. However, the cytotoxicity associated with PAMAM has always been a significant concern, particularly for cationic PAMAM dendrimers such as PAMAM-NH₂ [209], which exhibit relatively higher toxicity and therefore necessitate meticulous consideration during their application. Serum albumin, the predominant protein in plasma, plays a pivotal role in maintaining blood osmotic pressure and facilitating the transportation of small molecules [210]. Bovine serum albumin (BSA), which shares similarities with human serum albumin, is extensively employed in drug delivery technology due to its non-cytotoxic nature [211]. Furthermore, BSA demonstrates the capability to undergo a phase transition resulting in the formation of amyloid-like structures [212]. These dense protein membranes possess remarkable stabilization and substrate adhesion properties attributed to their abundance of functional groups. The cationic surface-active antimicrobial compound octenidine (OCT) has been selected as an antimicrobial ingredient due to its absence of systemic or local toxicity [213]. BSA forms a complex with OCT through hydrogen bonding, electrostatic interactions, and hydrophobicity that undergoes a phase transition to generate a nanofilm (Figure 13) [9]. In vivo experiments have demonstrated that this nanofilm exhibits antimicrobial and mineralization sealing effects, effectively preventing both primary and secondary caries. These findings support the proposal of IDC.

Figure 13:

PTB-OCT coating for managing dental caries. (A) Schematic representation of PTB-OCT coating formation and its ability to prevent primary and secondary caries. (B) Evaluation of the effect of the PTB-OCT coating on enamel and dentin remineralization in vitro (image source: Lu et al. [9] with permission).

Collectively, these remarkable findings emphasize the practicality of caries management strategies under the IDC concept. In conclusion, the strategy of combining remineralization technology with antimicrobial technology, complemented by good penetration properties, offers a compelling opportunity to develop the next generation of functional biomaterials for caries management.