Potential Beneficial Effects of Hydroxyapatite Nanoparticles on Caries Lesions In Vitro—A Review of the Literature

Dental caries is one of the most common human diseases which can occur in both primary and permanent dentitions throughout the life of an individual. Hydroxyapatite is the major inorganic component of human teeth, consequently, nanosized hydroxyapatite (nHAP) has recently attracted researchers’ attention due to its unique properties and potential for caries management. This article provides a contemporary review of the potential beneficial effects of nHAP on caries lesions demonstrated in in vitro studies. Data showed that nHAP has potential to promote mineralization in initial caries, by being incorporated into the porous tooth structure, which resulted from the caries process, and subsequently increased mineral content and hardness. Notably, it is the particle size of nHAP which plays an important role in the mineralization process. Antimicrobial effects of nHAP can also be achieved by metal substitution in nHAP. Dual action property (mineralizing and antimicrobial) and enhanced chemical stability and bioactivity of nHAP can potentially be obtained using metal-substituted fluorhydroxyapatite nanoparticles. This provides a promising synergistic strategy which should be explored in further clinical research to enable the development of dental therapeutics for use in the treatment and management of caries.


Introduction
Dental caries is one of the most prevalent and ubiquitous non-communicable diseases worldwide. It is characterised by the progression of demineralization of the dental hard tissue, resulting in an imbalance in the equilibrium between the tooth mineral and biofilm [1]. Previously, the management of caries was driven by the understanding that it was a purely infectious disease and could only be managed invasively by removing all demineralized tissue. However, contemporary treatment strategies to manage caries have changed from the traditional surgical approach to a medical model, which now often includes dietary analysis and advice, oral hygiene instruction, placement of fissure sealants, and mineralization control by topical application of fluoride or other mineralising agents [2]. Among the available mineralization control strategies, fluoride-based treatments have the highest level of supporting evidence. Their widespread use is generally considered the main reason for reducing the incidence of dental caries in most populations. The released fluoride prevents enamel demineralization and reduces caries susceptibility by its incorporation into enamel hydroxyapatite, therefore, stabilizing the crystal structure by forming fluorapatite and lowering the solubility product constant (Ksp) [3]. Consequently, this increases resistance to acid demineralization. However, currently available fluoride therapies have limited efficacy in some individuals, and at the population level, the effect of fluoride in reducing dental caries prevalence is reaching a plateau [4]. More recently, investigators have been developing new mineralization approaches to close this gap in efficacy. Calcium and phosphate-based treatment systems are designed to ensure a constant supply of these ions and aim to directly increase mineral concentration in the environment around the caries lesions [5]. These systems include the use of amorphous calcium phosphate (ACP), functionalized tricalcium phosphate (fTCP), bioactive glass containing calcium sodium phosphosilicate and hydroxyapatite nanoparticles (nHAP) [6].
The enamel rod (prism) is the basic unit of human tooth enamel. Enamel rods are tightly packed hydroxyapatite (HAP) crystals structures which are hexagonal in shape, with sizes up to 70 nm in width and 30 nm in thickness. They provide rigidity to the enamel rod and provide significant strengthen to the enamel [7]. HAP is a mineral form of calcium apatite with the molecular formula Ca 10 (PO 4 ) 6 (OH) 2 and a calcium-to-phosphorus molar ratio of 1:67. It has a crystal lattice and its chemical composition is most similar to the apatite crystals of the human enamel. HAP also exhibits good biocompatibility and bioactivity [8]. Nanoparticles have a large surface area to volume ratio, which results in them exhibiting different biomedical activities compared with materials of increased size, furthermore, they can possess unique physical properties that make them desirable for use in biology and material science [9]. The synthesis of hydroxyapatite as nanosized particles has the potential to enable an improved physical and chemical characteristic by the enlargement of the reactive surface area. With respect to morphology and structure, the crystal lattice of nHAP is predicted to resemble the tooth enamel better and consequently would have beneficial effects for hard tissues repair.
Although there are a few previous reviews reporting nHAP in dentistry, they are either not contemporary [10] nor discuss its use within a broad scope of dental application [11,12]. One published systematic review reports on the efficacy of nHAP in caries prevention, however, no strong conclusions were made due to only a very limited number of in vivo and in situ studies being evaluated [13]. A significant number of in vitro studies have now investigated the effect of nHAP on dental hard tissues and cariogenic bacteria and biofilms have been conducted. Consequently, this review aims to provide recent novel information and reports on the action of nHAP in caries management, focussing on its effects on the dental hard tissues and cariogenic bacteria.

Literature Search Strategy
A literature search was undertaken in PubMed to identify English language publications from 2010 to 2022. The keywords included: (nano-HAP OR HAP-NPs OR nano-hydroxyapatite OR nanohydroxyapatite OR hydroxyapatite OR Hydroxyapatite nanoparticles) AND (caries OR carious OR cariogenic OR tooth decay).
(2) Studies related to antimicrobial or biocompatibility effect of nHAP on cariogenic species.
(3) Studies related to the mineralization effect of nHAP on tooth hard tissue (enamel, dentine, cementum) (this includes remineralization and modulation of demineralization on hard tissue).

Exclusion Criteria
(1) Studies not reported using the English language.
(2) Studies on HAP that were not in the nano-scale range.
(3) Studies not related to dental caries and abstracts without the associated full papers.
(4) Case reports, conference papers, book chapters, patents, letters to the editor, systematic reviews, meta-analyses, and literature review papers.

Results
A total of 1052 potentially eligible articles published up to February 2022 were identified using the search terms described (Figure 1). After screening titles and abstracts, 125 articles remained for further analysis. Full-text reading was undertaken and 38 articles were finalized for inclusion in this review. Among these articles, twenty-nine studies investigated the effects of nHAP on enamel mineralization (Table 1), five studies were reported on dentine remineralization and one study on cementum (Table 2), and three studies investigated the antimicrobial effect of nHAP on cariogenic bacteria (Table 3). Twenty-two studies used toothpaste containing nHAP, nine studies used an aqueous slurry of nHAP; nHAP was also incorporated in other formats such as tooth cream, tooth gel, and resin infiltration in three studies.
A total of 1052 potentially eligible articles published up to February 2022 were i tified using the search terms described (Figure 1). After screening titles and abstracts articles remained for further analysis. Full-text reading was undertaken and 38 art were finalized for inclusion in this review. Among these articles, twenty-nine studie vestigated the effects of nHAP on enamel mineralization (Table 1), five studies wer ported on dentine remineralization and one study on cementum (Table 2), and three s ies investigated the antimicrobial effect of nHAP on cariogenic bacteria (Table 3). Twe two studies used toothpaste containing nHAP, nine studies used an aqueous slurr nHAP; nHAP was also incorporated in other formats such as tooth cream, tooth gel resin infiltration in three studies.  Table 1 presents the main findings of the studies which investigated the role of nH   Table 1 presents the main findings of the studies which investigated the role of nHAP on enamel mineralization. Enamel specimens were prepared using either bovine or human teeth; the majority (28 out of 29) of studies generated early artificial caries lesions by chemical means on enamel specimens prior to treatment to mimic incipient enamel caries. These caries lesions were then treated with a range of treatments including nHAP formulations. The format of the nHAP containing products included toothpastes (18 studies) [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], slurries prepared with nHAP powder (eight studies) [26,[32][33][34][35][36][37][38], resin infiltration (one study) [39], cream (one study) [40], and gel (one study) [41]. Nineteen studies employed microhardness to evaluate the effect of nHAP on remineralization. Eighteen of these studies found that products with a nHAP component demonstrated increased enamel surface microhardness when compared with control groups. Five studies measured the mineral recovery from the enamel lesion after nHAP treatment, with all studies reporting that the nHAP treatment groups exhibited higher mineral recovery when compared with negative controls. Other studies also demonstrated the repair of surface defects and a smoother uniform distribution of Ca/P particles in the surface area exposed to nHAP when compared with other treatment groups [14][15][16]. A few studies reported nHAP used in combination with fluoride revealed a significant reduction in lesion depth in contrast to other agents [16,17]. Table 2 presents the key findings from the six studies which investigated the role of nHAP on dentine or cementum mineralization, with inconsistent findings being reported. The format of the nHAP products used were toothpaste (four studies) [19,20,42,43], slurry prepared using nHAP powder (one study) [44], and gel (one study) [41]. One study identified a significant increase in surface microhardness following nHAP treatment of cementum when compared with fluoride treatment [41]. While another study demonstrated formation of a partially filled remineralized layer after nHAP treatment in contrast to the group treated with 30% pre-reacted glass ionomer (S-PRG) filler, which resulted in formation of a fully remineralized layer on dentine [42]. Similarly, a further study showed reduction in lesion depth when nHAP was used in combination with fluoride [18]. Notably, one study claimed that nHAP was unable to reduce microhardness loss [19]. Table 3 presents the key findings of studies which investigated the effect of nHAP on cariogenic bacteria and biofilms. One study showed that pure nHAP tends to enhance Streptococcus mutans biofilm formation; the mechanisms behind could be that nHAP was capable to increase glucosyltransferase transcription which resulted in an increase in production of insoluble glucans [45]. However, there were different results when calcium ions were substituted partially with metal ions. A study investigated the effect of loading zinc ions with nHAP in combination with alendronate grafted poly-acrylic acid, concluding that zinc ions significantly enhanced the materials' antibacterial effect against S. mutans in contrast to samples where nHAP was used alone [32]. In another study, toothpaste with zinc substituted nHAP was also found to be less antimicrobial against S. mutans when compared to the one with strontium (Sr), magnesium (Mg), and fluoride (F) substituted nHAP [46]. • 7% nHAP toothpaste demonstrated less mineral loss on enamel lesions in contrast to 0.14% Amineflouride.

•
The surface layer of the lesion showed remineralization. • A significant higher surface mineral recovery content was observed in the specimens when treated with nHAP in contrast to sodium fluoride. • Crystal nucleation in the demineralized pores along with the formation of a uniform thick layer was observed when the specimens were exposed to nHAP. • Particle size: not reported. • Format: nHAPF: (1% nHAP and 1450 ppm NaF) was obtained in toothpaste form and diluted with water in the ratio 1:3 to form a dentifrice slurry.
• nHAPF group revealed significant increase in surface microhardness as compared to the fluoride group. • Surface roughness significant decrease in the nHAPF group as compared with the Fluoride group.  • Particle size/shape: Hexagonal in shape and possessed rod-shaped morphology were prepared using a hydrothermal method. • Format: nHAP was incorporated in the resin infiltrates.
• Micro-hardness tests revealed higher enamel resistance of nHAP-2h and nHAP-5h against demineralization compared to control nHAP-free and Hap-0 h infiltration. • Enamel from two nHAP + resin infiltrate groups demonstrated higher crystallinity than control groups.    • The lesion depth decreased significantly by 10.56% in nHAP group, 6.73% in 5% Calcium sodium phosphosilicate group, and 9.58% in Amine fluoride group.   • nHAP treatment was unable to reduce the loss of dentine surface hardness regardless of fluoride addition.
Besinis et al., 2014 [44] • Sound dentine (control 1) • Substrate: Dentine specimens were prepared from human premolars, and completely demineralized to dentine collagen matrix. • nHAP treatment increased Ca and P levels of the local scaffolds of dentine matrix. • nHAP treatment was unable to recover the minerals from the dentine collagen matrix, however, it reduced the porosity of the dentine collagen matrix. • nHAP when used in combination with 5000 µgF/g, showed a smaller dentine lesion as compared to the placebo group.

Discussion
While a recent systematic review, which was based on in vivo and in situ studies of nHAP in caries prevention, has been undertaken with a proper risk of bias evaluation [10], only four studies were included in the analysis. The major reasons for exclusion of studies were lack of a control group, a short study duration (less than 1 month), and not reporting an Institutional Review Board (IRB) approval. This very low number of included studies resulted in inconclusive evidence for the efficacy of nHAP. This highlights the need for more well controlled clinical trials to be undertaken in the future. Ideally, a risk of bias of the included studies should also be provided in the current review. In particular, studies that provide no details of the nHAP particle size and concentrations compromise the quality of the review.
A wide range of nHAP structures, such as nano-rods, microspheres, or hierarchically nano-structures, are reported in the articles reviewed here, and these potentially influence the effects exerted by nHAP. Indeed, it has been previously demonstrated that the morphology of the nanoparticles will affect the speed of Ca 2+ release, as Ca 2+ in microspheres appears to release more rapidly than hierarchically nano-structures [48]. The synthesized nHAP in the reviewed studies was prepared by using several different techniques, including a hydrothermal method, a sol-gel method, and a wet-chemistry method, which are believed to be able to provide high phase purity and grain sizes of between 20-50 nm [49]. Aqueous nHAP slurry in distilled water provides a relatively simple approach to assess the effect of the nanoparticles on the caries lesion. However, it may not be highly clinically relevant. Furthermore, nHAP was incorporated into several different formats of dental products, including toothpastes, creams, and restorative materials. Studies used either commercialized dental products, which contained nHAP, or incorporated nHAP into existing toothpastes by a certain weight percentage. One study attempted to incorporate nHAP into the resin infiltration, and they found that this test material caused higher enamel resistance against demineralization compared with the control nHAP-free infiltration [39].
The most common substrate used in the studies was enamel with chemically-induced artificial early caries to mimic the clinical situation of incipient caries (non-cavitied) presented on the coronal side of the tooth. The human enamel includes morphologically aligned, parallel, up to 70 nm in width and 30 nm in thickness, micron-long carbonated hydroxyapatite nanocrystals, bundled either into 5-µm-wide rods (prisms) or a space-filling interrod arrangement [50]. When caries (demineralization) are initiated, the enamel prism junctions are enlarged and destruction of the interprismatic structure is observed [51], while the size of nHAP enhances its penetration into the interprismatic enamel space [52]. Moreover, a few matrix proteins remain embedded in the enamel's organic component and these proteins operate as a scaffold for conduction of nHAPs so they can be easily deposited within the nano-gaps [53]. These proteins enable the capture of the minerals and subsequently facilitate mineral apatite formation [52]. In comparison, fluoride has been shown to predominately integrate into the surface of the initial caries lesion, while being less effective in deep sub-surface lesions and this effect has been found alongside both high fluoride and low fluoride treatment [54]. This lamination effect results in a limitation in the dose-dependent effectiveness of fluoride products, therefore, creating a negative effect for complete remineralization into the deeper zones and subsequently limiting application [55]. An in situ study, which compared the effectiveness of two toothpastes containing nHAP or fluoride in promoting remineralization of initial enamel caries, found that while nHAP achieved comparable efficacy with 500 ppm fluoride, it enabled a more homogenous lesion of remineralization when compared with the fluoride induced lesion surface lamination following transverse microradiography analysis [56]. Therefore, nHAP should be developed and applied to enhance the effect of existing fluoride therapies rather than to replace them, but it still provides an alternative option to those who are reluctant to use fluoride.
Root dentine rather than cementum is the most frequently used substrate when investigating root caries. This is due to the cementum being relatively thin (20 to 50 microns near the cemento-enamel junction) [57]. Therefore, the dentine beneath the cementum is considered the major component in root caries progression. The results of the studies on dentine are somewhat inconsistent, which might be due to the different experimental conditions and different particle sizes used. Comar et al. [19] found that an nHAP paste was unable to reduce the loss of dentine surface hardness, which may be due to the relatively large size of the nHAP particle used in their study, which was 100 nm and already reached the maximum size due to the definition of a nanomaterial. It is plausible that a mechanical imbrication of the paste into the inter-tubular dentine space due to the size of the particles occurred. Besinis et al. [44] reduced fully demineralized dentine blocks to a collagen matrix prior to the nHAP treatment. They found that the nHAP with a particles size of 20 nm was able to locally infiltrate into the scaffolds with increased levels of calcium and phosphate deposition. Both Tschoppe et al. [20] and Leal et al. [43] demonstrated an increased remineralizing effect on dentine when nHAP was used alone or with fluoride; the particles sizes of their studies were less than 20 nm. Juntavee et al. [41] showed that nHAP containing gels had a higher capability for the remineralization of cementum when compared with fluoride varnish, which was possibly associated with the nanoparticle size as it was capable of constructive interdigitation with the cementum structure. Future studies should focus on the mechanisms of nHAP-induced remineralization at a more in-depth level to determine the role of nHAP in the calcium phosphate formation process and to determine how the size of the nHAP affects this process.
The antimicrobial effect of nHAP was negligeable given its high biocompatibility and low toxicity. In addition, it was also found that the formation of S. mutan biofilm was enhanced when S. mutans was co-cultured with the presence of 5% nHAP suspension [45]. Caries result from an ecological imbalance in the physiological equilibrium between oral microbial biofilms and tooth minerals. Maintaining tooth minerals and controlling oral microbial biofilms are essential for the control of caries [58]. Aimed at a dual action effect nHAP in both mineralization and antimicrobial effect, studies attempted to develop a hybrid nHAP by substituting calcium ions with metallic ions. Silver is a well-known antimicrobial agent due to its broad spectrum, low toxicity, and lack of cross-spectrum bacterial resistance [59]. Silver can be incorporated into the crystal structure of hydroxyapatite to produce silver-containing hydroxyapatite [60] to give a formula of Ca 10−x Ag x (PO4) 6 (OH) 2 with 0.0 ≤ x ≤ 0.5. Notably, a relatively small number of calcium ions are substituted with silver ions [61,62]. This silver-containing hydroxyapatite has been shown to reduce bacterial adhesion and to have minimal tissue cytotoxicity [60], however, aesthetic effects may be compromised due to the fact that silver has a black coloration when it is oxidized, which readily occurs in air [63]. Zinc is another commonly used metal which can substitute for the calcium ion in nHAP. In the orthopaedic field, it is regarded as an essential trace element that promotes bone formation and exhibits antimicrobial effects [3]. Several studies involving Zn-substituted apatite containing a low amount of zinc (<1 wt%) have been reported, and demonstrate the material to exhibit good bioactivity with antibacterial properties [64][65][66]. Indeed, Chung et al. [67] and Stanic et al. [66] observed a reduction of bacterial strains Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Candida albicans (C. albicans), and S. mutans cultured on Zn-substituted HAP. Furthermore, strontium is a divalent cation that is located in the same column of the periodic table as calcium. Consequently, strontium has chemical properties that are similar to those of calcium, and it can partly substitute for calcium and be incorporated into the crystal lattice structure of hydroxyapatite. Sr-substituted HAP also demonstrates good bioactivity and can directly bond to bony tissue under non-weight-bearing conditions [68]. A synergistic effect of strontium and fluoride on hydroxyapatite formation has been demonstrated in previous studies, and strontium was able to aid remineralization of a carious lesion in the presence of fluoride [69,70]. Sr-substituted nHAP has been shown to display enhanced biocompatibility compared with pure nHAP [26]. However, reports on the application of nanosized metal-substituted HAP in caries management are limited. One previous study found that the inhibition ratio of synthesized Zn-substituted nHAP against S. mutans was significantly higher compared with the pure nHAP group [32]. A further study used a toothpaste con-taining Mg-Sr-F substituted hydroxyapatite and demonstrated that it exerted a decreased rate of early colonisation of S. mutans in comparison with those containing Zn-substituted nHAP after 12 h. This may be due to the synergistic effect of fluoride and strontium in caries management [46,69,70]. Notably, instead of calcium being substituted by metal cations, fluorohydroxyapatite (Ca 5 (PO 4 ) 3 (OH) 1−x F x ) could be formed by exchanging fluoride ions for the hydroxyl group in the HAP; the isotropic distribution of the charge on fluoride anions allows for a better fit in the lattice structure compared with the larger asymmetric hydroxyl ions. This produces a more ordered apatite structure, which is characterized by increased thermal and chemical stability compared with HAP [63,71]. Future studies on metal-substituted fluorohydroxyapatite nanoparticles could identify this as a promising synergistic strategy for caries management.

Conclusions
This review of the current literature concludes that nHAP can promote mineralization in early caries lesions, potentially by being incorporated into the porous tooth structure caused by the caries process and thereby increasing its mineral content and hardness. The particle size of nHAP plays an important role in this remineralization process. The antimicrobial effect of nHAP can be potentially achieved by using metal substituted nHAP. All the above properties highlight nHAP as a promising bioactive material for potential use in the management of dental caries, which should be explored in further clinical research.