Modes of Action and Clinical Efficacy of Particulate Hydroxyapatite in Preventive Oral Health Care − State of the Art

3.1.1. Studies

(1) The first clinical trial using HAP in the field of caries prophylaxis was published in 1989 [34]. Two different schools with at least 200 fourth-graders were included in the study. The study duration was three years. One group used a (fluoride-free) toothpaste containing 5% HAP, and the other group used a placebo-toothpaste without any active ingredients. Every year, the dmft/DMFT was acquired. While on the level of the primary dentition only minor effects could be observed, the caries reduction on the level of newly-erupted teeth was statistically significant in the HAP-group compared to placebo (mean caries inhibition: about 45%) [34].

(2) These above results were confirmed by a randomized clinical trial (non-inferiority-trial) from Schlagenhauf et al. [79]. This study was performed at five German university-hospitals using high-caries risk-patients undergoing orthodontic treatment. Since it is well known that these patients develop caries within four weeks [80, 81], the study was conducted over a period of six months and the 147 patients were randomly distributed using either a (fluoride-free) toothpaste containing 10% microcrystalline HAP, and a toothpaste containing amine fluoride/stannous(II)fluoride (1400 ppm fluoride). Caries incidence was measured using ICDAS-scores 1 and 2. Out of the 133 participants overall who finished the study per protocol, the HAP-group was not inferior to the fluoride-group [79].

(3) Lelli et al. used extracted teeth from an in vivo study (so called ex-in vivo study) to investigate whether a calcium-layer can be observed after using a HAP-toothpaste. Due to orthodontic or prosthetic reasons, teeth had to be extracted. Participants had either brushed their teeth with a fluoride-toothpaste or with a HAP-toothpaste for at least 8 weeks. The extracted teeth were analyzed using scanning electron microscopy (SEM) to visualize a possibly formed superficial layer and energy dispersive X-ray spectroscopy (EDX) to analyze its elemental composition. While in the fluoride-group no additional layer could be observed, the use of HAP-toothpaste leads to the formation of a calcium phosphate-containing layer on top of the natural enamel [48].

(4) Najibfard et al. conducted a double-blind in situ study with thirty participants using enamel slabs with and without artificial carious lesions [35]. Using a crossover-design (washout-phase in between the phases was seven days), participants brushed for at least 28 days with a fluoride-toothpaste (1100 ppm fluoride) or a HAP-toothpaste (5% HAP and 10% HAP, respectively). The slabs were analyzed using microradiography to quantify mineral loss and lesion depth. The results showed that HAP-toothpaste prevented the enamel from demineralization. Additionally, remineralization of enamel was not different between the toothpastes containing fluoride (1100 ppm fluoride), and HAP-toothpaste (5% and 10% HAP) [35].

(5) A clinical trial compared three different mouth rinses containing either HAP, CHX or fluoride with respect to plaque accumulation, gingival indices, and remineralization properties [41]. 81 children used the respective mouth rinse twice a day under parental supervision for two weeks. The children were examined after 1, 2, 4, and 6 weeks. With respect to caries (remineralization), HAP performed equally compared to fluoride and was also effective in reducing dental plaque and improving gingival health [41].

(6), (7) Two in situ studies investigated the bacterial adhesion to enamel surfaces after rinsing with an (6) aqueous dispersion containing HAP-particles [23] and (7) a mouth rinse containing HAP-particles [47]. Participants wore enamel slabs and rinsed with the respective dispersions. As positive control, CHX-containing mouth rinse was used, and negative control was also studied. Enamel slabs were carried in the oral cavity for six to twelve hours and then analyzed using bacterial staining and microscopy-techniques. Additionally, in vitro experiments regarding antimicrobial effects were also carried out. HAP does not kill the bacteria but leads to a decrease of bacterial attachment on enamel surfaces. The reduction of initial bacterial colonization to enamel surfaces in situ was comparable with the reduction of the antimicrobial agent chlorhexidine [23, 47].

Several in vitro studies have investigated the remineralization-potential of particulate HAP [71, 72, 82-84]. Most of the evaluated studies used demineralized enamel or dentin, and applied HAP-particles on these slabs [71, 83, 84]. Two studies, however, used pH-cycling models [72, 82]. Out of these, Esteves-Oliveira et al. did not detect a caries-preventive effect of HAP in vitro [82], while de Carvalho et al. described caries-inhibiting effects of HAP [72].

3.1.2. Mechanisms

The effects described in the results of the analyzed studies imply a number of different mechanisms where HAP plays a major role. As caries is a biofilm-associated disease, this biofilm should consequentially be reduced. HAP has already been shown to reduce the bacterial biofilm on the teeth [23]. Bacteria need a rough surface for attachment [85].

HAP in oral care products might help smoothen the surface through two different effects: Firstly, particulate HAP attaching to the enamel surface may preferentially deposit in small depressions such as scratches and superficial defects because it can more firmly attach and may be harder to remove by either abrasive food particles or brushing than on the smooth enamel surface areas, thus leveling the surface on the microscopic scale. Secondly, the remineralization effect and the resulting bio-chemically induced formation of a protective layer may also be more pronounced in surface depressions of the outer enamel. Both effects lead to a smoother surface that consequently decelerates the attachment of potentially harmful bacteria.

Furthermore, bacteria can erratically attach to individual HAP particles that are either loosely attached to the enamel surface or float in the oral cavity, thus inducing bacteria co-aggregation. In consequence, bacterial load will be reduced by spitting out the residual HAP after thorough tooth brushing. Despite all routine daily oral hygiene, bacteria can attach on the teeth after a certain time. If a protective layer of HAP particles is present and regularly sustained, this biofilm is formed on its surface instead of the calcium phosphate of the original enamel. When acids are produced within this biofilm, this protective layer might be dissolved, and calcium and phosphate ions will be released. This can have two main effects: On the one hand, the biofilm formation might be disrupted by influencing the energy metabolism of oral bacteria [86]. On the other hand, the excess of released calcium and phosphate ions become available for other, different effects. They might for instance help and promote remineralization of demineralized tooth surfaces and support the natural remineralization-mechanisms of saliva, i.e. shifting the equilibrium from de- to remineralization. If residual plaque is present (i.e. plaque that has not been removed by toothbrushing), HAP may also be incorporated into the biofilm as demonstrated for other calcium phosphates before [6, 87].

When acids are produced by bacteria, the dissociation products of HAP may act as buffering-solution. Particularly the phosphate ions are able to neutralize acids to a certain level, likewise the salivary phosphate-buffer. If present in excess, the released calcium and phosphate ions can also switch the solubility product balance back to remineralization.

In addition to the mechanisms described above, HAP can remineralize early caries-lesions directly which was shown under in situ conditions (Fig. 4) [35]. From a histological point of view, initial caries-lesions show larger distances between the rod-shaped HAP crystallites of the enamel than healthy portions. Small HAP particles from oral-care products might be able to penetrate through these gaps between enamel-rods and thus fill these initial lesions.

The HAP particles might bind to enamel/pellicle due to electrochemical forces, because of partial electronegativity: Zeta-potential of enamel and HAP-particles show a negative net surface potential of -15 to -30 mV, and basic proteins (i.e. from saliva/pellicle) a positive net surface potential of +20 to + 30 mV [88, 89]. HAP particles might also act as crystal nuclei by attracting calcium and phosphate from saliva [32]. Local concentration gradients can induce the formation of calcium phosphate through crystal growth on the enamel surface.

Since HAP is a calcium phosphate source itself, the described mechanisms do not only rely on ions provided with the salivary flow. As soon as bacteria are able to attach to a HAP-layer on tooth surfaces and acids are produced, calcium and phosphate ions will be released which may have a positive influence on the remineralization process (Fig. 5A-C).

HAP has a wide range of actions with respect to caries prevention. The main advantage of HAP compared to other oral care active ingredients is its high biocompatibility, i.e. no potentially toxically side effects like fluorosis. In consequence to this, there is no regulation in dosage [42, 43].

3.2.1. Studies

Several clinical trials report an improvement of gingival health after having used HAP-containing oral care products. (8) Harks et al. tested in a randomized clinical trial the differences in the plaque-formation-rate between a HAP-toothpaste and an antibacterial amine fluoride/stannous (II) fluoride-toothpaste. Sixty-seven patients with mild periodontitis completed the whole double-blind 12 weeks-trial. Besides plaque-formation-rate, several other clinical parameters were tested (i.e. bleeding on probing). The two groups used their respective toothpaste for 4 weeks without any therapy at home. After these 4 weeks supragingival cleaning was performed and the toothpastes were used for another 8 weeks. Plaque-formation rate did not change within that time. However, gingival health improved within both groups after 4 and 12 weeks. There were no differences between the antibacterial toothpaste and the HAP-toothpaste [25].

(9) Plaque samples from this study were also investigated using 16S rRNA amplicons for next generation sequencing. No differences of the plaque-composition could be observed between these two groups [90]. While mainly Fusobacterium and Prevotella species were observed in the interproximal and subgingival sites, buccal and lingual surfaces were dominated by Streptococcus and Veillonella species. However, the use of either antibacterial or anti-adhesive toothpastes did not change the composition noticeably indicating the stability of the ecological niche. Interestingly, gingival bleeding was reduced in both groups showing an effect on periodontal health of both treatments [25].

(10) Additionally, Cosola et al. tested a mouth rinse containing HAP (and zinc-PCA) with patients after surgical procedures [40]. Twenty-six patients were randomly allocated to either CHX-group or HAP-group. All patients received oral surgery. As postoperative treatment, patients rinsed with the mouth rinse. After their removal the suture threads from both groups, CHX and HAP (and zinc-PCA) were tested in vitro for colony-forming units. Despite different treatment, threads from both groups were found to have the same effect on bacterial growth. Since prevention of periodontitis relies on controlling bacterial growth, it is important to note that this study shows HAP (with addition of zinc-PCA) to reduce bacterial growth on suture threads. This goes in line with the in vivo (5) and in situ (6), (7) studies presented before [23, 25, 41].

3.2.2. Mechanisms

The results of these studies provide a number of useful hints with regard to HAP’s possible involvement in the prevention of periodontitis.

Dental plaque bacteria can interact with extrinsic HAP when present in the oral cavity. Thus, early colonizers, such as streptococci bind to free HAP derived from oral-care products instead of the tooth surface. The HAP can be either free particles in the oral cavity or particles bound to the tooth surfaces thereby creating a protective film of alternative substrate. As the early colonizers are needed for further biofilm-formation, the biofilm will be consequentially reduced either by directly spitting out of the free particles or by abrasion of the HAP layer through oral hygiene related cleaning activity.

It is known that gingivitis and periodontitis are promoted by a dysbiotic dental plaque if it is not removed. Hindering the settlement of early colonizers and reducing growth of the dental plaque can slow and potentially even prevent reaching a dysbiotic state.

The mode of action of HAP with respect to periodontitis is indirect. HAP is not able to directly interact with the immune system. Nevertheless, by reducing bacterial load and controlling the biofilm-formation, periodontal pathogens (i.e. Porphyromonas gingivalis and Tannerella forsythia) will not be able to lead to a shift in the taxonomic composition of the biofilm, as they are late colonizers and with that they are dependent of the early colonizers and bridge microorganisms (i.e. Fusobacterium nucleatum) [52, 62].

HAP also acts as an biomimetic cleaning agent (same hardness as the tooth) in toothpaste formulations, directly reducing dental plaque [32, 91].

The authors hypothesize that a HAP-layer applied to a tooth surface directly after mechanical dental plaque removal might lead to an aggravated biofilm formation [48]. Formulations with the addition of zinc salts or lactoferrin may boost the effect of HAP, especially against already existing or developing biofilms [30, 40, 92]. Zinc-substituted HAP, where Zn²⁺ions replace a part of the Ca²⁺ ions [5, 48], might also be dissolved in acidic environments, and the released zinc might act as an antimicrobial agent. This might promote the positive effects of HAP with respect to gingival health [25, 40, 47, 92].

In addition to that, HAP might also interact with proteins derived from microorganisms. Proteins are known to act as virulence factors, i.e. arginine deiminase arc A from P. gingivalis [59]. HAP particles present in the vicinity of the bacteria might bind these proteins [93]. Being immobilized, the virulence factors will not be able to interact with the human immune system.

Finally, HAP does not show unwanted side effects like discoloration of teeth or irritation of taste and can be used on a daily basis, in contrast to substances like chlorhexidine [23, 30, 47].

Furthermore, HAP does not kill bacteria (in contrast to e.g. stannous ions and chlorhexidine) but controls them by its physical presence or by passively withdrawing potentially harmful metabolic products from the oral cavity environment. Modern approaches focus on controlling the oral bacteria rather than killing them [94], since it is known that the use of antibacterial agents might lead to resistance or tolerance of bacteria against these agents. Biomimetic concepts have been shown to be promising alternatives or supplements in promoting periodontal health.

3.3.1. Studies

Clinical studies in the field of erosion are rare due to a feasible study population and an acceptable study duration for the participants and ethical issues [95]. Therefore, in situ and in vitro studies using a sophisticated study design are much more important. (3) An ex-in vivo study by Lelli et al. has shown the formation of a (protective) calcium phosphate layer on top of the teeth in the HAP-group [48]. While some in vitro studies were not able to show a positive effect of HAP-toothpastes on erosion [96, 97], other studies were able to show protective effects of HAP-toothpastes [24, 71, 73, 74, 98, 99].

(11) Fabritius-Vilpoux et al. tested in vitro, if HAP-particles from an aqueous dispersion can attach to (mechanically and chemically eroded) enamel-surfaces [24]. Bovine enamel slabs were pre-treated with phosphoric acid. Afterwards, these slabs were dipped into agitated aqueous dispersions containing different HAP-concentrations. HAP particle attachment and surface-coverage was determined using SEM. Even a dispersion containing only 1% of HAP resulted in an area coverage of 10% of the eroded enamel surface. A dispersion with 10% HAP covered more than 30% of the enamel after one-time application [24].

(12) Another in vitro study used also enamel slabs and tested Vickers-hardness after erosive challenges and application of toothpastes [74]. Enamel slabs were erosively challenged four consecutive times (0, 8, 24, and 32 hrs.) for two minutes. After each challenge, toothpaste was applied on the surface. Vickers-hardness measurements revealed that acidic attacks significantly reduce enamel surface hardness. However, HAP-containing toothpastes caused significant re-hardening of the surface, indicating the occurrence of remineralization [74].

(13) The same group also used human enamel slabs in another study [99]. Here, the slabs were pre-treated with the respective toothpastes. Several fluoride-containing and calcium-containing toothpastes were tested besides HAP-toothpaste. These pre-treated slabs were then dipped into an acidic soft-drink for a maximum of 32 min. As measurement for erosive challenge, loss of weight of the slabs was determined. The HAP-toothpaste showed significantly less loss of mineral compared to the other products. This indicates a protective effect of HAP against erosive challenges [99].

(14) Colombo et al. used a visual rating system imaged by SEM to evaluate the enamel surface after application of a HAP-toothpaste [73]. Human incisor specimens were prepared and underwent an erosive challenge. In four different groups (positive control with no erosive treatment, negative control without toothpaste-application, fluoride toothpaste, HAP-toothpaste), different toothpastes were applied for 3 min. and rinsed off with distilled water at 0, 8, 24, and 36 hrs time intervals. In between the applications, specimens were stored in artificial saliva. Using a systematic assessment-method by SEM, enamel damage was recorded by four observers. The HAP-toothpaste was the only tested toothpaste where mineral deposition was observed after the erosive challenges. The grade of damage was consistently lowest in the HAP-group [73].

3.3.2. Mechanisms

From to above studies, one can deduce the following mechanisms: HAP forms, after application, a protective layer on the tooth surface. The protective layer might act as expendable shield that protects the tooth material from acidic attacks. Consequently, the protective HAP layer will be dissolved, releasing calcium- and phosphate ions that can also act as buffer system:

Ca₅(PO₄)₃(OH) + 4 H⁺ → 5 Ca²⁺ + 3 HPO₄^2- + H₂O

Furthermore, it is known that the content of calcium within an erosive surrounding might lead to a shift of the equilibrium from dissolution to homeostasis. When parts of the teeth are eroded by acids, HAP is potentially able to remineralize attacked surfaces. Dental erosion and dental caries are both diseases caused by acids. However, the amount of acids and the origin of the acids is different. Consequently, pathogenesis of these two acid-driven diseases differs. Erosion is a fast process compared to caries: Acids produced by bacteria slowly penetrate through the outer layers of enamel to create a subsurface demineralization (caries), while extrinsic acids lead to a dissolution of the enamel’s outer layers (erosion). Enamel crystallites and prisms can be analyzed, when using SEM [73]. In erosion, HAP can cover the demineralized surfaces and might also directly remineralize the underlying demineralized tissue. In vitro experiments using clean enamel surfaces indicate that HAP particles attach to the surface of eroded teeth not only by electrochemical forces, but directly form solid interfaces between HAP crystallites from the particles and the enamel [24]. Hornby et al. performed several experiments where they used HAP in combination with fluoride treatment [100]. However, one experiment was performed using ⁴⁵Ca-labelled HAP to determine Ca-uptake from a HAP slurry after an erosive attack. While the Ca²⁺-uptake from HAP with sound surfaces was 1.05 mg/mm², Ca²⁺-uptake with demineralized enamel was three times higher (3.16 mg/mm²). The authors concluded that this indicates an availability of Ca²⁺-ions from HAP for remineralization [100].

In conclusion, HAP can form a protective layer on tooth surfaces and remineralize eroded enamel and dentin [48, 71, 73, 74, 99]. By forming a “sacrificial layer”, acidic attacks will not directly demineralize the teeth. Additionally, HAP leads to a shift of the solubility equilibrium (Fig. 6). Regular use of HAP is needed to cope with regular acidic challenges.

3.4.1. Studies

(15) Hu et al. conducted a systematic review and a meta-analysis that proved the effect of HAP in relieving dentin hypersensitivity [27]. The authors searched five databases for randomized controlled trials investigating dentin hypersensitivity pain relief. Risk of bias was assessed following the Cochrane guidelines. Confidence intervals and evidence were also calculated in this study. Particulate HAP was evidentially proven in relieving dentin hypersensitivity [27].

Amaechi et al. performed an (16)in situ study [28] and an (17) in vivo study [29] in the field of dentin hypersensitivity using HAP. (16) The in situ study tested the occlusion of dentin tubules after application of either 10% or 15% HAP [28]. Overall, 20 participants per group were recruited to wear human dentine blocks for at least 14 days. Additionally, a toothpaste containing fluoride and another containing NovaMin were also tested. Untreated blocks were used as control. After 7 and 14 days, respectively, dentine occlusion was visualized using SEM. Both tested concentrations showed after 7 and 14 days a higher degree (up to 50%) of completely occluded dentin tubules than the non-HAP toothpastes. In the 15% HAP-group, the test surfaces were 100% covered with a precipitate layer [28]. (17) These results were confirmed by the clinical trial from the same group where reduction of dentin hypersensitivity was investigated [29].

(18) Huettemann and Doenges published already in the year 1987 a double blind clinical trial using different toothpastes with HAP particles of different diameters [101]. 140 patients with dentine hypersensitivity were recruited. Study duration was four weeks and sensitivity was clinically tested using standardized tests (i.e. cold stimulus). The test pastes containing HAP (diameter 2 µm and 6 µm) were compared to a placebo. While in the placebo-group no improvement could be measured, 90% of the HAP-group reported an improvement of dentin hypersensitivity already after 3-5 days. 50% of the HAP-group were pain-free within the study-period (four weeks) [101]. These results were confirmed by several other clinical trials using HAP-containing formulations for relieving dentin hypersensitivity [75, 76, 78, 102].

(19) Hiller et al. tested the in vitro permeability of dentin after application of particulate HAP [77]. For this, they used bovine dentin slabs where a HAP-toothpaste was applied. Subsequently, the hydraulic conductance was tested and used as a measure for the degree of occlusion of the dentin tubules. Permeability was significantly reduced after application of all tested toothpastes, including the HAP-application [77].

3.4.2. Mechanisms

The mechanisms of HAP in preventing dentin hypersensitivity can be summarized as follows: In exposed dentin the dentinal tubules are open towards the oral cavity and to the pulp. Thus, external stimuli can propagate through the dentinal fluid directly to the nerve tissue in the pulp via the odontoblastic processes located in dentinal tubules. Since the applied HAP-particles have a high polarity, they are able to bind both to collagen and hydroxyapatite from dentin. Thus, these particles will attach to dentinal surfaces and eventually occlude exposed dentinal tubule openings if they are smaller than the tubule diameter. The size of tubules/diameter close to the tooth surface or the DEJ, respectively, is about 3.5 µm [103]. Most oral care products contain HAP-particles of sizes between 0.1 and 10 µm [38]. Particles need to have diameters <5 µm to reliably occlude dentinal tubules [38]. HAP particles close the tubules by being pressed into the tubule openings during for instance brushing. Within a certain time, HAP-particles that have occluded tubules will bio-chemically bind to the collagen-rich dentin and bio-chemically fuse with the inner mineral lining of the tubule. Deposited HAP soon gets mineralized by attracting calcium and phosphate ions from saliva. Mineralization will lead to fewer open tubules, and consequently less possibilities for external stimuli to induce pain. When using HAP containing oral care products regularly, dentinal tubules can be completely occluded due to the filling effect building up over time and the concurrent formation of a protective layer on the dentin. As positive side effect, dentin might also be protected from acidic attacks by the mineralized deposited HAP that can act as sacrificial layer.

In conclusion, HAP is known for decades to reduce dentin hypersensitivity. Several studies have shown a significant improvement of clinical parameters when using HAP-based oral care products, which is confirmed by a recently published meta-analysis [27].

3.5.1. Studies

Tooth whitening becomes more and more popular, because the social demand for whiter and brighter smiles is increasing [104]. Beside in-office bleaching, several oral care products for home use show whitening properties [105-108]. Most whitening toothpastes on the market are characterized by high abrasiveness. Their effect relies on removing the outermost stained layer of enamel which may lead to increased roughness or other side effects, such as dentin hypersensitivity [91, 104, 105, 109]. In addition to an increased amount of abrasives, toothpastes often use phosphate-systems for stain removal [106, 107]. However, only extrinsic stain can be removed when using whitening toothpastes. (20), (21), (22), (23), (24) There are studies showing an effect of HAP with respect to tooth whitening [110-114] and their results are promising. Basically, HAP acts as cleaning agent, but in contrast to other abrasives used for tooth whitening products (i.e. perlite and alumina) it has the same hardness as enamel [91]. Consequently, HAP does not lead to excessive enamel and dentin abrasion. (20) Dabanoglu et al. showed whitening properties of HAP. HAP-particles remained stable on the tooth surface after application of hydrodynamic shear force [110]. (21), (22), (23) The whitening-effect can be attributed to an attachment of HAP to the tooth surface, rather than a polishing process [111-113]. (23) The whitening effect of HAP could also originate from physical properties related to its particulate structure [112]. (24) In vivo results showed whitening effects of HAP-particles [114]. These results were verified with in vitro testing: Whitening properties of HAP are mainly based on diffuse reflection leading to optical whitening effects [114].

3.5.2. Mechanisms

Besides removing stain mechanically from tooth surfaces during routine oral hygiene, HAP particles might also bind proteins which often lead to discolorations. These would then be removed together with the particles during the cleaning process. The protective layer formed by HAP particles on tooth surfaces appears inherently white in colour as long as it remains firmly attached to the tooth surface. The size and irregular orientation of the HAP crystallites constituting the particles makes them ideal scatterers within the wavelength range of visible light. The resulting reflection of light makes the tooth surface appear even brighter white than natural, untreated tooth surfaces, especially if they are naturally darker tainted or contain incorporated amounts of extrinsic stains.

In conclusion, HAP particles are promising agents for tooth whitening, as they do not lead to tooth abrasion. HAP particles might remove staining proteins, cover tooth surfaces and enhance the perceived whiteness by scattering and reflection of light.