Enamel and Its Interaction with the Oral Environment

Abstract

Enamel, comprised of hydroxyapatite (HAP) units forming crystallites and rods, constitutes the structure of teeth. HAP is represented by the stoichiometric formula Ca₁₀(PO₄)₆(OH)₂. However, biological HAP, found in enamel, deviates from this stoichiometry due to deficiencies in Ca²⁺, PO₄^3–, and OH^–, and contamination with CO₃^2– and HPO₄^2–, and trace elements within their lattice. Its integrity is influenced by saliva, oral bacteria, fluoride from oral care products, and dietary acids. Salivary glycoproteins form an acquired salivary pellicle on enamel, while oral microorganisms create dental biofilm, which can become cariogenic with increased sucrose levels. The cariogenic biofilm generates acids, which reduces hydroxyl and phosphate groups surrounding enamel, thereby lowering the ion activity product (Ip) of the dental biofilm fluid and saliva and resulting in enamel demineralization. Initial caries appear as subsurface lesions with crystallite dissolution, mitigated by topical fluoride promoting the formation of calcium fluoride-like reservoirs on tooth surfaces and within dental biofilm. Enamel becomes susceptible to irreversible wear with frequent and prolonged dietary acid exposure. Dental erosion, chemically induced below pH 4.5, dissolves fluorapatite and hydroxyapatite predominantly on the surface layer, without subsurface lesions. Understanding these processes is crucial for preventive strategies against dental caries and erosion.

1. Introduction

Enamel is an incredibly remarkable mineralized tissue that not only covers the exterior surfaces of teeth but also significantly contributes to our overall appearance and plays a vital role in digestion. Its organizational complexity spans multiple dimensions, ranging from the atomic level with hydroxyapatite (HAP) units to structures at the micrometer level such as crystallites, up to the millimeter level including enamel rods, and finally encompassing the entirety of the tooth surface. In the oral cavity, enamel interacts with the surrounding aqueous environment, which consists of saliva, oral bacteria, fluoride sourced from oral care products, and dietary acids. Consequently, the conditions within the oral cavity can be quite harsh environmentally, presenting biological and chemical challenges to the integrity of enamel. As a crucial component in the initial stages of digestion, the enamel is exposed to a variety of both protective and harmful agents as part of the dentition’s function.

The acquired salivary pellicle is mainly formed by the selective adsorption of salivary glycoproteins and proteins from various sources, primarily saliva. In addition to providing protection against enamel demineralization, the pellicle also serves as a foundation for the subsequent development of dental biofilm. The mouth harbors a diverse range of oral microorganisms that form biofilms on dental and mucosal surfaces. These resident oral bacteria contribute to host defenses by hindering the establishment of many exogenous microorganisms. The development of dental biofilm can be categorized into several stages, with numerous non-specific and specific interactions occurring between the pellicle and bacterial cells, ultimately determining the success of attachment and colonization.

Despite dental biofilms being natural and contributing to host well-being, on occasion, this symbiotic relationship can deteriorate, leading to oral diseases. Several hypotheses regarding the development of dental caries and periodontal diseases were proposed during the nineteenth and twentieth centuries. However, the Ecological Plaque Hypothesis (EPH) has gained widespread acceptance. According to this hypothesis, the disease results from an imbalance in the total microbiota due to ecological stress, leading to an enrichment of certain oral pathogens or disease-related microorganisms in dental biofilm. Dental caries is a multifactorial disease greatly influenced by the host’s diet, making it a fitting example for the EPH. Individuals who frequently consume significant amounts of fermentable carbohydrates are selected for acidogenic and aciduric bacteria that lead to acid production. In the cariogenic environment, both OH⁻ and PO₄³⁻ ions are reduced, consequently lowering the Ip to a value often below the solubility product constant (Ksp), resulting in enamel dissolution. Remineralization, an important natural repair process, counteracts cariogenic challenges to maintain the balance between mineral loss and gain. Therefore, the caries process entails the accumulation of numerous episodes of demineralization and remineralization, rather than a unidirectional demineralization process.

Currently, it is known that fluoride ions fit well into the structure of a HAP crystal, even better than the hydroxyl group, resulting in lower solubility of fluorapatite (FAP) compared to HAP. However, many studies indicate that a compound similar to calcium fluoride (CaF₂) is an important source of fluoride for oral fluids. This compound forms when the fluoride concentration in the solution surrounding the enamel surface exceeds 100 ppmF. Therefore, given the high fluoride concentration in oral care products, it is expected that a calcium fluoride-like reservoir forms on tooth surfaces, primarily within dental biofilm. Finally, it should be noted that fluoride ions can adsorb to the surface of sound enamel and/or partially demineralized crystallites, attracting calcium ions and enhancing remineralization.

Enamel has inherent mechanisms to counteract daily exposure to dietary acids. Consequently, repeated contact with dietary acids, particularly over prolonged durations, can result in dental erosion. Dental erosion is identified by the deterioration of dental hard tissue via a non-bacterial chemical process. This degradation can be initiated by either hydrogen ions or chelating agents. Typically, dental erosion happens when the pH of fluids around the tooth drops below 4.5, leading to the dissolution of both FAP and HAP in such acidic environments.

2. Chemical compositions and structural hierarchy of enamel

The mineral content of enamel includes HAP, calcium carbonate, calcium fluoride, and magnesium phosphate, making up around 89, 4, 2, and 1.5% of its makeup, respectively [1]. Enamel is composed of 17.7 wt % phosphorus, 36.5 wt % calcium, 3.5 wt % carbonate, 0.5 wt % sodium, 0.44 wt % magnesium, 0.3 wt % chloride, and 0.01% fluoride [2]. Three of the classic enamel proteins including amelogenin, ameloblastin, and enamelin are known to be essential to form tooth enamel. However, a small amount of protein remains in tooth enamel after mineralization is completed and teeth are erupted [3]. Enamel, the toughest biological material found in humans, is formed by specialized cells known as ameloblasts. Within its histological structure, enamel comprises enamel rods (also called prisms) and interrods, serving as the basic building blocks of tooth enamel (Figure 1). Enamel rods, cylindrical-like structures measuring 5–8 μm in diameter, traverse the entire width of the enamel from the dentino-enamel junction (DEJ) to the outer surface of the tooth. A thin rod sheath surrounds each rod and separates it from adjacent rods by an interrod substance [4]. The rod is shaped somewhat like a cylinder and made up of HAP crystallites or crystals (Figure 2).

Figure 1.

A light micrograph of forming enamel rods. Hematoxylin and eosin staining shows the row of numbered enamel rods forming as a wavy course. One part is cut longitudinally (blue arrow), while another part is cut cross-sectionally (black arrow) (the photograph was kindly provided by the Department of Oral Biology, Faculty of Dentistry, Thammasat University, Thailand).

Figure 2.

Longitudinal-sectioned scanning electron microscopy (SEM) image of human enamel rods. (A) Enamel is constructed via cylindrical enamel rods (R) and interrod enamel (IR). Each enamel rod is composed of numerous HAP crystallites (original magnification x 2000). (B) Densely packed HAP crystallites were organized into enamel rods (R) and interrods (IR). Notably, the interrod surrounds each rod, and its crystallites are oriented in a direction different from those making up the enamel rod (original magnification x 2000) (Courtesy: Dr. Apiwat Ridhabhaya, International College of Dentistry, Walailak University, Bangkok, Thailand).

The crystal is the true structural unit of enamel. The smallest space unit of the HAP crystals is called the hydroxyapatite unit cell [Ca₁₀(PO₄)₆(OH)₂], containing 10 calcium ions, 6 phosphate ions, and 2 hydroxyl ions [5]. Every enamel rod is composed of millions of HAP crystallites, measuring approximately 70 nm in width and 30 nm in thickness, and varying in length. A distinguishing characteristic of mature enamel crystallites is their significant size compared to those found in dentine, cementum, and bone [6, 7, 8]. When viewed in cross section, enamel rods display a pattern resembling fish scales or keyholes, comprising a head and a tail (Figure 3). The head of each rod is defined by a slender proteinaceous sheath, approximately 0.5 μm in thickness. Densely packed HAP crystals were organized into enamel rods. Crystallites in the head region align along the long axis of the rod, while those in the tail align perpendicular to the head (Figure 4) [7, 9, 10]. It is crucial to highlight that the “keyhole” pattern is considered within the context of the classic concept. Notably, the rod tail exhibits less distinct boundaries and seamlessly transitions into the interrod enamel [11]. Fundamentally, in longitudinal section, enamel prisms are cylindrical, making the term “rods” more accurate [9, 12]. Despite the enamel rod not strictly conforming to a prismatic outline, the term “enamel prism” has gained acceptance through widespread usage [6].

Figure 3.

Schematic representation of the keyhole concept of enamel rod. The diagram shows the six-sided ameloblasts overlying keyhole-shaped enamel. Body (head), neck, and tail of the enamel rod are also shown in the diagram.

Figure 4.

Cross-sectioned SEM image of human enamel rods. The image was obtained by SEM (original magnification X 5000). Head (H) and tail (T) in keyhole-like patterns of enamel rods were observed in this figure. Notably, interrod enamel (IR) is located around each enamel rod, and it enhances the “keyhole” appearance of enamel rods by acting as its border (Courtesy: Dr. Apiwat Ridhabhaya, International College of Dentistry, Walailak University, Bangkok, Thailand).

The crystals within the enamel rod exhibit no variation in size, shape, and composition when compared to those in the interrod region. When crystallites are generated from the distal end of the Tomes’ process, they align parallel to the rod axis. Nonetheless, the alignment of crystallites varies when they originate from the proximal part of the Tomes’ process (Figure 5) [13]. Given that the distal and proximal ends of the Tomes’ process are angled at approximately 30 degrees, the crystallites in both the rod and interrod enamel also display this angular orientation [8, 9, 14]. Each individual crystallite is stacked atop another and held together by a thin layer, approximately 2 nm thick, of hydrated protein. These enamel crystallites, in turn, constitute a self-assembled agglomeration of HAP nanoparticles, ranging in size from 20 to 40 nm, which serve as the fundamental building blocks of enamel. The HAP nanoparticles maintain cohesion through more rigid, interfacial molecular bonds, including hydrogen bonds facilitated by surface-hydrated water molecules (Figure 6) [15, 16, 17].

Figure 5.

Schematic representation of secretory ameloblasts. The organization of secretory ameloblasts as would be revealed in a section along their long axis. Prismatic enamel rod is produced by the distal end of Tomes’ process, and interprismatic enamel (“interrod”) by proximal portions of the Tomes’ process. Red, purple, and green dots in the Tomes’ process represented secretory granules in ameloblasts.

Figure 6.

Schematic representation of the nanostructure of enamel crystallites. Each enamel rod (4–8 μm in size) consists of many crystallites approximately 60–70 nm wide. Individual crystallite is stacked one on top of another and “glued” together by a thin layer (approximately 1–2 nm thick) of hydrated protein. Each enamel crystallite is, in turn, a self-assembled agglomeration of HAP nanoparticles (20–40 nm), which are the basic building blocks of enamel. The HAP nanoparticles hold together with more rigid, interfacial molecular bonds including hydrogen bonds (Adapted from Ref. [15]).

The building block unit of enamel, dentine, cementum, and bone corresponded to an apatite structure. Generally, stoichiometric HAP, which is Ca₁₀(PO₄)₆(OH)_2, has been used as a model of tooth and bone minerals [18]. Ten Ca²⁺ ions (20 positive charges), six PO₄^3– ions (18 negative charges), and two OH⁻ ions (2 negative charges) in Ca₁₀(PO₄)₆(OH)₂ hold together by electrostatic interactions [19, 20]. The spatial arrangement of HAP atoms is intricate, and a detailed configuration is beyond the scope of this chapter. Here, we present only the fundamental elements of the structure. Basically, isolated HAP unit cells cannot exist independently and are associated with numerous repeating units in a crystallite. Figure 7 illustrates a three-dimensional concept of the HAP unit cell within a crystal. The a-b axes lie in the same plane, forming the floor and ceiling of a parallelogram, with each side measuring 0.942 nm and two angles each of 60 and 120°. The height of the unit cell (0.688 nm) aligns in the c-axis, perpendicular to the a-b plane and parallel to the long axis of the crystal [5, 21].

Figure 7.

Schematic representation of hydroxyapatite unit cell. (A) The unit cell of HAP projected along a-axis. The a- and b-axes form a parallelogram with each side 0.942 nm. The height represents the c-axes and is 0.688 nm. Note two hydroxyl groups are located at 0.25c and 0.75c of the height of unit cells. Calcium ions (II) are also located in the screw axis at 0.25c and 0.75c in the form of equilateral triangles around the two hydroxyl groups. Calcium ions (I) are located inside the unit cell at the a-b plane (0.0c) and one-half the distance between the two a-b planes (0.5c). (B) The unit cell of HAP projected along c-axes. Diagram illustration of stacking of Ca-II ions in the form of equilateral triangles around the hydroxyl groups. The adjacent triangle does not line at the same plane but are rotated by 180 degrees with respect to each other. Notably, when viewing along the c-axis, the hydroxyl ions are exactly superimposed. Phosphate groups are omitted in the diagrams for clarity.

The phosphates within each HAP unit cell are separated into two layers, with heights of 0.25c and 0.75c, as illustrated in Figure 8. These layers predominantly fill the space and form the essential framework of the unit cell. According to the difference in calcium ion position, the general formula of HAP is Ca-I₄Ca-II₆(PO₄)₆(OH)₂, where Ca-I and Ca-II are two different crystallographic positions for 10 calcium atoms. Four of them are situated in the Ca-I position, and the other six calcium atoms are placed in the Ca-II site. Although one hydroxyl ion, three Ca-II, and three phosphates positions are at each corner of the unit cell, only 1/4 of them belong to each corner. Therefore, one HAP unit cell consists of two hydroxyl ions, six Ca-II, and six phosphates at 0.25c and 0.75c, whereas there are four Ca-I at 0.0c and 0.5c [5, 21, 22, 23].

Figure 8.

Schematic representation of the position of phosphate tetrahedral groups with respect to the c-axis. Hydroxyl ions located in the central c-axis at 0.25c and 0.75c are surrounded by calcium-II, phosphates, and Ca-I. Calcium-II ions surrounded the screw axis at 0.25c and 0.75c. Six Ca-II ions are situated on two oppositely directed triangles perpendicular to the c-axis and separated by 0.5c. At a slightly greater distance, one finds phosphate groups similarly arranged. Notably, hydroxyl ions, Ca-II ions, and phosphate groups are shown in this figure, whereas Ca-I ions are omitted for clarity.

The essential feature of HAP is the hexagonal framework of calcium atoms and phosphate groups surrounding a column of hydroxyl groups. From a crystallographic point of view, the arrangement of calcium atoms, phosphate, and hydroxyl groups in the crystal can be modeled as in Figure 9 [24, 25]. Within the atomic lattices of HAP, two distinct unconnected channels can be delineated: a small channel and a large channel. The first channel (type I) possesses a diameter of 0.25 nm and is surrounded by Ca-I. This channel exhibits coordination 9 with the oxygen atoms of the phosphate tetrahedrons, resulting in the formation of a polyhedron. The second channel (type II) holds significance in the properties of HAP, featuring a larger diameter than the preceding one (ranging from 0.3 to 0.45 nm). It includes six additional Ca atoms, denoted as Ca-II, positioned at the periphery of the channel (Figure 10) [26, 27]. Various atom substitutions can take place in the large channel. For instance, hydroxyl groups can undergo substitution by anions such as fluoride (Figure 11), and phosphate groups can be substituted by other anions, such as carbonate [23, 28, 29].

Figure 9.

Hydroxyapatite lattice structure projected along the c-axis. The number written in the symbol is z parameter (direction perpendicular to the page) (Adapted from Ref. [24]).

Figure 10.

Schematic picture of the two channels of HAP unit cells. The projection of the structure Ca₄(1)Ca₆(II)(PO₄)₆(OH)₂ along the c-axis. In this atomic lattice, small (type I) and large (type II) channels can be described (Adapted from Ref. [26]) (see in-text explanations for details).

Figure 11.

A view of fluorapatite (FAP) projecting perpendicular to the c-axis. In the central c-axis, there is a substitution of the hydroxyl ion by a fluoride ion. Fluoride ions replace hydroxyl ions on a 1:1 basis and are randomly dispersed along the c-axis. Calcium-II ions surround the screw axis at 0.25c and 0.75c. The fluoride ions are positioned within the triangular Ca-II configuration at 0.25c and 0.75c. It is important to note that this figure illustrates fluoride ions and Ca-II ions, with Ca-I ions and phosphates omitted for clarity.

3. Biological hydroxyapatites

Biologically, HAP in enamel is highly non-stoichiometric. The molar ratio of calcium to phosphorus in enamel ranged from 1.50 to 1.67, whereas the ratio in stoichiometric HAP remains constant at 1.67. The presence of non-stoichiometric HAP is due to the loss of Ca²⁺ ions, resulting in an electrical imbalance that is rectified by the inclusion of H⁺ ions and a reduction in OH⁻ ions. This phenomenon is represented by the formula Ca_10-Z(HPO₄)_Z(PO₄)_6-Z(OH)_2-Z; where 0 < Z < 1 [26, 30]. Consequently, enamel mineral differs from ideal HAP, incorporating ions such as HPO₄²⁻, CO₃²⁻, Na⁺, F⁻, and others into its apatite lattice. Within the HAP structure of enamel, the carbonate group occupies two distinct positions: the hydroxide position (A) and the phosphate position (B). Approximately 11 weight% of the total carbonate in deciduous enamel is situated in position A, while 10 weight% of the total carbonate in permanent enamel is found in the same position. Carbonates play a crucial role in enamel maturation and may contribute to the initial phase of dental caries formation [31].

4. Surface properties of enamel

The Stern-Gouy electrical double-layer theory is used to describe the microscopic structure at the solid surface [32]. The outer enamel layer (less than 1 μm from the anatomical surface) is most likely a non-stoichiometric apatite. The outer surface of enamel exhibits surface area fractions of calcium, phosphates, and hydroxyl ions about 16.5, 78.4, and 5.1%, respectively [33]. Phosphoric acid displays three pKa values (2.1, 7.2, and 12.7). While PO₄³⁻, HPO₄²⁻, H₂PO⁴⁻, and H₃PO₄ can all be found in aqueous solutions, HPO₄²⁻ (62%) and H₂PO₄⁻ (38%) are predominantly present at neutral pH [34]. Consequently, the outer surface of enamel acquires a negative charge due to the presence of HPO₄²⁻ and H₂PO⁴⁻groups. This negative charge facilitates the formation of the Stern layer, also recognized as the calcium-rich layer, which firmly adheres to the enamel surfaces (Figure 12) [33, 35].

Figure 12.

A schematic representation of the solid-liquid interface of enamel crystallites. Typically, solid enamel carries a negative charge attributed to the presence of HPO₄²⁻. This negative charge results in the formation of the Stern layer, also known as the calcium-rich layer, which tightly adheres to the enamel surfaces. This layer is integral and inseparable from the enamel. In the solution phase, the remaining ions present in saliva, including calcium ions, acid phosphates, and fluorides, are dispersed by electrostatic interactions, forming what is known as the diffuse layer. The diffuse layer represents the distribution of these ions in the solution surrounding the enamel.

5. The oral environment

The tooth surface is continually immersed in either saliva or gingival crevicular fluid. Saliva plays an important role in maintaining enamel integrity by regulating the demineralization and remineralization of enamel minerals. The active concentrations of calcium, phosphate, and fluoride ions in saliva are key factors influencing the stability of enamel hydroxyapatite, along with salivary pH. Elevated concentrations of calcium and phosphate in saliva facilitate ionic exchanges directed toward the tooth surfaces, initiating post-eruptive maturation from the time of tooth eruption [36]. As soon as saliva contacts tooth surfaces, salivary glycoproteins selectively adsorb to the tooth surface to form an acquired salivary pellicle. The adsorbed salivary components not only sustain high levels of calcium and phosphate within saliva but also enhance the adherence of oral bacteria to tooth surfaces [37].

The mouth contains a wide range of microorganisms, which is more diverse than those found in other parts of the body. It is likely that the oral cavity offers various ecological niches [38]. Most oral bacteria are attached to the numerous surfaces in the oral cavity as oral biofilm (e.g., dental biofilm on tooth surfaces), where they are constantly bathed in either saliva or gingival crevicular fluid, providing hydration to the oral biofilm as well as nutrient to oral bacteria [39]. Dental biofilm contributes to the development of oral diseases such as dental caries and periodontal diseases. However, the consistent presence of fluoride ions in saliva and plaque fluids is crucial for protecting enamel from demineralization when facing caries threats [40]. Alongside the cariogenic environment, enamel can also be exposed to acidic conditions, particularly through the consumption of acidic foods and beverages. Dental wear, such as dental erosion, is frequently observed in individuals who regularly consume sports and/or soft drinks [41].

6. Acquired salivary pellicle formation, composition, and function

An acquired salivary pellicle forms when saliva comes into contact with the tooth [42]. This pellicle is comprised of proteins, lipids, carbohydrates, and other macromolecules. The components of the acquired salivary pellicle originate from various sources, including salivary gland secretion, gingival crevicular fluid, blood, products from the oral mucosa, oral bacteria, and diet [43]. Primarily, the acquired salivary pellicle is composed of adsorbed salivary proteins. Examples include albumin, amylase, carbonic anhydrase I-II, VI, cystatins, fibrinogen, fibronectin, histatins, immunoglobulins, lactoferrin, lysozyme, salivary mucins MG1 (MUC5B) and MG2 (MUC7), myeloperoxidase, proline-rich proteins (PRPs), secretory IgA, and statherin [44, 45]. Additional macromolecules in the acquired salivary pellicle include lipids and carbohydrates, encompassing glycolipids, phospholipids, and glycoproteins [43]. Importantly, the composition of the acquired salivary pellicle varies among individuals [46]. Figure 13 shows the acquired salivary pellicle structure with the atomic force microscopy (AFM) technique [43, 47]. The acquired salivary pellicle forms by dynamic and selective processes of adsorbed and desorbed salivary proteins on the enamel surface [42]. Transmission electron micrography shows that the acquired salivary pellicle structure contains an electron-dense basal layer and an outer globular layer [43, 48].

Figure 13.

AFM image of the acquired pellicle structure. AFM image of in vitro acquired salivary pellicle morphology on enamel block after 2-h formation time. This view highlights the three-dimensional topographic differences as a result of the acquired pellicle layer adsorbed onto the enamel surface.

The initial stage of acquired salivary pellicle occurs within seconds to a couple of minutes. The adsorbed salivary proteins attach to the enamel surface to form the electron-dense basal layer via electrostatic interactions [44, 49]. The electron-dense basal layer consists of a densely globular structure of phosphate and calcium-binding peptides, precursors of acquired salivary pellicle, and acid-resistance proteins [42, 50], such as PRPs, histatins, mucins, and statherin [51, 52, 53]. This layer is characterized by high acid resistance and strongly adheres to enamel crystals [45, 51]. The thickness of the electron-dense basal layer at the initial stage is approximately 10–20 nm [54]. It takes 60–120 minutes to reach the maturation stage and stays at a steady level for several hours [42, 54]. The thickness of the outer globular layer at the maturation stage is approximately 100–1000 nm [54]. The outer globular layer consists of other macromolecules and adsorbed proteins such as albumin, amylase, carbonic anhydrase, and mucins [45, 50]. This outer globular layer is composed of different sizes of micelle-like globular and granular structures. It exhibits knotted structures and globular-like structure [42, 55]. The formation mechanism of the acquired pellicle is shown in Figure 14 [43]. The functions of the acquired salivary pellicle have been approved for determining the primary colonizers of oral bacteria [56], lubricating the tooth surface during mastication and speech [43], maintaining mineral homeostasis [47], and protecting the tooth surface against dental caries and dental erosion [49, 53].

Figure 14.

Schematic representation of pellicle formation. (A) Enamel surface, (B) electron-dense basal layer at initial stage: attachment of pellicle precursor proteins, (C) outer globular layer at maturation stage: equilibrium between adsorption and desorption of macromolecules. This image is adapted from a courtesy image by Chawhuaveang et al. [43].

7. Dental biofilm (dental plaque)

7.1 Dental biofilm formation

As mentioned above, the enamel surfaces of teeth are generally covered by the acquired salivary pellicle, which is essentially free of bacteria. Following tooth cleaning, some bacteria attach to the pellicle shortly afterward. While remnants of dental biofilm left due to incomplete removal by oral hygiene procedures may account for some bacteria, the majority of newly deposited bacteria come directly from oral fluids [57]. Biofilm is a microbial community embedded in a matrix and attached to different surfaces or environments. Bacteria form biofilms in every substratum and their associated infections in plants, animals, and humans [58]. In human body, bacterial biofilms can attach to various clinical devices and microflora-exposed tissues including tooth surfaces [58, 59]. Biofilms offer a habitat for bacteria and prevent them from pH varying and nutrient deficiency. Bacteria in a biofilm are more resistant to antibacterial or chemical treatments compared with that of planktonic bacteria [60].

The term “dental plaque” was probably first described by G.V. Black in 1886 when he collected a group of microorganisms on early carious lesions. Therefore, dental plaque is the biofilm on the tooth surface. Dental biofilms have common properties, that is, the presence of an extracellular polysaccharide substances (EPS) matrix and drug resistance [61]. The dental biofilm has advantages over the planktonic bacteria in saliva in terms of preventing elimination from rinsing or swallowing, defending against pathogen invasion and host mediators. Oral biofilms are classified by the composition and differ at distinct locations, including (1) supragingival biofilm and (2) subgingival biofilm. The supragingival biofilm is located on tooth surfaces, mostly contains Gram-positive and facultatively anaerobic bacteria (especially Streptococcus spp.), and is associated with dental caries. In contrast, the subgingival biofilm is located in gingival crevice, mostly contains Gram-negative and obligately anaerobic bacteria, that is, Porphyromonas gingivalis, and correlates with periodontitis [59].

Dental biofilm formation occurs within a few hours in a sequential event consisting of four stages (Figure 15): (1) initial colonization of tooth surfaces covered with an acquired salivary pellicle, (2) rapid bacterial growth and EPS production, (3) bacterial coadhesion and coaggregation, and (4) maturation and dispersion [62]. At stage 1, the tooth surfaces exposed to the oral environment are steadily covered by a conditioning film or acquired salivary pellicle, which is mostly derived from the adsorption of organic and inorganic molecules in saliva. Some proteins in the pellicle offer binding sites for adhesion of the planktonic initial bacteria, mainly from the genera Streptococcus and Actinomyces [63]. The first attachment of bacteria to the tooth surface covered by the pellicle is facilitated by fimbriae, as well as by various forces at different ranges, resulting in specific interactions between receptors and adhesins. These forces can be either attractive (involving hydrophobic interactions, van der Waals forces, hydrogen bonds, and calcium bridges), repulsive (involving electrostatic interactions), or a combination of both (involving acid-base interactions) (Figure 16) [64, 65].

Figure 15.

Dental biofilm formation. (1) primary colonizers adhere to a tooth surface covered with acquired salivary pellicle; (2) rapid cell growth, division, and production of EPS resulting in microcolony formation; (3) coadhesion of single bacterial cell, coaggregated bacteria, and groups of identical bacteria into the young multispecies dental biofilm; and (4) maturation and the development of clonal mosaics within the multispecies dental biofilm.

Figure 16.

Schematic representation of initial bacterial adherence to tooth surfaces. Initial bacterial approach and adhesion to a pellicle-coated tooth surface are mediated by (A) van der Waals forces, (B) electrostatic attraction, (C) calcium bridging, (D) hydrogen bond, (E) hydrophobic interactions, and (F) adhesion-receptor interactions. Notably, the primary colonizers provided new specific receptors for the coadhesion of secondary colonizers.

Initially, dental biofilm forms through the interactions of both adhered and planktonic bacteria, with its further maturation being dominated by the proliferation and differentiation of the adhered bacteria [66]. The bacterial adhesion to an acquired salivary pellicle represents one of the most important factors in the bacterial colonization of tooth surfaces. Some bacteria, known as early or primary colonizers, are capable of colonizing on tooth surfaces coated by the acquired salivary pellicle. The primary colonizers create new specific receptors for late or secondary colonizers that are unable to directly bind to the pellicle. The early colonizers include Streptococci spp., Actinomyces spp., Capnocytophaga spp., Eikenella spp., Haemophilus spp., and Veillonella spp. However, 60–80% of primary colonizers are streptococci. Then, the early adherent streptococci on tooth surfaces recruit secondary colonizers to form a biofilm through an autoaggregation or coaggregation process. Subsequent attached bacterial species include Fusobacterium nucleatum, Treponema spp., Porphylomonas gingivalis, and Aggregatibacter actinomycetemcomitans (Figure 17) [67, 68, 69].

Figure 17.

Schematic representation of oral bacterial colonization. Bacterial colonization starts with the adhesion of early colonizers (such as Streptococci spp., Actinomyces spp., Capnocytophaga spp., Eikenella spp., Haemophilus spp., and Veillonella spp.) to host-derived proteins in acquired salivary pellicle. Consequently, the early colonizers recruit other oral bacteria to produce late colonizers. The late colonizers include Fusobacterium nucleatum, Treponema spp., Porphylomonas gingivalis, and Aggregatibacter actinomycetemcomitans (Adapted from Ref. [67]).

In Stage 2, after the initial bacteria have been successfully colonized, they consequently enter a logarithmic growth phase, and the biofilm will start differentiating as a highly structured community. Interspecies communication occurs by using the quorum-sensing system. This system facilitates biofilm differentiation, bacterial metabolism, EPS production, and biofilm maturation (Figure 18) [68, 70]. In Stage 3, the growth of early colonizers on the dental surface introduces new binding sites for other species. This binding process fosters coadhesion and coaggregation, leading to the formation of a multi-species biofilm (Figure 19) [71]. In Stage 4, synergistic and antagonistic interactions within the biofilm contribute to food and oxygen exchange. This exchange strengthens the biofilm matrix, facilitates equilibrated homeostasis, and culminates in the maturation of the biofilm. Eventually, bacteria are released from the matured biofilm, undergoing reorganization on other surfaces [63].

Figure 18.

SEM of dental biofilm. Dental biofilm accumulated on the stainless steel ligature (SSL) after 6 weeks of permanence in the mouth of an orthodontic patient. Bacterial cells (B) firmly adhere to the SSL surface via EPS (yellow arrow). The EPS also mediated cell-cell (green arrow) accumulation in biofilm formation (original magnification x 30,000) (Figure courtesy of Drs. Thanakorn Saengphen and Apiwat Ridhabhaya, International College of Dentistry, Walailak University, Bangkok, Thailand).

Figure 19.

SEM micrograph of matured dental plaque. Plaque was developed on the SSL (green arrow) after 6 weeks of permanence in the mouth of an orthodontic patient. Mass of biofilm comprised of aggregated coccoid (blue arrow) intertwining with rod- and filament-shaped bacteria (yellow arrow) (original magnification X 3000) (Figure courtesy of Drs. Thanakorn Saengphen and Apiwat Ridhabhaya, International College of Dentistry, Walailak University, Bangkok, Thailand).

Drug resistance is one of the effective synergistic interactions within dental biofilm. Chlorhexidine is a common antiseptic mouthwash used for dental plaque controlling [72]. An in vitro study using live/dead staining and confocal laser scanning microscopy demonstrated that chlorhexidine exhibits bactericidal penetration (red color) into dental biofilm surface (approximately 50% of total biofilm structure) (Figure 20). In contrast, the living bacteria (green color) were observed in the middle to bottom of the biofilm. This confirmed the resistant capability against chlorhexidine of some bacteria in dental plaque.

Figure 20.

In vitro effects of chlorhexidine on bacterial biofilm. In vitro S. mutans biofilm is developed on a saliva-coated HAP surface and incubated for 48 h. Chlorhexidine (1 μg/ml) is used to treat the biofilm. Live or dead bacteria are studied using Live/Dead® bacterial BacLight™ Bacterial Viability Kit (Invitrogen Molecular Probes, Carlsbad, CA, USA). Live and dead bacteria are stained by SYTO 9—green fluorescent and propidium iodide—red fluorescent, respectively. The stained biofilm is scanned and visualized from top to bottom (each layer thickness is approximately 5 μm) using confocal laser scanning microscope (EclipseTi; Nikon Instrument Inc., Tokyo, Japan).

7.2 Sugar metabolism and acid production

Among dietary carbohydrates, sucrose can induce significant biochemical and physiological changes during dental biofilm formation, rendering it one of the most cariogenic carbohydrates. The glycolysis pathway metabolizes sucrose to acids, with lactic acid produced at high sugar concentrations and acetic acid, formic acid, and ethanol generated at low sugar concentrations. Energy generated during glycolysis is essential for bacterial growth. Sucrose also functions as a substrate for the synthesis of intracellular polysaccharides (IPS) and EPS in dental biofilms. IPS can be catabolized to acids in the absence of dietary sugars. EPS plays a crucial role in dental biofilm formation. It fosters the development of a matrix, improving bacterial attachment and the subsequent buildup of bacteria within dental biofilms. Moreover, it contributes to the structural strength and volume of dental biofilms, heightening the acidity of the dental biofilm matrix [66, 73, 74, 75].

The dental biofilm maintains homeostasis to promote oral health, providing advantages to the normal flora and the host. In ordinary biofilm, S. mitis, S. sanguinis, and S. cristatus use arginine deiminase to suppress adhesion and the expression of virulent factors in P. gingivalis [76, 77]. In addition, S. salivarius, S. mitis, S. gordonii, S. sanguinis, S. cristatus, and A. naeslundii use salivary lactoperoxidase system to buffer the acid produced by certain cariogenic bacteria. The lactoperoxidase system also increases H₂O₂, which is toxic to S. mutans. This evidence confirms that bacterial interaction promotes biofilm neutralization and maintains the demineralization/remineralization balance to prevent dental caries [77, 78].

The “Ecological plaque hypothesis” (EPH) has clearly explained ecological shift from normal dental plaque forward to caries risk plaque [79]. When there is a high frequent consumption of carbohydrates and fermentable sugars, most of the bacteria ferment the sugar and produce the acids as by-products. This decreases the pH in the dental biofilm from relatively neutral conditions (pH 6.0–7.0) to a more acidic pH (5.5), which reduces the buffering capacity of saliva [80]. The acidic environment favors the rapid growth of cariogenic bacteria including mutans streptococci (MS), Actinomyces, and Lactobacillus. MS, especially S. mutans, has relatively high acidogenic potential compared with the non-mutans group in terms of sugar uptake and metabolism. The cariogenic bacteria ferment carbohydrates by degrading glucose into pyruvate and lactate/acetate/formate, creating the cariogenic environment (Figure 21) [81, 82].

Figure 21.

EPH and caries. The diagram depicts the dynamic relationship that exists between the dental plaque and the local environment, according to EPH (Adapted from Ref. [81]).

Based on EPH, a disclosing staining agent has been developed to assess caries risk in patient’s dental biofilm. Currently, the 3-tone staining gel (GC Tri-plaque ID Gel™, GC Corporation, Japan) is one of the practical tools used for evaluating status and acidic change in dental biofilm. The gel contains sucrose and pigments (blue and red) that can penetrate and stain the dental biofilm, resulting in three colors based on the dental biofilm status. The pink color indicates newly formed dental biofilm in its initiation stage without EPS. Meanwhile, blue/purple blues represent old dental biofilm, with light blue denoting extra high-risk or cariogenic dental biofilm (Figure 22) [83]. The environmental change in dental biofilm due to high frequent sugar consumption provides an acidic environment and ecological shift to cariogenic bacteria accumulation, and disrupts the demineralization/remineralization balance in enamel and initiating carious lesions [79].

Figure 22.

Dental biofilm staining with GC Tri-plaque ID Gel^TM. Dental biofilm is stained according to the company’s instructions. The result is shown in three colors based on the dental biofilm conditions: new dental biofilm (pink), old dental biofilm (blue/purple), and extra high-risk dental biofilm (light blue).

8. Demineralization and remineralization

Demineralization of HAP is initiated by protonated ions of phosphates (PO₄³⁻ + H⁺; HPO₄²⁻) and hydroxyl groups (OH⁻+ H⁺; H₂O) in the apatite lattices. According to Coulomb’s law, calcium ions in HAP will therefore not be bound to an adequate extent but rather will be lost. It is worth noting that the attractive forces between ions in water are significantly weaker, about 1/80th, compared to those in a vacuum. As per Coulomb’s law, this suggests that HAP is prone to dissolve in water [19]. As mentioned above, organic acids, especially lactic acid, are generated by cariogenic bacteria in dental biofilm, particularly, in the presence of high sugar concentrations. In theory, when the pH of dental biofilm decreases, there comes a point where enamel HAP starts to dissolve. This particular point is often termed as the critical pH. The physiological concentration of salivary calcium is about 1.0 mmol/L. This makes the critical pH, which is the tipping point between enamel de- and remineralization, to be about pH 5.5, that is for healthy resting adults producing normal saliva [19, 84, 85]. Notably, PO₄³⁻ and OH⁻ in the HAP are protonated by H⁺ if the pH drops below critical pH (pH 5.5). Then, the phosphate and hydroxyl ions lose one negative charge, which is important for binding calcium ions. This causes a loss of minerals from the apatite lattices (Figure 23) [20].

Figure 23.

Schematic representation of HAP dissolution in the acid environment. (A) HAP is stable at pH 7. (B) The PO₄³⁻ and OH⁻ ions from the HAP are protonated by H⁺ ions if the pH drops below critical pH (pH 5.5). Then, the phosphate and hydroxyl ions lose one negative charge, which is important to bind Ca²⁺. This causes a loss of minerals from the HAP lattices (Adapted from Ref. [20]).

In essence, ionic solids, such as HAP, undergo constant reactions with the surrounding media. All minerals exhibit an inherent and fixed solubility in water at any given temperature. Water molecules are essential in removing ions from the crystal surface by diminishing the electrostatic interactions between positively and negatively charged ions, which is facilitated by water’s high dielectric constant. Additionally, water molecules create hydration shells around the ions that are released. The energy of these hydration shells exceeds the lattice energy holding the crystal together [86]. When an ionic solid (s) is in water, it will release an equal number of constituent ions into the aqueous (aq) solution at equilibrium. When solid HAP is dissolved in a solution, it breaks down into Ca²⁺, PO₄³⁻, and OH⁻ as follows:

$${\mathrm{Ca}}_{10}{\left({\mathrm{PO}}_4\right)}_6{\left(\mathrm{OH}\right)}_2\left(s\right)\leftrightarrow 10{\mathrm{Ca}}^{2+}\left(\mathit{aq}\right)+6{\left({\mathrm{PO}}_4\right)}^{3-}\left(\mathit{aq}\right)+{\text{2OH}}^{-}\left(\mathit{aq}\right).$$

This type of chemical equilibrium is described by the solubility product constant (Ksp). The Ksp of HAP is simply expressed to:

$$\mathrm{Ksp}={\left[{\mathrm{Ca}}^{2+}\right]}^{10}\mathrm{x}{\left[{{\mathrm{PO}}_4}^{3-}\right]}^6\mathrm{x}{\left[{\mathrm{OH}}^{-}\right]}^2\text{or}{\left[{\mathrm{Ca}}^{2+}\right]}^5\mathrm{x}{\left[{{\mathrm{PO}}_4}^{3-}\right]}^3\mathrm{x}\left[{\mathrm{OH}}^{-}\right].$$

It should be noted that the Ksp for a particular crystal is approximated to be a constant that is characteristic of that crystal. A crystal with a low Ksp is less soluble than a high Ksp. For example, the Ksp for HAP [Ca₁₀(PO₄)₆(OH)₂] is approximately 5.5x10⁻¹¹⁸ (mol.L⁻¹)⁹. HAP has a low Ksp because of the near close-packed structure, which minimizes the distance of charge separation in Coulomb’s law. The Ksp for FAP [Ca₁₀(PO₄)₆F₂] is about 5.0x10^–123 (mol.L⁻¹)⁹. This indicates the comparatively denser arrangement of ions within the lattice and the slightly smaller size of unit cells in FAP when compared to HAP [19].

In various solutions like saliva, dental biofilm, or soft drinks, the ion activity product (Ip) associated with HAP is calculated using the same formula as Ksp, but with concentrations of reactants and products taken from any specific point in the reaction, not solely at the equilibrium point. When Ip equals Ksp, the solution is saturated with respect to HAP. If Ip is lower than Ksp, the solution is unsaturated. Conversely, if Ip exceeds Ksp, the solution is supersaturated [87]. It should be noted that, basically, human salivary calcium and phosphate ions are supersaturated with respect to HAP. However, spontaneous precipitation of HAP does not occur under physiological conditions. This phenomenon has been attributed to the inhibitory activities of statherin and the acidic proline-rich phosphoproteins (PRPs) in acquired salivary pellicle [88, 89]. Notably, salivary proteins, particularly statherin, act like a “negative” catalyst, or inhibitor, to slow down the process of demineralization. It has been reported that this can be by up to 50%. This occurs when the proteins modify the chemistry of the dissolution process at the enamel surface (Figure 24) [84].

Figure 24.

Schematic representation of biodemineralization and bioremineralization of enamel. (A) Chemical reaction conditions corresponding to saturation (Ip=Ksp) of enamel coated with statherin and PRPs result in the equilibrium between HAP degradation and formation. (B) Biodemineralization of enamel due to acidic where Ip is less than Ksp. When the pH decreases, hydrogen ions from the acid bind with the negative phosphate and hydroxyl ions from the acquired salivary pellicle and plaque fluids. Consequently, the Ip in the pellicle layer becomes unsaturated, causing a shift that favors demineralization. This results in the loss of calcium and phosphate ions from the crystal until a solubility equilibrium is reached. (C) If the concentration of protons adjacent to the enamel surface decreases and/or the concentration of calcium and phosphate ions increases, the Ip of the surrounding solution will be greater than Ksp for HAP. This results in a supersaturated condition that resists biodemineralization and favors biomineralization of enamel.

9. Enamel dissolution in the cariogenic environment

HAP dissolution increases about 10-fold for a change of 1 pH unit [87]. Two reasons account for the heightened solubility of HAP when subjected to acid attack. Initially, H⁺ ions resulting from the metabolic activity of cariogenic biofilms will combine with OH⁻ ions encircling HAP, particularly within plaque fluids, resulting in the formation of H₂O molecules. For example, if the pH of the aqueous solution drops from 7 to 5, the hydrogen ions will increase to 10⁻⁵ M, and OH⁻ falls to 10⁻⁹ M, or to 1% of its previous concentration. Second, the proportions of the four phosphate species (H₃PO₄, H₂PO₄⁻, HPO₄²⁻, and PO₄^3–) in plaque fluid and saliva vary with pH, although the total phosphate concentration is 5 × 10⁻³ mol/L, as is typical of saliva. The H⁺ ions protonated PO₄³⁻ in the plaque fluid to HPO₄²⁻ and H₂PO₄⁻. Therefore, as the pH of the plaque decreases, the concentration of PO₄³⁻ in the fluid, and potentially in saliva, decreases as well. With both PO₄³⁻ and OH⁻ concentrations in plaque fluid diminishing at lower pH levels, it is anticipated that both ions will dissolve out of the enamel HAP to maintain equilibrium in the surrounding liquid solutions. This process is geared toward preserving neutrality and ultimately results in the dissolution of calcium from enamel HAP. However, in a cariogenic environment, the concentration of calcium remains relatively unaffected initially. Conversely, the concentrations of both OH⁻ and PO₄³⁻ undergo significant reduction, leading to a decrease in Ip to a value below the Ksp [19, 86, 87, 90]. Subsequently, the decrease in ionic attraction between cations and anions due to acid attack, as previously mentioned [19, 20], along with a decrease in Ip of surrounding fluids, results in mineral loss from enamel [86, 87, 90].

In the process of tooth decay, there is a cycle of demineralization and remineralization. During demineralization, dissolved Ca²⁺ and HPO₄^2– move actively from the advancing lesion front to the outer enamel surface. This movement is facilitated by the Galvani potential, which is created by the inward diffusion of protons. Upon reaching the tooth surface, the demineralized calcium and phosphate ions may combine with available fluoride, resulting in the formation of a mineral-rich layer at the lesion surface (Figure 25) [91, 92]. When the cariogenic conditions in the dental plaque subside, typically when the plaque pH rises above the critical pH of approximately 5.5, the remineralization phase begins. Unlike during demineralization, there is no outward movement of minerals from inside the lesion due to the absence of Galvani potential. Instead, calcium and phosphate concentrations at the tooth surface are higher than those in the lesion. Consequently, both ions from enamel demineralization, plaque fluids, and saliva passively diffuse into the enamel through micropores of the intact surface layer and particularly, intercrystallic spaces of the lesion. It is worth noting that the most effective remineralization initially occurs at the lesion boundary and then progresses toward the tooth’s surface (Figure 26) [91, 92].

Figure 25.

Schematic illustration of demineralization mechanism. An acid attack causes the Galvani potential, followed by active diffusion of calcium and phosphate ions out of the tooth (Modified from Ref. [91]).

Figure 26.

Schematic illustration of remineralization. Calcium and phosphate ions diffuse from a higher concentration outside of the tooth into the carious lesion, where precipitation occurs on existing apatite minerals at the lesion boundary, progressing to the enamel (Modified from Ref. [91]).

It is essential to note that the demineralized lesion in the initial carious stage is considered reversible to a certain extent through remineralization. However, predominant demineralization leads to the formation of irreversible cavities. Figure 27 outlines the hypothetical chain reactions occurring during demineralization/remineralization cycling. As demineralization initiates, a portion of the original tooth mineral dissolves, resulting in the formation of dicalcium phosphate dihydrate (DCPD, CaHPO₄.2H₂O) and octacalcium phosphate [OCP, Ca₈(HPO₄)₂(PO₄)₄·5H₂O] under acidic conditions. When the acidogenic attack has subsided, and a neutral pH is returned, both DCPD and OCP may form apatite or carbonated hydroxyapatite [93, 94].

Figure 27.

The postulated phase transition in the caries process. For the sake of simplicity, we here two assumptions: (i) the original tooth mineral is expressed as Ca-deficient apatites substituting carbonate at both PO₄ and OH lattice sites and (ii) the involvement of octacalcium phosphate [OCP, Ca₈(HPO₄)₂(PO₄)₄·5H₂O] as an intermediate phase is excluded (Adapted from Ref. [93]).

10. Initial caries

The first noticeable indication of enamel demineralization is the emergence of a white spot lesion (WSL). These lesions have a chalky white appearance, attributed to the increased porosity in the subsurface zone resulting from demineralization (Figure 28). The entrapment of air or saliva within the pores of demineralized enamel impacts light transmission due to variations in the refractive indices among enamel, air, and saliva [95, 96]. In the routine oral environment, demineralization and remineralization are dynamic processes that occur frequently, influencing the development or reversal of carious lesions. Analysis through scanning microradiography of naturally occurring initial carious enamel has revealed an average reduction of 30% mineral mass in the subsurface lesion [97, 98]. Examinations of initial carious lesions using various imbibition solvents and a polarized light microscope have identified four distinct zones: a relatively intact surface layer, the lesion body, a dark zone, and a translucent zone signifying the advancing front of the lesion (Figure 29) [99].

Figure 28.

A clinical photograph of WSLs. WSLs (black arrows) are located at the incisor cervical regions. They are chalky white in appearance and feel slightly rough upon gentle probing.

Figure 29.

Diagram of histological changes and the pore volume distribution (%) in initial caries. The surface layer is due to mineral re-precipitations. In the subsurface, the body of the lesion is triangular in shape, with a summit oriented toward the DEJ. The striae of Retzius are well-marked in the body of lesion. The subsurface lesion displays two borders: one dark zone that is in progress and a reaction zone (limiting or translucent zone).

The transparent zone marks the innermost section of the advancing lesion front and shows minimal impact from caries attack. It demonstrates a pore volume ranging from 1 to 5%, indicating a slightly higher pore density compared to sound enamel, which generally maintains a pore volume of about 0.1%. Adjacent to the transparent zone is the dark zone, which, according to polarized light studies, displays a pore volume ranging from 2 to 4%. Sitting between the dark zone and the relatively unaffected surface layer, the body of the lesion represents the largest segment of initial carious enamel. This region undergoes the most significant demineralization, with a pore volume ranging from 5 to 25%. Notably, the striae of Retzius are prominently visible in this area, suggesting demineralization preferentially occurs in regions with higher porosity. The outermost “unaltered” surface remains relatively unaffected by the caries attack. It exhibits a slightly elevated pore volume (1%) compared to normal enamel [98, 100].

It is important to recognize that the development of enamel caries does not necessarily start with subsurface issues. Processes involving dissolution, precipitation, and protection from adsorbed proteins in an acquired salivary pellicle contribute to understanding the formation of porosity or solubility gradients. A white spot lesion typically signifies demineralization beneath the surface with heightened enamel porosity and porous outer tissues. Nonetheless, certain proteins like PRPs and others with protective properties can shield the surface from further demineralization and hinder crystal growth. Initially, due to their size, adsorbed proteins can penetrate and diffuse only into the deeper enamel layers at an intermediate stage of caries progression. Yet, as caries advance and pore sizes increase, smaller pellicle precursor and acid-resistant proteins may also infiltrate the subsurface lesion. Consequently, salivary proteins of varying sizes remain at different levels within the lesion, forming a protective coating during the demineralization process [101].

Scanning electron microscopy of the outer surface of initial caries showed two main structural features, accentuation of the perikymata and deep focal holes (Figures 30 and 31) [102, 103, 104]. Broadening of interprismatic spaces was commonly observed in affected areas. Mineral dissolution of prism cores was markedly observed when compared to that of prism borders (Figure 32). Demineralization of enamel shows loosely packed globular crystals with a few areas of the residual healthy enamel. These non-affected areas show no globular crystals (Figure 33) [104].

Figure 30.

SEM of WSLs. Perikymata (red arrows): are prominent. The borders of the perikymata are flattened with an irregular shelf-like border. There are numerous deep focal holes (yellow arrows) (original magnification x 200; bar = 100 μm).

Figure 31.

SEM of focal holes. Deep funnel-shaped focal holes (red arrows) were observed in the enamel surface of definite WSLs (original magnification x 3500; bar = 5 μm).

Figure 32.

SEM of affected and unaffected areas. Broadening of interprismatic spaces (green arrows) was commonly observed in demineralized areas. Mineral dissolution is more advanced in prism cores (red arrows) compared to prism borders (blue arrows). Apparently, the intact areas (orange arrow) show relatively smooth surfaces (original magnification x 2000; bar = 1o μm).

Figure 33.

SEM of demineralized crystals. At higher magnification (x 7500; bar = 1 μm), the lesional surface shows loosely packed globular crystals (red arrows) with a few areas of the residual smooth enamel surface (blue arrows) of non-affected enamel. These non-affected areas show no globular crystals.

11. The interaction between enamel and fluoride in the oral environment

11.1 Formation of fluorapatite and calcium fluoride

Most toothpaste products available in stores typically contain 1000–1500 ppmF, typically as sodium fluoride (NaF) or sodium monofluorophosphate (MFP) [105]. Generally, the concentration of fluoride in the saliva is around 0.01 ppmF [106]. After using a fluoride product, the remaining fluoride in the mouth gets diluted by saliva. Salivary fluoride is distributed across various compartments in the oral cavity. Fluoride ions can be found in both saliva and dental plaque fluid. Fluorides in bound forms are present in dental plaque, calcium fluoride, enamel, and oral mucosa. Importantly, fluorides adsorbed onto the oral mucosal surface act as a significant reservoir for the slow release of fluoride. Fluoride that is swallowed is partially deposited in bones; however, it is not released back into the oral cavity (Figure 34) [107].

Figure 34.

The fate of fluoride after using a fluoride product. Fluoride in saliva is exchanged with tooth, plaque, and mucosa. Fluoride is loss from saliva by swallowing and splitting (Adapted from Ref. [107]).

Fluoride presented in the oral fluid could interact with enamel HAP as follows [108]:

1. Ionic exchange reaction between F and OH in HAP

   $${\mathrm{Ca}}_{10}{\left({\mathrm{PO}}_4\right)}_6{\left(\mathrm{OH}\right)}_2+{\text{2F}}^{-}\to {\mathrm{Ca}}_{10}{\left({\mathrm{PO}}_4\right)}_6{\mathrm{F}}_2+{\text{2OH}}^{-}$$E1

2. FAP formation from supersaturated solutions:

   $$10{\mathrm{Ca}}^{2+}+{{\text{6PO}}_4}^{3-}+{\text{2F}}^{-}\to {\mathrm{Ca}}_{10}{\left({\mathrm{PO}}_4\right)}_6{\mathrm{F}}_2$$E2

3. HAP dissolution and CaF₂ formation

   $${\mathrm{Ca}}_{10}{\left({\mathrm{PO}}_4\right)}_6{\left(\mathrm{OH}\right)}_2+20{\mathrm{F}}^{-}\to 10{\mathrm{CaF}}_2+{{\text{6PO}}_4}^{3-}+{\text{2OH}}^{-}$$E3

The first and second reactions occur when the fluoride concentration in the oral fluid is low (typically between 0.01 and 10 ppmF). These reactions lead to the formation of “firmly bound fluoride” as fluoride integrates into the HAP lattice. Fluoride fits snugly into the HAP crystal structure, even better than the hydroxyl group, resulting in lower solubility of the new compound namely FAP compared to HAP. The critical pH (4.5) for densely packed crystals like FAP is slightly lower than the critical pH (5.5) for original HAP. However, in a cariogenic environment, plaque pH levels may drop to 4.5 or lower, and this reduced solubility is insufficient to prevent tooth demineralization [90, 108, 109, 110, 111]. Furthermore, studies have demonstrated the occurrence of caries in vitro [112] and in vivo [113] in shark enameloid, which consists mostly of FAP, even with complete substitution of F for OH in FAP. This supports the notion that a continuous supply of fluoride in oral fluids surrounding tooth enamel during the caries process is more crucial than a high fluoride content in the enamel [114, 115].

The third reaction reveals that in the oral environment with high fluoride concentrations (ranging from 100 to 10,000 ppmF), significant amounts of calcium fluoride (CaF₂) or CaF₂-like material, rather than FAP, are the predominant products. These high fluoride concentrations are present in topical fluoride treatments, including professional gels and varnishes, fluoride toothpastes, and fluoride mouthwashes [110, 116, 117]. Calcium fluoride formation involves a two-step process. Firstly, calcium ions dissolve from the enamel. Secondly, the dissolved calcium ions bind with applied fluoride, resulting in the formation of calcium fluoride globules [110]. These globules form not only on intact enamel surfaces but also on dental biofilm, acquired salivary pellicle, and demineralized surfaces. The pertinent chemical reactions are depicted in Figure 35. In summary, calcium fluoride acts as a pH-controlled fluoride and calcium reservoir that can release free fluoride into the fluids surrounding the tooth, thereby promoting remineralization and hindering demineralization [109, 118].

Figure 35.

CaF₂ and FAP formation in the oral cavity. FAP was formed during long-term exposure to low fluoride levels (<100 ppmF) in the oral fluid. However, CaF₂ is the major or probably the only reaction product on dental hard tissues from short treatments with highly concentrated fluoride (≥ 100 ppmF) agents. Therefore, calcium fluoride acts as a pH-controlled fluoride (and calcium) reservoir (Adapted from Ref. [109]).

In summary, while pure CaF₂ dissolves readily in water, it does not exist in the oral cavity due to being coated with HPO₄²⁻, proteins, and other saliva-derived substances. This coating enhances the stability of CaF₂-like materials by forming a protective film that inhibits solubility. When subjected to acid attacks like those from caries, this protective layer dissolves, releasing calcium and fluoride. Therefore, CaF₂-like materials serve as a reservoir for fluoride, releasing it at low pH levels during acid attacks and maintaining stability on tooth surfaces for longer periods at neutral pH levels (Figure 36) [90, 119].

Figure 36.

Formation and decomposition of calcium fluoride-like materials. The calcium fluoride is stabilized by surface adsorption of HPO₄²⁻ and/or also proteins from saliva. This phase can dissolve at pH ≤ 5 and provide fluoride and calcium ions (Adapted from Ref. [90]).

11.2 Cariostatic mechanisms of fluoride

Currently, it is widely acknowledged that fluorides incorporated into enamel have minimal impact on its solubility. Conversely, small concentrations of fluoride in the vicinity of the tooth exhibit a more potent ability to prevent demineralization compared to fluorides incorporated within the enamel. In such scenarios, fluoride ions partially adsorb to the surface of HAP crystals and maintain a dynamic equilibrium with fluoride ions present in the surrounding fluid. This results in an equilibrium or oversaturation concerning FAP. Adsorbed fluoride ions can attract free calcium ions, primarily sourced from the Stern layer. As a result, these calcium ions bind to available phosphate ions (Figure 37), leading to the creation of a remineralized layer akin to FAP on the crystal surface. This veneered surface becomes considerably less soluble than its original state. Importantly, fluoride often facilitates the redeposition of calcium and phosphate ions onto existing crystal remnants rather than the formation of new crystals (Figure 38). Additionally, adsorbed fluorides on the HAP crystals are thought to directly shield against demineralization [107, 120]. Due to their significant electronegativity, fluoride ions not only aid in remineralization, as discussed, but can also bind to H⁺ ions, thereby inhibiting plaque acidity and enamel demineralization [20].

Figure 37.

Enhancement effect of fluoride on calcium phosphate precipitation. Initially, fluoride ions adsorbed to the enamel surface. Then, adsorbed fluoride ions can attract free calcium ions, primarily sourced from the Stern layer. These calcium ions bind to available phosphate ions, leading to the creation of remineralization (Modified from Ref. [120]).

Figure 38.

Schematic representation of demineralization followed by FAP formation. First, fluoride ions adsorbed to the demineralized apatite crystal surface. Consequently, adsorbed fluoride ions attract calcium ions, which attract phosphate ions later, starting to form a FAP-like remineralized veneer on the demineralized crystal surface (Modified from Ref. [107]).

12. Dental erosion

12.1 Chemical aspects of erosion

Enamel, which typically withstands exposure to dietary acids, becomes susceptible to dental erosion when subjected to frequent and extended acid exposure. This erosion results from the interaction between tooth surfaces and acids or chelating agents. Primarily, this phenomenon occurs in the absence of plaque [121]. When HAP interacts with erosive solutions, tooth minerals dissolve following the reaction:

$${\mathrm{Ca}}_{10}{\left({\mathrm{PO}}_4\right)}_6{\left(\mathrm{OH}\right)}_2+\left(2+3x+2y+z\right){\mathrm{H}}^{+}\to 10{\mathrm{Ca}}^{2+}+x{\mathrm{H}}_3{\mathrm{PO}}_4+y{\mathrm{H}}_2{{\mathrm{PO}}_4}^{-}+z{{\mathrm{HPO}}_4}^{2-}+{\text{2H}}_2\mathrm{O}.$$

$$\left(x+y+z=6\right)$$

During erosive challenges, hydrogen ions originating from dietary acids initially target the minerals found in HAP, such as phosphate, hydroxyl, and carbonate ions. This process causes the dissolution of HAP crystallites, releasing calcium ions. Apart from the acid assault resulting from dietary acid exposure, dental erosion can also occur due to the formation of complexes between anions from chelating agents and calcium ions within HAP. For instance, in the citrate ion, two or three COOH groups may have lost their protons, leading to negatively charged sites that attract calcium ions within HAP. Consequently, this results in the dissolution of calcium from the crystal surface [122, 123, 124].

The acquired salivary pellicle is a semipermeable membrane [42, 125]. The outer globular layer is the first line of defense against erosive demineralization [126]. Both electron-dense basal and outer globular layers of the pellicle act as a physical barrier to inhibit direct contact between the enamel surface and erosive acids [54, 127]. The semipermeable membrane of an acquired salivary pellicle can delay interaction between erosive acids and the enamel surface [42, 128]. Moreover, the acquired salivary pellicle can modify the transportation of calcium and phosphate ions out and proton ions in at the enamel surface during acid challenge [128, 129]. Therefore, the acid diffusion and enamel dissolution rate are delayed [130]. If the erosive challenge continues, the erosive acids can damage the acquired salivary pellicle and enamel surface [52]. Consequently, proton ions from erosive acids can diffuse through the pellicle membrane [42]. Meanwhile, calcium and phosphate ions at the tooth surface move into the surrounding area [54, 128].

The adsorbed salivary proteins in acquired salivary pellicles, such as PRPs, histatins, carbonic anhydrase, and statherin, have a buffer capacity property [42, 45]. Some salivary proteins have desorbed from the enamel surface to the oral cavity during the erosive challenge for neutralizing erosive acids [131]. Furthermore, these adsorbed salivary proteins, especially statherin, could maintain the level of calcium ions by binding calcium domains of the enamel surface [45, 50]. The acquired salivary pellicle is gradually dissolved after exposure to erosive acids from the outer globular layer to the electron-dense basal layer [42]. Consequently, the thickness of the acquired salivary pellicle is considerably decreased [52]. Therefore, the acquired salivary pellicle acts as physical and chemical protection on the enamel surface against dental erosion [42, 126]. However, the acquired salivary pellicle cannot protect the tooth surface when faced with severe erosive acidity or prolonged exposure time [42].

The acquired salivary pellicle exhibits partial damage after erosive demineralization [56]. The outer globular layer of the acquired salivary pellicle is widely dissolved [52]. Meanwhile, the electron-dense basal layer is partially eroded [52]. The acquired salivary pellicle would be reestablished immediately after erosive demineralization by adsorbed salivary proteins in the oral cavity again [131]. After erosive demineralization, the ultrastructure of acquired salivary pellicle-coated enamel appears noticeably superficially dislodged with localized eroded areas and partially dissolved enamel crystallites [49]. The consequences of enamel erosion are calcium and phosphate loss, surface roughness, and surface microhardness loss in enamel [132]. The preventive effect of the acquired salivary pellicle depends on the maturation stage, the thickness and compositions of the acquired salivary pellicle, the concentration of erosive acids, and the exposure time of erosive acids [43, 130, 131, 133].

12.2 Histological features of erosive enamel

Between pH levels of 4.5–5.5, HAP is prone to dissolution, potentially leading to the formation of FAP and the initiation of early carious lesions. Conversely, when the pH drops below the critical value for FAP (pH ≤ 4.5), both HAP and FAP may dissolve, resulting in dental erosion. Unlike the process of initial caries, where demineralization creates a subsurface lesion with a relatively intact surface initially, dental erosion predominantly impacts the surface layer (Figure 39) [86, 121, 134, 135]. Ultrastructure of the naturally eroded enamel showed small, shallow, irregular depressions with a characteristic honeycomb-like or pitted appearance (Figure 40A). At higher magnification, small globules occurring singly or as aggregates were seen in erosive areas (Figure 40B) [136]. These appearances were also found in the experimental erosive enamel. After 7–15 days of immersion in the cola-type soft drink (pH 2.35), there was an accentuated demineralization of the entire enamel surface, being more pronounced on the interprismatic portion [137].

Figure 39.

Schematic illustration of the difference between caries and erosion lesions. At pH levels between 4.5 and 5.5, HAP tends to dissolve, whereas FAP will be formed. Thus, the subsurface lesion is observed in the initial carious lesion. In contrast, surface-softened lesions (erosion) occur when the pH level is lower than 4.5 due to the dissolution of both HAP and FAP (Adapted from Ref. [134]).

Figure 40.

SEM of naturally eroded enamel at the initial stage. (A) The eroded area shows a rough and irregular surface in association with a honeycomb-like surface (red arrow) (original magnification x 1000; bar = 10 μm). (B) At higher magnification, the eroded enamel shows loosely packed globular crystals (green arrow), whereas the adjacent normal-appearing enamel surface consists of densely packed globular crystals (yellow arrow). Original magnification x 5000; bar = 5 μm.

13. Concluding remarks

Enamel’s structural integrity relies on a meticulously organized hierarchical arrangement, spanning various dimensions, from the atomic level with HAP, to nanoscale crystallites, to microscale enamel rods, and ultimately to the tooth’s entire surface. While HAP is composed of 10 calcium ions, 6 phosphate ions, and 2 hydroxyl ions [Ca₁₀(PO₄)₆(OH)₂], enamel deviates from stoichiometric HAP due to deficiencies in primary constituents such as Ca²⁺, PO₄³⁻, and OH⁻, and the inclusion of ions such as CO₃²⁻, HPO₄²⁻, and trace elements. Under normal conditions, the tooth surface is exposed to saliva, oral bacteria, fluoride from oral products, and dietary acids. Upon contact with saliva, tooth surfaces adsorb salivary proteins, forming an acquired salivary pellicle. The oral cavity hosts diverse oral microorganisms, forming biofilms on dental and mucosal surfaces. Salivary components within the acquired salivary pellicle play a pivotal role in initial bacterial adherence. While dental biofilms contribute to host well-being, disruptions in this relationship can lead to oral diseases such as dental caries and periodontal diseases. According to the Ecological Plaque Hypothesis, dental caries result from microbiota imbalance due to ecological stress, leading to an abundance of cariogenic bacteria. In cariogenic conditions, reduced concentrations of OH⁻ and PO₄³⁻ in plaque fluids, and potentially saliva, cause the Ip in plaque fluids to fall below the Ksp of HAP, initiating enamel dissolution. However, it is crucial to recognize that the caries process involves cycles of demineralization and remineralization. Initial carious lesions manifest as subsurface lesions with a clinical appearance resembling white spot lesions. SEM of initial caries exhibits accentuation of the perikymata, deep focal holes, and broadening of interprismatic spaces. Mineral dissolution was markedly observed at prism cores, compared to prism borders.

Currently, it is widely acknowledged that CaF₂ is vital in caries prevention. Formation occurs when fluoride concentration around enamel surpasses 100 ppmF. However, the ability to form calcium fluoride-like materials during brief intraoral exposure is limited, and these compounds are swiftly lost after using fluoride-containing oral products. The tooth surface may be exposed to acidic diets, with prolonged exposure leading to dental erosion. Erosion involves the chemical loss of dental hard tissue, unrelated to bacterial action, induced by hydrogen ions and/or chelating agents. It typically occurs when fluid pH around the tooth surface drops below 4.5, causing FAP and HAP dissolution under acidic conditions. Additionally, several proteins in saliva and the acquired salivary pellicle may offer protection against dental erosion.

Acknowledgments

The authors would like to thank Christian Estacio, International College of Dentistry, Walailak University, for critically reading the text.