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© Woodhead Publishing Limited, 2013

20

2Biomineralization and biomimicry

of tooth enamel

V U K U S KO KOV I C, University of California, USA

DOI: 10.1533/9780857096432.1.20

Abstract: This critical review sommarizes the basics of biomineralization of tooth enamel and scrutinizes attempts to replicate this intricate biological process in vitro. Special emphasis is given to the author’s results, obtained during studies on the formation of enamel by biomimetic means. Fundamental insights found regarding the latter process are presented. Some paradigmatically accepted aspects of the mechanism of amelogenesis, that is, biomineralization of enamel, are challenged. Amelogenin, the major protein of the developing enamel matrix, is thus claimed to be a mineralization inductor, rather than an inhibitor, presumably acting as a channel between the ionic growth units in the protein matrix and the uniaxially growing crystals of apatite. The role of water and other minor constituents of enamel is questioned, as well as the biologically active morphology of amelogenin aggregates and the reliability of recombinant proteins in studying amelogenesis in vitro. Appropriate crystal growth rates, the Ostwald–Lussac law, Tomes’ process and mineralization of dentin present other aspects of amelogenesis discussed here. It is also claimed that three fundamental facets of amelogenesis ought to be coordinated in parallel for successful biomimetic replication of the given process in the laboratory: protein assembly, proteolytic digestion and crystal growth.

Key words: amelogenesis, biomimicry, biomineralization, enamel, self-assembly.

2.1 Introduction

Tooth enamel presents a prototype of a miniscule and yet extraordinarily intricate segment of the vertebrate body, research into which bears poten-tial signifi cance not only for dental science and orofacial therapies, but for understanding the essence of biomineralization processes per se. This explains why it attracts the attention of scientists from a wide array of fi elds. Not only is the complexity of the formation of this tissue such that its understanding and thorough investigation require knowledge of various materials science and life science fi elds, but insights obtained by research open trajectories for numerous other biomaterial- and biomedicine-related areas of knowledge. In addition to these fundamental merits, understanding biomineralization of tooth enamel carries an important medical signifi -cance. For, fi nding soft chemical ways to disinfect and remineralize the

Biomineralization and biomimicry of tooth enamel 21


diseased enamel is the fi rst step in ensuring less invasive and more biocom-patible ways of treating enamel dissolution that occurs by attacks by cario-genic bacteria.

This chapter starts with a description of biomineralization of tooth enamel, followed by questions that touch upon some currently disputable or plainly unknown details about this biomineralization. In this way, impor-tant questions will be raised around which future research in this fi eld will be based.

2.2 Structure of enamel

Tooth enamel is crowned in the realm of biominerals as the hardest of its members. Another of its peculiar attributes is that it is the only epithelium-derived mineralized tissue. It is also the only biomineral among vertebrates to be almost fully deprived of soft organic components, as 96–98 wt% is accounted for by mineral content only. Despite its almost purely mineral composition, tooth enamel is unlike typical ceramics, as it is typifi ed by an exceptional toughness and only moderate brittleness, all owing to its extraordinarily complex microstructure. Namely, enamel is composed of apatite fi bers, 40–60 nm wide and up to several hundred micrometers long, assembled in bundles, that is, rod-shaped aggregates 4–8 μm in width (Fig. 2.1). Having length-to-width aspect ratios of up to 3 × 104, apatite crystals in enamel are 1000 times longer than those found in bone (50 × 20 × 3 nm on average). This is made possible since the maintenance of this tissue does not depend on intrinsic cell proliferation and vascularization, as is the case with collagenous hard tissues that comprise bone.1 Approximately 1000 apatite fi bers are bundled within each enamel rod, 5–12 million of which are found on a single tooth crown, lined up parallel to each other. The long axis of the enamel rod is, within each row, generally perpendicular to the

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2.1 Histological section of the developing human tooth in the maturation stage (a) and a micrograph showing parallel arrangement of enamel rods (b). 1, ameloblasts; 2, enamel; 3, dentin; 4, odontoblasts; 5, pulp.

22 Non-metallic biomaterials for tooth repair and replacement


underlying dentin, the only exception being that enamel rods near the cement–enamel junction (CEJ) in permanent teeth tilt slightly toward the root of the tooth.

2.3 Amelogenesis at the molecular scale

The biological formation of enamel is known as amelogenesis, a process that lasts for about four years at an appositional crystal growth rate of about 4 μm per day in humans. The slow timescale of the process, making it lengthier than embryogenesis, implicitly speaks in favor of its extraordinary complexity. Besides specifi c cells, ameloblasts, the process engages numer-ous macromolecular species, divisible into families of proteins, proteases and protease inhibitors. Although the chronology of amelogenesis is typi-cally divided into three stages – the secretory, the processing and the matu-ration stage – in view of the pronounced overlap of these stages at the molecular scale, the correctness of this classifi cation can be questioned. For example, the key components involved in protein assembly are secreted during the processing stage, while no precise boundary has been established between the end of processing and the beginning of maturation. Also, although maturation stage can be thought of being the one during which the grown crystals are merely refi ned, concentration of the mineral phase in the developing enamel is estimated to increase in this stage from 15–20 % to its fi nal percentage.2 Two-thirds of the time spent in the process of amelogenesis thus belongs to the maturation stage rather than to the processing one. Therefore, the following three events, taking place in paral-lel during amelogenesis, may be said to describe this process more accu-rately at the nanoscale.

2.3.1 Protein expression, secretion and assembly

The process of amelogenesis can be said to begin with ameloblasts express-ing and secreting proteins that make up the enamel matrix, 90% of which are composed of a single protein, amelogenin. The remaining 10% consists of other proteins: ameloblastin, enamelin, serum albumin (not expressed by ameloblasts and thought to arrive by diffusion through the extracellular matrix (ECM) from the adjacent soft tissues), amelotin and proteolytic enzymes. Together, they assemble into a scaffold that acts as a template for the growth of uniaxially oriented apatite crystals.

2.3.2 Nucleation and crystal growth

Proteins involved in amelogenesis are typically divided to predomi-nantly hydrophilic and predominantly hydrophobic ones. The epithet



‘predominantly’ is attached here to both kinds of species because, strictly speaking, there are neither perfectly hydrophilic nor perfectly hydrophobic proteins. They are both hydrophobic and hydrophilic in their molecular nature; if this were not so, protein globules would either swiftly unfold had they been completely hydrophilic or wind down into dysfunctional clumps had they been fully hydrophobic. Still, some proteins involved in amelogen-esis, including enamelin and ameloblastin, can be considered comparatively hydrophilic in nature; as such, they are supposed to act as nucleation sites for crystallization of apatite.3,4 This hypothesis is supported by the fact that enamelin is expressed only in the secretory stage, whereas its expression is halted during the maturation stage.5 Initial enamel crystals also nucleate along the dentin–enamel junction (DEJ) at an early time point when amelo-genin is hardly present at all in the protein matrix, which is mainly com-posed of enamelin and ameloblastin. On the other hand, amelogenin, a low-molecular weight protein, the full-length isoform of which contains between 160 and 200 amino acids, depending on the species in question, is comparatively hydrophobic and has been assumed to inhibit apatite growth.6 It contains only one phosphorylated site (16Ser), which makes it different from the highly phosphorylated matrix macromolecules that control biomineralization in bone and dentin or the acidic glycoproteins of mollusk shells.7 It also contains a short, 12-carboxyl-terminal residue sequence of hydrophilic amino acids at the C-terminal (Fig. 2.2), which makes its molec-ular structure arguably amphiphilic.

Of course, as shown in Fig. 2.2, although amelogenin, usually endowed with the epithet ‘hydrophobic’, is indeed more hydrophobic than most proteins, it is more hydrophilic than human hemoglobin alpha chain, for example. Still, owing to a large content of proline residues (25–30% of all the amino acids in the peptide chain of amelogenin) as well as those of histidine, glutamine and leucine, amelogenin is considerably hydrophobic, which explains its tendency to form aggregates in contact with a polar solvent even at extremely low concentrations (<0.01 mg ml−1). For the very same reason, attempts to crystallize amelogenin molecules have failed owing to their resistance to standing at a periodic distance from each other, leaving their secondary structure still in the domain of the unknown. Nevertheless, the current model of crystal growth during enamel formation presupposes that amelogenin proteins self-assemble into poly-disperse nanospheres ∼20 nm in size (Fig. 2.3) (comprising about 40–60 molecules per spherical aggregate of this size), which then align into beaded strings and adhere onto the (hk0) faces of the apatite crystals, promoting their growth in the direction of the crystallographic [001] axis. As such, they are hypothesized to prevent the growth and fusion of crys-tals perpendicular thereto, while aligning them approximately parallel to each other.



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2.2 Hydrophobicity plots obtained using ExPASy ProtScale Kyte & Doolittle model (window size = 9; linear weight variation model) for human amelogenin (solid line) and human hemoglobin alpha chain (dashed line). The positive score on the diagram denotes hydrophobic sequences.

= Amelogenin nanosphere

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2.3 Schematic depiction of crystal growth during amelogenesis, at least according to the current questionable popular paradigm. The schematic structure of amelogenin nanosphere was adapted from Snead.8



2.3.3 Proteolysis

The action of proteases, including matrix metalloproteinase-20 (MMP-20, also known as enamelysin), enamel matrix serine protease 1 (EMSP1, also known as kallikrein-4), and cathepsin B, in hydrolysis of amelogenins and other ECM proteins presents a crucial segment of amelogenesis.9 Owing to its high selectivity of the cleaved peptide bonds, MMP-20 is usually considered as the major protease in this process.10 By controlling the modu-lation of full-length proteins, it is supposed to act as a regulator that controls the functionality of amelogenins. Since enamel is a 95 wt% mineralized tissue, it is clear that proteolytic degradation and removal of the protein matrix has to be orchestrated in synchrony with the lateral growth of apatite crystals.

Following proteolysis, amelogenins and ameloblastins are removed from mature enamel, leaving behind predominantly enamelins and tuftelin in trace amounts. Before the eruption of the tooth, but after the maturation stage, ameloblasts are also broken down and, consequently, enamel, unlike other tissue in the body (even dentin is able to partially remineralize itself, as the pulp cells form layers of reparative dentin whenever bacterial deg-radation of teeth reaches the pulp), has no way to regenerate itself ‘from the inside’.

Finally, there is increasing evidence that these three aspects of amelogen-esis – crystal growth, proteolysis and protein assembly – mutually affect each other, so that understanding any one of them individually is condi-tioned by the simultaneous understanding of the other two. Cooperative assembly of macromolecular species and crystal formation, probably fi rst proposed by Eastoe in 1963,11 rather than hierarchical and sequential con-ductance of crystal growth by a pre-assembled protein matrix is thus increasingly used as the hypothesis for describing amelogenesis at the molecular scale.12–14 Henceforth, successive imitation of enamel growth in vitro can be imagined as a peak of a pyramid fi rmly based in the knowledge of all three given aspects of amelogenesis (Fig. 2.4).

2.4 Key issues in biomineralization and biomimicry

of tooth enamel

2.4.1 Are the minor amounts of lipids, proteins and water accidentally remnant in enamel or structurally incorporated with a mechanical purpose?

The old school of thinking tends to consider the miniscule amounts of organic matter in enamel as mere non-functional impurities; however, some research groups are beginning to treat enamel as a composite ceramic



Enamel in vitro

CrystallizationProtein

self-assembly

Proteolysis

2.4 Biomimicry of amelogenesis is based on and utilizes three well understood and essential aspects of the process: protein self-assembly, proteolysis and crystallization.

material despite its low content of organic matter,15 claiming that entrap-ment of such a small concentration of macromolecules increases the tough-ness and strength of what would be an otherwise brittle ceramic material enamel without them.16 The biological material often referred to support this argument is the spine of sea urchin, which contains only 0.02 wt% of glycoprotein (∼10 proteins per 106 unit cells). The amount is, however, large enough to absorb energy effi ciently from propagating cracks and thus mark-edly enhances the resistance of the material to fracture.17

2.4.2 Is amelogenin acting as an inhibitor or promoter of nucleation, or both?

As shown in Fig. 2.5(a), recombinant human amelogenin is quite effi cient in decreasing the nucleation lag time of metastable calcium phosphate solu-tions in direct proportion to its concentration. A set of biomimetic experi-ments based on slow titration of amelogenin sols with calcium and phosphate ions has also yielded conditions under which no crystal growth of apatite substrates was observed in the absence of amelogenin (Fig. 2.5(c),(d)), while the presence of amelogenin accelerated crystal formation in direct propor-tion to its concentration (Fig. 2.5(b)).18 The results thus confi rmed that the substrate-specifi c growth of apatite is conditioned by adsorption of amelo-genin on the growing crystal surface.19 Both of these insights have clearly suggested that amelogenins can act as effective nucleators for precipitation of calcium phosphates. In view of this, other recent reports on the ability of amelogenin to promote nucleation of apatite are not surprising.14 Tarasev-ich et al. have shown that the nucleation promoting/inhibiting effect of amelogenin largely depends on its concentration,20 confi rming a well-known fact that additives may often exert diametrically opposite effects depending



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2.5 (a) Nucleation lag time decaying in proportion to the concentration of rH174, human recombinant amelogenin obtained from Escherichia coli, in the concentration range 0–0.84 mg ml−1. (b) Crystal growth increasing in proportion to the concentration of rH174 as a function of titration volume. (c) Crystal formation absent from apatite/glass substrates at zero amelogenin concentration. (d) Plate-shaped calcium phosphate crystals obtained in the presence of amelogenin from metastable calcium phosphate solutions. Partially adapted from Uskokovic et al.18 and reprinted with permission from Elsevier.

on their concentration.21 The crucial question today is no longer whether amelogenin can promote nucleation of calcium phosphates, but whether it can act both as a nucleator and inhibitor of nucleation depending on its conformation, morphology, concentration and cooperative assembly involv-ing other protein species present in the developing enamel matrix.

By showing that adsorption of amelogenin is the fi rst step in inducing controlled crystal growth we confi rm the idea that evidence of adsorption does not necessarily imply protein–mineral interactions that hinder the crystal growth on the binding sites. For example, osteocalcin, one of the proteins involved in mineralization of bone, despite aligning with and binding to some of the growing crystal planes, does not constrain crystal



growth along these directions.22 Protein assemblies adsorbed on crystal surfaces were thus proposed to act as channels or bridges that transfer ions from the solution and promote their anchoring onto the growing faces. By reversing the old paradigm which has stated that the role of amelogenin assemblies is to block the approach of ions to the growing crystals, we have often joked that we have literally torn down the walls of the old way of thinking and transformed its steely gates into wonderful bridges, bringing forth a more inspiring picture of the way Nature crafts its materials (Fig. 2.6).

2.4.3 What is the biologically active morphology of amelogenin aggregates?

Unlike non-polar acetonitrile, where amelogenin can exist as a monomer within a certain concentration window,24 in water, the medium of biological

Enamel rod

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2.6 Scheme describing a model of the protein-controlled crystal growth in amelogenesis. Amelogenin assemblies, such as nanospheres or nanofi bers, are adsorbed on the surface of apatite crystals where they channel their building blocks of either calcium and phosphate ions from the solution or amorphous calcium phosphate entities. These amorphous units may form by precise coordination of Ca2+ bound to the protein and phosphate ions diffusing in the hydrodynamic layer of the protein particles. Reprinted with permission from Uskokovic.23



importance, amelogenin readily aggregates and adopts one of two different morphologies: nanospheres or nanofi bers of various length (Fig. 2.7). The latter have been shown to form owing to controlled aggregation of nano-spheres as primary subunits.25 However, observations of spherically shaped nanosized amelogenin aggregates in vivo have been far from convincing and questions regarding the biologically active morphology of amelogenin assemblies lie still unanswered. Biological molecules, especially the lengthy ones, such as proteins, comprise many active points on their surface which can engage in weak chemical interactions that are, in turn, responsible for their assembly into exciting morphological units.26 However, without dem-onstrating the ability of these supramolecular symmetries to act in a biologi-cally functional manner, their relevance within in vivo contexts is predestined to remain only a hypothesis.

2.4.4 Can recombinant proteins be reliable models for studying amelogenesis in vitro?

The fi rst time amelogenin was expressed in vitro as a recombinant protein was in 1994.27 Since then, most studies using this protein have been carried out with its recombinant versions rather than those isolated from animals, typically porcine, mouse or bovine. However, the structural difference between recombinant amelogenins and the nascent ones expressed by ameloblasts, although seemingly minor, may pose far greater limitations than routinely thought. For example, amelogenins expressed by ameloblasts

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2.7 AFM images of recombinant full-length human amelogenin nanospheres 20–40 nm in size (right) and protofi brous nanostrings (left) formed by mixing amelogenin and water in the presence of calcium and phosphate ions.



are phosphorylated at one residue (16Ser), whereas the recombinant pro-teins expressed by E. coli do not comprise any post-translational modifi ca-tions. This causes concerns about the functional discrepancies that may occur owing to this slight structural variation, particularly in view of the fact that it is well known that phosphorylated groups are especially important in the formation of calcium phosphate minerals. The mineralization of dentin is, for example, directed by the phosphophoryn protein family typi-cally with numerous repeats of the sequences Asp-Ser(P)-Ser(P)- and Ser(P)-Asp.28 Studies have indicated that single-residue phosphorylation of amelogenins is absolutely crucial to ensure molecular conditions for the proper development of enamel.29,30 Moreover, recombinant amelogenins dispossess the fi rst residue at the N-terminal, which may also cause drastic differences in the mechanism of their folding.

Single-point mutations have been shown to lead to far greater implica-tions, both in vivo and in vitro, than is insinuated by the magnitude of these structural perturbations. Aside from innumerable cases wherein single-point mutations disrupt the functionality of proteins (e.g. substitution of valine with glutamic acid in the β-chain of hemoglobin resulting in sickle cell anemia), single-point mutations have been shown to modify the peptide self-assembly too.31 A single-point mutation in the amelogenin-coding gene thus resulted in a single amino acid substitution (Pro-41→Thr) in the primary structure of amelogenin and, hence, to a specifi c type of amelogen-esis imperfecta, related to severe dental enamel malformation.32,33 A similar single-point mutation (Pro-70→Thr) in recombinant full-length human amelogenin has been shown to result in signifi cantly lower rates of apatite growth compared to that observed in the presence of the wild-type,34 as demonstrated in Fig. 2.8. Moreover, mutations not only in amelogenin genes, but in those that encode MMP-20 cause amelogenesis imperfecta, a pathological state typifi ed by abnormal and signifi cantly weakened enamel.35,36 The mutation g.2142G>A on the gene coding for this KLK-4 has also been shown to cause abnormal enzymatic activity, resulting in enamel crystals of normal length but insuffi cient thickness.37

2.4.5 What is the role of proteolysis in crystal formation?

While numerous studies have been carried out to assess the specifi city of the proteolytic cleavage of amelogenins and other proteins in the enamel matrix and the morphogenetic effects of alterations in the native structures of these polypeptide reactants, the effects proteolysis exerts on crystal growth has not been tackled to a similar extent. As shown in Fig. 2.9, pro-teolytic digestion by means of MMP-20 can be suspected of playing an additional nucleation-promoting role compared to that played by amelo-genin per se, as mentioned earlier. Proteolysis has been shown to cleave the



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2.8 Signifi cantly lower average calcium phosphate crystal height deposited on fl uoroapatite substrates during titration of single-point Pro-70→Thr mutated human full-length recombinant amelogenin (P70T) in sol than that deposited in the presence of wild-type amelogenin (rH174), at different supersaturation ratios (a and b). Also, less specifi c crystal growth observed for mutated amelogenin (c) in comparison with the wild-type one (d). The size of both AMF micrographs is 10 × 10 mm. Partially adapted and reproduced with permission from Zhu et al.34

hydrophilic C-terminal of amelogenin and thus promote intensive aggrega-tion and ripening of its nanospheres, detectable as an increase in the particle size during dynamic light scattering analysis. Consequently, the protein increasingly deposits onto the growing crystals and, since it supposedly acts as a bridge that facilitates the fl ow of ionic growing units from the solution to the crystalline faces, crystal growth becomes kinetically favored. In this



way, MMP-20 can be said to lower the energy of activation required for heterogeneous nucleation of calcium phosphate and serve as a potent kinetic factor that infl uences the thermodynamics of apatite growth.38

2.4.6 What is the role of micro-ions and less abundant protein species?

The role of many ions present in the enamel matrix in minor but controlled amounts are currently only the subject of hypotheses. For example, a steady, although probably carefully monitored and modulated infl ow of zinc ions is known to exist owing to its role in regulating the activity of MMP-20. Fluoride is also present in natural enamel, where it has the effect of strength-ening the apatite lattice. Still, in overly large amounts it leads to a counter-effect: increased porosity and weakening of the enamel structure.39 It was also shown that increased levels of fl uoride in developing enamel decrease the activity of MMP-20,40 resulting in the condition known as fl uorosis. The role of fl uoride ions in promoting elongation of apatite crystals has, however,

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2.9 An increase in the average particle size of amelogenin following its proteolysis by MMP-20 (MMP-20/rH174 1 : 1000 weight ratio), starting at t = 0 (a). A moderate amount of crystal formation is observed during titration of amelogenin sols with calcium and phosphate ions (b) and a markedly greater amount of calcium phosphate precipitate forms under identical conditions when proteolytic reaction is coupled to the titration (c).



been well documented.41,42 In a set of experiments, only a combination of amelogenin and fl uoride led to formation of rod-like apatite crystals, while only octacalcium phosphate precipitated in the absence of fl uoride.43,44

The role of other ions, including magnesium and sodium, known to be present in the natural enamel, should not be underestimated, especially in view of their inhibiting effect on apatite growth rates. Since crystal growth rates and the aspect ratio of grown crystals often stand in inverse propor-tion, it is possible that these growth-inhibiting ions may exert a pivotal infl uence on the overall process. Finally, variations in pH may also be crucial, particularly in view of the specifi city of interaction between amelo-genin and apatite, depending on the surface charge sign and magnitude.45

For example, as shown in Fig. 2.10, the fact the particle size measured by means of dynamic light scattering after mixing hydroxyapatite and amelo-genin nanoparticles corresponds to that of their sum, while the resulting zeta potential is equal to the positive value of circa +5 mV, which amelo-genin possesses at the given pH, clearly suggests that adsorption of the protein onto the mineral surface proceeds momentarily when their surface charges carry opposite signs.45 The defi nitive role of pH changes is still not clear, although ameloblasts have been shown to modulate the pH of the enamel fl uid rhythmically. Therefore, covering the micro-distance from smooth to ruffl ed endings of the cells is accompanied by a local pH change in the surrounding enamel fl uid from nearly physiological (7.2–7.4) to slightly acidic (6.1–6.8) values.46

Reports on the role of enamel matrix proteins other than amelogenin in enamel formation have been relatively scarce to date.47 Still, even simple requirements that entail proper crystal growth, such as controlling and modulating pH, are known to depend on a plethora of protein species secreted by ameloblasts and transitorily present in the enamel matrix.48 The role of other protein species is still not even partially elucidated, especially not in synergy with other components of the developing enamel matrix. Studying these in parallel, however, introduces a lot more variables into the system than can be coped with on the timescale of a normal scientifi c project, especially considering the time-consuming kinetics required to replicate the naturally slow enamel-yielding reactions. However, signs still point to the essential role of these less abundant protein species in amelogenesis. For example, mutations on the enamelin-coding gene have been shown to result in severe phenotypic amelogenesis imperfecta.12 This highly acidic glycoprotein is observed to form a sheath around the growing apatite crystals (their c-axes being perpendicular to the β sheets of the sur-rounding enamelin), which is in compliance with the current model of extracellular mineralization. According to this model, hydrophobic pro-teins, such as amelogenin, collagen or cellulose, are assumed to be involved in the buildup of the insoluble macromolecular matrix of the developing



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2.10 (a) Average hydrodynamic diameter and ζ-potential of the colloidal mixture of HAP and rH174 at [rH174] = 0.16 mM when rH174 was abruptly added to the HAP sol comprising 150 mM KCl, 20 mM Tris/HCl, at 25°C and pH 4.50 +−0.02, and continuously measured over 11 h aging time: (b) Transmission electron micrograph demonstrating the adsorption of amelogenin onto hydroxyapatite nanoparticles Partially adapted and reprinted with permission from Uskokovic.45



hard tissue, whereas hydrophilic proteins are involved in attracting precur-sor ions and providing the nucleation surfaces.

Even though the low concentration of enamelin in the enamel matrix can be thought of as a sign of its low importance, that need not be necessarily so. For, there are many examples of macromolecular or amphiphilic addi-tives that exhibit a cooperative effect on the assembly of the precipitated phase at low concentrations only.6 The morphological specifi city, such as preferential adsorption of the additive molecules along specifi c planes of the crystalline phase, in these cases rapidly diminishes at higher concentra-tions. Ameloblastin is also presumed to carry out a signifi cant function, not only because of its localization at the secretory end of ameloblasts where the crystal growth is initiated, but because we know that both an elevated and hindered expression of ameloblastin results in amelogenesis imper-fecta.12 The roles of even less abundant components of the enamel matrix, such as KLK4, keratin K14, DLX3 or biglycan protein, the mutant expres-sions of which are also known to produce the conditions of amelogenesis imperfecta,49 have also not been investigated thoroughly.

2.4.7 Can artifi cial enamel be synthesized at comparatively high growth rates?

Low metastable levels of supersaturation appear to be crucial for providing the right conditions for protein-guided crystal growth.50 Low rates of nucle-ation and crystal growth naturally favor the formation of elongated crystals. In contrast, rapid mixing of reactants, yielding high initial concentrations of nuclei is a routine recipe for fabrication of ultrafi ne particles that are not to exceed a few nanometers in diameter. For example, when controlled degradation of urea is used slowly to increase alkalinity of the solution and provide conditions for precipitation, the apatite crystals formed turn out to be either plate-shaped or needle-shaped.51 Single-crystal apatite fi bers 20–60 μm in length and 100–300 nm in diameter were thus obtained by precipitation using decomposition of urea.52 Attempts to initiate nucleation and crystal growth at a higher rate by increasing the supersaturation ratio would presumably deprive amelogenins of their ability to direct the crystal-lization events.50

Eventually, it is possible that an informal conclusion will be arrived at, stating that without slow growth comparable to the extraordinary slowness of amelogenesis, artifi cial replicas of enamel-like structures could not be synthesized. Even if a satisfactory degree of morphological semblance is reached, structural differences at the fi ne scale may possibly be large enough to render the given material mechanically inferior to natural enamel.

One thing is certain though. Namely, the elongated morphology and highly crystalline nature of apatite in enamel in comparison to smaller,



nanosized and much less crystalline apatite particles formed in bone and dentin implies that different models of growth should be applicable in these two cases of biomineralization.23 Indeed, a model involving template-based catalyzed nucleation and limited growth by hydrophilic proteins, such as osteocalcin, which is valid for bone and dentin, can thus be claimed not to be applicable to enamel. Instead, a model based on (a) slow crystal growth, (b) gradual increase of supersaturation levels and (c) the role of amelogenin in channeling the growth units onto the growing apatite surface, is given here as an alternative to the standard models of biomineralization that depict nucleation events as taking place on foreign organic surfaces, gov-erned by their hydrophilic character and precisely matching lattice spacing, and crystal growth as proceeding while being inhibited by the adsorbing bioorganic particles.

2.4.8 To what extent does the Ostwald–Lussac rule apply to the mechanism of formation of apatite in enamel?

Crystallization of apatite under simple precipitation conditions in aqueous media is known to obey the Ostwald–Lussac rule, which dictates that pre-cipitation of apatite as the most stable calcium phosphate phase has to be preceded by precipitation of the less soluble phases formable at low tem-peratures and in hydrated local environments (which excludes oxide phases: tricalcium phosphate polymorphs and tetracalcium phosphate). This is illus-trated in Fig. 2.11: namely, a system undergoing a phase transition will tend

ΔG

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2.11 Free energy of a system undergoing phase transition through a multitude of transitory states.



to make multiple steps over transitory states separated from each other by a smaller energy barrier than the one posed between the initial and the fi nal state.

Consequently, the precipitation of apatite at pH >7 should follow the following route in the absence of kinetic factors capable of inducing tran-scendence in some or all the stages in the process:

amorphous calcium phosphate → monocalcium phosphate → dicalcium phosphate → octacalcium phosphate → hydroxyapatite

In general, the higher the pH, the more rapid the transition from one stage to another and the less is the probability of halting the precipitation at an intermediate stage and detecting one of these transitory phases. At suffi ciently high pH values, the solution will also be virtually undersaturated with respect to phases that dominate the low pH range, including monocal-cium phosphate and dicalcium phosphates (brushite and monetite), which implies that they are missing in this stepwise progression of the precipitate toward the crystal structure of apatite. Still, opinion among biomineraliza-tion experts widely varies in terms of whether ECM proteins and other biointerfaces can provide suffi ciently potent kinetic forces to enable transi-tion of hydrated solutes directly to the most stable biomineral phase, apatite, following the onset of precipitation. Ever since a dark octacalcium phos-phate line was found during a transmission election microscopy (TEM) analysis positioned in the center of enamel apatite fi bers,53 the belief that apatite is the phase that forms directly during amelogenesis has been inten-sively questioned. Since then, many studies have demonstrated that the amorphous calcium phosphate presents a transient phase in the course of amelogenesis.54,55

2.4.9 How crucial are the underlying Tomes’ process and the mineralization of dentin for proper amelogenesis?

As can be seen from Fig. 2.1(a), the growing dentin and enamel are adjacent to each other. Although odontoblasts responsible for directing the growth of dentin, coming from mesenchymal tissue, and ameloblasts responsible for directing the growth of enamel, coming from epithelial tissue, are thor-oughly separated after the mineral growth begins, these two types of cells are engaged in intensive communication prior to initiation of an almost simultaneous crystallization of dentin and enamel. Ameloblasts enter their fi rst formative state after the fi rst layer of dentin is formed, secreting the enamel matrix and at the same time retreating away from the DEJ, leaving the matrix to mineralize by itself.56 The question that remains is to what extent does this initial dentin surface exert an effect on the nature of enamel



growth. For, just as in many other aspects of life, the fi rst steps made during surface-specifi c growth greatly determine the direction which it will assume and, therefore, the nature and structure of the fi nal product. After all, the smoothness and stiffness of the surface, as well as the nature and density of chemical groups residing on it, all crucially determine its activity and potential to be the starting point of growth of a new phase. This is nicely illustrated in Fig. 2.12, which shows how fi brous amelogenin assemblies form strictly on the fl at silica glass surface and not on the rigged fl uoroapa-tite crystals embedded in the glassy matrix. Furthermore, the underlying Tomes’ process, during which ameloblasts retreat from the growing fi bers and toward the epithelium, may be crucial in aligning the apatite fi bers, bundling them up within prisms composed of rod and inter-rod enamel and preventing their random tilting in space.

2.4.10 Do biomimetic studies present a reliable approximation of the biological process?

Having been played around with, DNA molecules were assembled into an array of attractive morphologies, from cubes to triangles to pentagons to hexagons and octahedrons.57 Yet, none of those probably have any signifi -cance for the biological domain, at least not in the evolutionary terms. Similarly, macrophage peptides have been intensively utilized to produce exciting material structures;58,59 still, this also cannot be taken as a sign that

500 nm

2.12 AFM image of fi brous amelogenin assemblies forming only on the fl at glassy surface and not on the apatite crystal.



somewhere in Nature they indeed act along these lines. As already pointed out, a high potential for molecular recognition predisposes organic mole-cules and particularly polypeptides to assemble into an endless variety of morphologies depending on the environmental conditions to which they are naturally structurally sensitive.60 This versatility of interactions is responsible for a variety of mutually contradictory conclusions, that abound in literature reports on attempts to replicate amelogenesis in vitro. For, insights derived under one set of conditions may easily turn out to be at odds with those inferred from another seemingly similar, but in reality drastically different set of conditions. If we add an immense sensitivity to the slightest changes in boundary conditions on the evolution of these systems in time, we come to an awareness of a yet deeper gap that should prevent experimentalists from coming up with omnipotent conclusions from their studies. Strictly speaking, what is observed in a simplifi ed biomi-metic set of conditions may present only an indication that similar struc-tures and mechanisms may occur in vivo as well.

Just as the absence of evidence is in no way equal to evidence of absence, so the evidence of an interesting effect in the laboratory setting does not imply that this effect is evidence of the principle investigated by labo-ratory mimicry. Whatever the insights obtained from biomimetic studies, precautionary measures ought to be taken against deriving premature and grandiose conclusions from them. For, what appears to be immanent in one context need not be as naturally derived from another. Some of the greatest blunders in science came not from cleverly framed propositions, but from pretentious broadening of their validity far beyond the scope proposed by their originators. A most striking example comes from Darwin’s theory of evolution and the concept of ‘survival of the fi ttest’ which he proposed in his Origins. Namely, even though he mentioned that it ought to be grasped in a ‘broad and metaphorical sense, so that it implies an interdependence of one creature on others, including not only individual lives, but the entire progeny’,61 the followers of his teaching should be blamed for irrational exaggeration and overly literal interpreta-tion of this phrase, which nowadays holds a quite different casual connota-tion from that of which Darwin conceived.62,63 Although this example may seem remote from the realm of dental science, it may not be so. For, when-ever an intriguing discovery is glimpsed, it takes considerable modesty to keep on claiming its validity only under the experimental settings rep-licated in the laboratory rather than in the biological circumstances mimicked.

This leads to another important question, on which the research fate of many biomimeticians depends. It is whether in vitro remineralization of enamel, the precursor of soft and non-invasive clinical therapy, can be accomplished in a cell-deprived manner. Or, as Sherlock Holmes would



have phrased it, ‘We cannot imitate Nature, we can only recreate it as a whole’. Future insights in this fi eld of research will certainly depend on how biomimeticians come to terms with this holistic saying of one of the world’s dearest detectives.

2.5 Conclusion

The general feeling is that only a tip of an iceberg has been glimpsed in our understanding of the fascinating biomineralization process called amelogenesis. Despite the sense of confi dence in what has already been verifi ed or merely theorized as certain in the circle of ‘enamel growers’, digging deeper into the nature of their fi ndings gives a sense that much more has been hypothesized than truly confi rmed. Hypotheses are cer-tainly not lacking in this fi eld since almost every idea that can occur can be traced back to a hypothesis already proposed. However, inventiveness in designing experimental methods that would contribute to validation of these assumptions in reality are what this fi eld needs in the foreseeable future.

Another trait of vital importance, on which the accomplishment of the aforementioned aims will depend, is the openness of the dental community to cross-disciplinary fertilization of ideas. Dental science has greatly bene-fi ted in the past from the constructive intrusion of physicists and physical chemists into its realm and although many may be skeptical, in view of the risks such interdisciplinary encounters bear, or may feel insecure about putting their research into hands of someone who will shape it into a dif-ferent and often barely intelligible language, these will be challenges that need to be overcome with intellectual courage by members of the dental community.

Finally, insights into the mechanism by which the protein matrix guides the growth of apatite crystals in enamel, such as those presented here based in part on previously published results,18,23,64,65 are not only relevant to dental researchers, educators and clinicians interested in amelogenesis. They are valuable for understanding the protein–mineral interactions that govern many other types of biomineralization. The broad relevance of fi nd-ings obtained with a focus on enamel growth may remind us once again that small is beautiful, that each fi eld of research hides universal meanings and that focusing on small things and details of physical reality may lead us to grasp answers to much greater secrets of the universe. Such an aware-ness of greatly potent fi ndings awaiting in seemingly small research fi elds can motivate scientists from a plethora of other fi elds to peek into the world of dentistry and fi nd either a temporary solace for their wonder therein or a safe academic nest.



2.6 Acknowledgments

The author acknowledges the NIH grants K99-DE021416, R01-DE17529 and R01-DE015821 for support.

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