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2008, 112, 7162–7165 Published on Web 05/27/2008
Anisotropic Demineralization and Oriented Assembly of Hydroxyapatite Crystals in Enamel: Smart Structures of Biominerals Haihua Pan,*,† Jinhui Tao,† Xinwei Yu,† Lei Fu,‡ Jiali Zhang,‡ Xiangxuan Zeng,§ Guohua Xu,§ and Ruikang Tang*,† Department of Chemistry and Center for Biomaterials and Biopathways, Zhejiang UniVersity, Hangzhou, 310027, China, First Affiliated Hospital, College of Medicine, Zhejiang UniVersity, Hangzhou, 310009, China, and Department of Chemical Engineering, Zhejiang UniVersity, Hangzhou, 310027, China ReceiVed: March 31, 2008; ReVised Manuscript ReceiVed: May 09, 2008
It is interesting to note that the demineralization of natural enamel does not happen as readily as that of the synthesized hydroxyapatite (HAP), although they share a similar chemical composition. We suggest that the hierarchical structure of enamel is an important factor in the preservation of the natural material against dissolution. The anisotropic demineralization of HAP is revealed experimentally, and this phenomenon is understood by the different interfacial structures of HAP-water at the atomic level. It is found that HAP {001} facets can be more resistant against dissolution than {100} under acidic conditions. Although {100} is the largest surface of the typical HAP crystal, it is {001}, the smallest habit face, that is chosen by the living organisms to build the outer surface of enamel by an oriented assembly of the rodlike crystals. We reveal that such a biological construction can confer on enamel protections against erosion, since {001} is relatively dissolution-insensitive. Thus, the spontaneous dissolution of enamel surface can be retarded in biological milieu by such a smart construction. The current study demonstrates the importance of hierarchical structures in the functional biomaterials. Enamel is the exterior coating of tooth, and its primary mineral component is hydroxyapatite (Ca10(PO4)6(OH)2, HAP).1 As the most highly mineralized (>95 wt %) tissue in vertebrates, enamel is composed of numerous rodlike apatite crystals, which are bundled in the ordered prisms. Despite the complicated structures of the biomineral,2 it is revealed that the basic building blocks of enamel are HAP nanoparticles.2,3 These tiny particles are assembled into nanofibril and then into fibril and fiber. Bundles of fibers form the prismatic and interprismatic enamel structures. The prism bundles grow from the dentino-enamel junction (DEJ) toward the outer enamel surface (OES). The preferential orientations of the fibers are consistent with the c-axis of HAP, and they are also parallel to the directions of the prism bundles. However, the fibers in interprisms are inclined. It is well-known that the remarkable mechanical characteristics are achieved by the biological enamel via such hierarchical constructions.2 The apatites in enamel are usually nonstoichiometrically substituted HAP.4 Since the synthesized HAP and the natural enamel apatite crystals share a similar morphology and dimension, HAP is widely used as a model compound for enamel in laboratories.5 Chemically, HAP is basic calcium phosphate and this inorganic compound can be dissolved readily in acidic solutions.6 The dissolution of enamel leads to dental erosion. In nature, dental caries is always caused by bacteria, which * Corresponding authors. Phone and Fax: +86-571-87953736. E-mail:
[email protected] (H.P.);
[email protected] (R.T.). † Department of Chemistry and Center for Biomaterials and Biopathways. ‡ First Affiliated Hospital, College of Medicine. § Department of Chemical Engineering.
10.1021/jp802739f CCC: $40.75
produces the acidic condition at the localized site to dissolve the calcium mineral. However, it is also noted that some diets (e.g., sour sweet, soft drinks, soda, juice, etc.) induce acidic oral environments too,7 which is able to initiate the demineralization of the mineral. However, the dissolution of enamel surface is extremely slow when the enamel is exposed to the undersaturated milieu. Actually, the spontaneous dissolution of enamel cannot occur readily in nature and most enamel can maintain hardness and health for decades. Oral environments, such as saliva8 and biofilm on the surface of enamel,9 are considered to play important roles in the biological protection of OES. Furthermore, recent studies have revealed that enamel can be kinetically preserved against demineralization, since it is biologically constructed by nanoparticles.3,10 Also, our nano dissolution model suggests that the active dissolution sites are difficult to be produced on the nano solid surfaces.3 As a result, the dissolution of the tiny HAP is slow and the reaction may even be suppressed when the particle sizes are only tens of nanometers. However, some vulnerable regions on the dissolution of enamel are still present; e.g., Voegel et al. have reported the presence of a dislocation line parallel to the c-axis of enamel apatite by transmission electron microscopy (TEM).11 However, in biological fluids, these imperfections of apatite crystals cannot lead to the fast dissolution of the crystals. Besides, according to the previous understanding,12 the erosion of enamel is started through the formation of the central core lesions, which extend anisotropically along the c-axis of apatite. The erosion then spreads laterally, and the development of the lateral side lesion parallels the {100} planes. Why does the erosion prefer the a/b directions rather than the c direction? Unfortunately, no reason 2008 American Chemical Society
Letters able explanation has been proposed to understand the mechanism of the anisotropic behavior of enamel dissolution. In this Letter, we study the anisotropic demineralization of natural enamel and synthesized HAP crystals. It is revealed that the {001} faces can be relatively stable under acidic conditions. This experimental phenomenon can be understood by the HAP-water interface at the atomic level. Interestingly, the OES is composed of numerous dissolution-insensitive {001} faces by an assembling of the apatite fibers along the c direction. Also, the demineralization resistances of the OES can be enhanced by this characteristic structure. Together with the protection effects from the biofilm9 and the preservation effect at the nanoscale,3 the smart construction of enamel can also confer on the biomaterials remarkable characteristics against demineralization at multiscale levels (both nano- and microlevels). Ten freshly extracted caries-free and filling-free human premolars were used in this study (ages of 18-28 years; the use of human tissue specimens followed a protocol that was approved by the ethical committee of the First Affiliated Hospital of College of Medicine of Zhejiang University and was also agreed upon by the patients). Each tooth was cut parallel to the natural OES. Samples were cleaned by ultrasonication in tripledistilled water for 5 min before and after a polish using silicon carbide papers (#800, #1500, and #2000). For the purposes of comparison, half of the enamel surface was covered by varnish and the rest was used in the demineralization experiment. HCl (20 µL, 0.02 M) was dropped onto the enamel surface for 20 min. The samples were washed using triple-distilled water, and the varnish was removed by acetone. The synthesized rodlike HAP crystals13 were used as the model system. The HAP suspensions (0.02 wt %) were dropped onto an amine modified silicon14 (the modification was applied to enhance the adhesion of HAP crystals on the substrate; details were given in the Supporting Information). After these were dried in air, HCl (20 µL, 0.02 M) was added to induce the demineralization of HAP. The erosion time of the synthesized HAP was only 30 s due to the rapid demineralization reaction. The detailed HAP-water interfacial structure was studied by molecular dynamics (MD) simulation to understand the physicochemical properties of HAP faces, which was performed by using the GROMACS 3.3 package (see http://www.gromacs.org).15,16 The force field parameters of Hauptmann et al. were applied for HAP.17 The explicit water model with a simple point charge (SPC) force field was used.18 The details of the HAP-water interface model were described in our previous work19 and are also described in the Supporting Information. The morphology changes of enamel and HAP crystals were characterized by scanning electron microscopy (SEM; SLR10N, FEI, Netherlands) and atomic force microscopy (AFM; Multimode with Nanoscope IVa Controller, Veeco, U.S.). Both prismatic (P) and interprismatic (IP) parts can be observed on the enamel surface (Figure 1a). The pattern of the assembled prism bands is of type 3 (also the key-hole type, marked by dashed lines).20 The rodlike apatite crystals in the prismatic enamel align in parallel and assemble in a compact fashion, and the interprismatic enamel has a different orientation and the fibers are disordered in the boundary region (Figure 1a). In our in Vitro experiments, the demineralization of enamel is significant under the acidic attack and the surface becomes rough due to the mineral loss (Figure 1b and d and Supporting Information Figure S2). Compared with the intact flat surface protected by varnish, some rodlike apatites are exposed after the acid treatment (Figure 1b and d and Supporting Information Figure S3). Heterogeneous demineralization of an enamel
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Figure 1. (a) SEM of human enamel surface. (b) Different enamel surfaces with and without the acid treatment. (c) Interprism (IP) and prism (P) regions on the enamel surface after the acid treatment. (d) AFM of the enamel surface after the acid treatment.
Figure 2. (a) AFM of the HAP crystal. (b) HAP crystal after the acid treatment. (c) SEM image of the acid treated HAP crystal. (d) Suggested structures of HAP crystal after the acid treatment.
surface is also observed on the natural enamel surface. The demineralization occurs more readily at the interprism-prism boundary and some sites in prism or interprism; thus, the grooves (deep gaps, marked by arrow heads) and small cavities (marked by arrows) resulted, respectively, during the acid treatment (Figure 1c). These results are also confirmed by the AFM studies (Figure 1d, the dissolved sites are marked by the arrow heads). The morphology of the synthesized HAP is shown in Figure 2a. The HAP crystal surface has relatively large and smooth terraces, and step dislocations (Supporting Information Figure S4). The crystallographic structures of the HAP facets are examined by using X-ray diffraction (XRD), TEM, and AFM.13 It is confirmed that the large flat surface in Figure 2a is the {100} face of HAP. The long axis of the rodlike HAP crystal is its c direction. The details of the surface erosion of HAP crystal after the acid treatment are characterized by using AFM
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Figure 3. (upper) Snapshots of the HAP-water interfacial structure of (001) and (100) faces; (lower) radical distribution of OH (oxygen of hydroxyl ion) and HW (hydrogen of water).
and SEM. It is found that many tiny hexagonal facets are newly formed on the dissolving HAP surface (Figure 2b; their corresponding height images are shown in Supporting Information Figures S5 and S6). The original edge of the crystal is smooth, but it becomes significantly rough during the demineralization (Figure 2c, SEM). The heterogeneous and anisotropic demineralization of the natural enamel originates from the hierarchical structures. During the formation of enamel, most of the organic “framework” disappears (degraded and removed) during the apatite maturation.20 The content of the remaining proteins is extremely low in the matured enamel (
