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Wear behavior of early carious enamel before and after remineralization
Wear 267 (2009) 726–733
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Wear journal homepage: www.elsevier.com/locate/wear
Wear behavior of early carious enamel before and after remineralization Shanshan Gao a , Shengbin Huang a , Linmao Qian b , Haiyang Yu a,∗ , Zhongrong Zhou b a b
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, PR China Tribology Research Institute, National Traction Power Laboratory, Southwest Jiaotong University, Chengdu 610031, PR China
a r t i c l e
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Article history: Received 28 August 2008 Received in revised form 22 November 2008 Accepted 23 November 2008 Keywords: Biotribology Wear mechanism Early carious enamel Remineralization
a b s t r a c t The purpose of the research was to evaluate the effect of remineralization treatment on the wear behavior of human early carious enamel. The degree of remineralization was observed with polarized light microscope and microhardness tester. Then the tribological properties of all the specimens were investigated by a CSEM nano-stratch tester system. The results showed that the hardness and density of carious enamel increased obviously, and the friction coefficient became higher after remineralization. It was easier to form cracks and debris on the remineralized enamel. It was concluded that the most effective remineralization agent (fluoride) in clinic could decrease the wear resistance of early carious enamel. There was no direct relationship between the content of minerals and the wear resistance of early carious enamel. The wear resistance of enamel could not be improved veritably by increasing the hardness. The main damage mechanisms of early carious enamel were the plastic deformation and adhesive wear while those of remineralized one were the combination of brittle cracks, plastic deformation and brittle delamination. New technique of remineralization is urgently needed clinically to improve the wear resistance. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Carious enamel is the susceptible dental enamel destructed locally by acidic by-products of carbohydrates [1,2]. The acid makes local pH values fall below a critical value (about 5.5), which causes the demineralization [3–5]. If the dissipation of calcium, phosphate, and carbonate continues, cavitation will eventually occur [6]. The process of demineralization can be reversed in its early stages by intake of calcium, phosphate, and fluoride [7]. Remineralization is a process of restoring mineral ions into the hydroxyapatite’s latticework structure [3]. Therefore remineralization, especially with fluoride, can repair damage caused by demineralization. It is of crucial importance to the prevention from cavitation [8]. Nowadays the researches on the remineralizing agents are mainly about fluoride, casein-phosphopeptide (CPP) with amorphous calcium phosphate (ACP), calcium carbonate (CaCO3 ), nano-hydroxyapatite, the Chinese herbal medicine such as Galla chinensis (G. chinensis) and so on [3,8–11]. Many of them have proved effective in preventing caries. Apart from new remineralizing agents, various methods have been used to assess the remineralization effect on early enamel caries. And they can be divided into two categories, direct and indirect technique. By direct one researcher detect the mineral intake and loss via microradiography, chemical analysis, and so on. By
∗ Corresponding author. Tel.: +86 28 85502869; fax: +86 28 85502869. E-mail address: [email protected] (H. Yu). 0043-1648/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.wear.2008.11.018
indirect one they determine the physical indexes of the enamel changing with mineral content, such as the microhardness, and the profilometry [12]. All the methods are mainly applied to assess the depth-resolved changes within remineralized enamel according to their mineral content, and histological features, such as their surface texture and appearances [13]. The most important function of teeth is to grind food through friction. Wear of teeth would take place inevitably during the process. Thus it is impossible to evaluate the effect of the remineralization correctly without the tribological researches. There were some researches on the wear resistance of remineralized enamel [14–16]. But most of them focused on the eroded enamel and proved that remineralization could increase the wear resistance of the eroded enamel [14,16]. Early carious enamel is different from the eroded enamel. Erosion had been described as a chronic, pathologic, localized loss of dental hard tissue etched away chemically from the tooth surface [16]. This chemical erosion might be caused by acidic substances and/or chelation without bacterial involvement. But early carious enamel owns apparently a relatively “intact” surface zone, rather than simple erosion [17,18]. And the researches on the wear resistance of the remineralized early carious enamel are rather scarce [14–16]. The early carious enamel after remineralization had better have a similar wear resistance with natural enamel. The inherent anisotropy of human tooth, such as mineral concentration gradient and consequent mechanical properties variation in enamel, influences its tribological behaviors as well. Early carious enamel before
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and after remineralization has different mineral concentration. So the different tribological properties may be embodied. An affective method providing a relatively rapid measure of wear mechanism of slender enamel is scratch test [19]. So the purpose of the research was to investigate the wear behavior of the remineralized enamel by nanoscratching test, to evaluate the wear resistance of the remineralizing agent, and to explore the tribological fundamentals of producing the remineralizing agents.
Table 1 The main compositions of demineralization solution.
2. Materials and methods
CaCl2 (mM)
KH2 PO4 (mM)
KCl (mM)
NaN3 (mM)
HEPES (mmol/L)
1.5
0.9
130
1
20
2.1. Preparation of early carious enamel specimens
Ca(NO3 )2 (mM)
KH2 PO4 (mM)
NaF (ppm)
Acetic acid (mM)
2.2
2.2
0.1
50
The PH of demineralization solution was adjusted to pH 4.5 using KOH.
Table 2 The main compositions of remineralization solution.
The PH of remineralization solution was adjusted to pH 7.0 using KOH.
Forty young’s premolars (11–14 years old) extracted for orthodontic treatment were collected immediately. Eight teeth with cracks, flaws or natural hetroplasia were excluded after investigation via microscope. Thirty two teeth were rinsed under tap water and kept in the water containing 0.05% thymol at four centigrade before they were taken for use [8]. All the teeth were sectioned carefully along the buccal-lingual direction. So there were sixty-four specimens altogether. These specimens were embedded into polymethyl methacrylate (PMMA). Tooth surfaces of the blocks were covered with PMMA except an exposed window about 4 × 4 mm. Then the surfaces were ground flat and polished with silicon carbide abrasive papers of a decreasing grit size, up to 2000 grit (Struers, Copenhagen, Denmark). Later they were ground to a mirror finish, thus the outer enamel layer of about 125 ± 23 m was removed (the thinnest thickness of normal premolar’s enamel is about 1.1 mm [20]). Finally, the specimens were ultrasonically cleaned in the distilled water for 5 min to remove the surface debris. The surface microhardness (SMH) of all the specimens were measured by a microhardness tester (Duramin-1/-2; Struers, Copenhagen, Denmark) with a Knoop indenter set at a load of 10 g for 15 s. Forty two enamel blocks with a baseline SMH between 256.1 KHN and 302.9 KHN were selected for the study [21].
Secondly, early carious enamel specimens were produced basically according to previous methods [8]. Each specimen was immersed in 8 mL of demineralization solution (Table 1) for 72 h at 37 ◦ C. Subsequently the SMH was measured again and recorded as SMH1. 2.2. Remineralization model Then half of each specimen was covered with an acid-resistant varnish to protect the early carious enamel. The cycling schedule was designed to imitate the oral environment’s pH dynamics [8]. The selected treatment solution was 1000 ppm NaF aqueous solution [8]. The main compositions of remineralization solution were listed in Table 2. 2.3. Surface microhardness analysis After the pH-cycling, SMH was measured again and recorded as SMH2. The measurement unit of hardness is kg/mm2 . The mean values at three different phases (baseline, after demineralization and after remineralization) were compared and the percentage SMH
Fig. 1. Design of whole test.
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S. Gao et al. / Wear 267 (2009) 726–733 Table 3 Surface microhardness analysis of enamel blocks at different phases. Treatment
Baseline SMH
Before pH-cycling
After pH-cycling
%SMHR
NaF
257.2 ± 13.0
80.5 ± 10.6
155.2 ± 18.8
42.1 ± 8.8
*
*
SMH values between before and after pH-cycling (p < 0.05). * Surface microhardness analysis of enamel blocks showed different surface microhardness.
were carefully observed via a Field Emission Scanning Electron Microscopy (INSPECT F, Czech Republic) to reveal the deformation and fracture patterns. 2.6. Statistical analysis Data were analyzed by applying SPSS 11.0 software. Student’s paired t-test was adopted to compare Knoop surface microhardness before and after remineralization. The statistical significance was considered only if p-value was less than 0.05 (p < 0.05).
Fig. 2. The selected diamond Brinell indenter with a nominal radius of 2 m.
3. Results 3.1. Surface microhardness
recovery [%SMHR = 100(SMH2 − SMH1)/(SMH1 − SMH)] was calculated. 2.4. Polarized microscopy examination Slices with the thickness of 500 m were cut from the central part of each specimen with a diamond blade, and all the sections were ground till becoming about 100 m thick with a watercooled diamond disc (Struers, Copenhagen, Denmark). Then they were examined under a Polarized Light Microscope (PLM) (ECLIPSE ME600L, Nikon, Tokyo, Japan) (Fig. 1). 2.5. Scratching tests After the treatment, the regions with the acid-resistant varnish were cleaned with acetone. Nanoscratch tests were conducted on the carious enamel and the remineralized enamel at room temperature. All scratch experiments had been performed on a CSEM nanoscratch tester apparatus (CSEM Instruments, Switzerland) in the same environmental conditions. A diamond Brinell indenter with a nominal radius of 2 m was used for scratch test (Fig. 2). The scratch test was performed, with a progressive load from 0.1 mN to 80 mN. The scratch velocity was set at 400 m/min. At least three scratches were conducted in one test region. The friction forces were recorded during the experiments. The remnant depths of the scratch grooves were measured with an AMBIOS XP-2 stylus profilometer (Ambios technology, Inc). All the morphologies
Table 3 shows the values of the SMH of enamel blocks at different stages. Statistically significant (p < 0.05) re-hardness was observed after remineralization. The %SMH of enamel blocks after remineralization improved 42.1 ± 8.6 compared with that of the early carious enamel. The results confirmed the remineralizing effect of fluoride on the early carious enamel. 3.2. PLM examination Polarized light evaluations of enamels are useful in describing the early carious enamel and the alteration of structure after remineralization [22]. Fig. 3 shows how early carious enamel developed before and after remineralization viewed with PLM. It could be easily observed that the intact enamel has a symmetrical higher density than early carious enamel (Fig. 3(a)). However, the early carious enamel has a surface with a relatively high density. Underneath the surface, the dark area represented the effect of demineralization. The mean depth of early carious enamel is 79 ± 14 m. It confirmed that the artificially induced carious enamel adopted in this study were similar to the natural caries enamel [18]. After remineralization, the density of the surface and the subsurface of enamel increased, and the surface became thicker (Fig. 3(b)). The depth (62 ± 11 m) of carious enamel decreased. Negative birefringent bands of the subsurface similar to that of the surface layer appeared.
Fig. 3. Polarized light microscopic pictures of enamel sections before and after the pH-cycling (a) before pH-cycling (b) after pH-cycling.
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Fig. 4. Evolution of friction coefficient versus displacement during scratching.
Fig. 6. Typical acquisition of remnant depth during the scratch experiment with an increasing normal load performed on the enamel before and after remineralization.
3.3. Coefficient of friction
rapidly. With the loads growing, the increasing rate decreased slightly. COF of the remineralized enamel were higher than that of the early carious enamel during the whole scratch process.
Fig. 4 shows the typical curves of friction coefficient versus displacement obtained during the scratching. Coefficient of friction (COF) was obtained from the ratio of the tangential force to the normal one. COF of early carious enamel increased linearly from 0 to 0.38 during the scratch process. As a comparison, COF of the remineralized enamel increased from 0 to 0.61, and the increasing rate was not constant. At the initiation of the scratching, COF increased
3.4. Micrographs The micrographs of the early carious enamel and the ones after remineralization are shown in Fig. 5(a) and (c). There were no obvious differences about the size and density of crystal. It
Fig. 5. Micrographs of enamel. (a) early carious enamel (b) early carious enamel after scratching (c) remineralized enamel (d) remineralized enamel after scratching.
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indicated that the crystals produced on the surface during the remineralization process were similar to those of the early carious enamel. The scratched regions are shown in Fig. 5(b) and (d). The arrangement of crystals became more compact, and the sizes of crystal diminished, which indicated that minerals in the early carious enamel and the new sediment were loose. The scratched surfaces before and after remineralization showed no obvious difference. Fig. 6 is the typical remnant depth curves after scratch experiment performed on two kinds of enamel (the early carious enamel
and the remineralized enamel). With the increase of the imposed load, the remnant depth increased accordingly. And the distinction of the remnant depths between two kinds of enamel became large. The remnant depth increased steadily before remineralization, while it fluctuated slightly after it. The remnant depths in the remineralized enamel were deeper. It indicated that the damages were severer in the remineralized enamel. The maximum damage depth would be less than 16 m in all specimens. The effect depth of remineralization was about 80 and the damage was located in the region of early carious enamel and the remineralized enamel. So the
Fig. 7. Micrographs of scratch before and after remineralization. (a–d) Before the remineralization; (e–h) after the remineralization.
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damage degree in the scratching test reflected the real wear resistance of the early carious enamel and the remineralized enamel. Typical micrographs of scratch conducted under ramping loads from 0.1 mN to 80 mN are presented in Fig. 7. Fig. 7(a)–(d) shows the early carious enamel, and Fig. 7(e)–(h) shows the remineralized enamel. On the surface of the early carious enamel, the width of the scratch increased with the loads (Fig. 7(a)). Traces of the plastically deformed grooves with slight rod dislodgement on the edges along the length were observed on the initial load levels (Fig. 7(b)). At the same time, there were some ripple-type deformations along the trace. The areas with dislodgement increased with the rising load, and some very slight delaminations occurred on the edge of the trace as the loads increased to about 60 mN (Fig. 7(c)). When loads reached about 70 mN, microcracks formed on some interfaces of ripple-type deformation (Fig. 7(d)). All the cracks focused on the trace, and the orientations of cracks were oblique to the scratching direction and formed an angle of 45◦ . Outside the scratching test areas, no cracks originated and propagated. The stress existed outside the trace would only produce the rod dislodgement. The enamel rods were compressed and deformed, which resulted in the dislocation on the interfaces of enamel rods. Severer damages occurred on the rematerialized surface (Fig. 7(e)–(h)). At the beginning of the scratching, only plastic deformation formed on the surface. Some closely spaced parallel lines (the white dash labeled in Fig. 7(f)) could be found in the trace, which also indicated the plastic deformation during the scratch process. The widths of the scratches became wider, and the interfaces of the enamel rods were not as evident as the cases before remineralization, which indicated that the dislocation deformation on the edge of the trace was not obvious. Cracks initiated on the lower level of load. When the loads increased to approximately 40 mN, cracks came into being at the two sides of the trace (Fig. 7(g)). Meanwhile, abundant delaminations occurred on the edge of the scratch. With the further increase of the loads, the distances between cracks shortened and the cracks lengthened. More brittle delaminations of the enamel tissue emerged around the scratches (Fig. 7(h)). The whole damage presented the character of a brittle damage. The distinct characteristics about wear resistance of the early carious enamel and of the enamel after remineralization would be explained in the part of discussion. Fig. 8 shows the delamination feature developed inside the scratch groove. After delamination, fresh tissues were exposed. Compared with the scratched area, the delamination in the early carious enamel was slight. Only part of enamel on the edge of trace was peeled off (Fig. 8(a)). Moreover the exposed fresh crystals seem to be looser and larger than those on the surface layer. It indicated
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that the crushed crystals only occurred on the surface. The crystals underneath were not affected by the scratching loads. The slip lines in the trace were still intact. While massive enamel tissues were delaminated under the same load after remineralization, the slip lines became the origins of the cracks, and formed the cracks in the trace (Fig. 8(b)). In addition, after remineralization, the size of the exposed crystal was smaller. 4. Discussion 4.1. Selection of remineralization agent In general, the early carious enamel is observed clinically as the white spot lesion, which is a small area of subsurface demineralization beneath the dental plaque [7]. The lesion is covered with a relatively intact, mineral-rich, porous surface layer, under which the mineral content is quite low [23]. Fluoride has been recommended and widely adopted as a preventive measure for caries, which proved to be the most effective remineralization agent in caries prevention to inhibit demineralization on the crystal surfaces and enhance remineralization on the crystal surfaces [24,25]. It is usually used as the positive control to evaluate other remineralization agents [8]. So fluoride was selected as the remineralization agent in this research. 4.2. The difference among the normal enamel, and the early carious enamel before and after remineralization The differences of wear between the early carious enamel and the remineralized enamel might lie in the effect of the minerals and protein. To understand it better, it was necessary to introduce the structures of the intact enamel, the early carious enamel and the remineralized enamel. Natural enamel is the highest mineralized tissue in the human body [26]. Crystallites of enamel are roughly rectangular in cross-section with an average width of 68 nm and an average thickness of 25 nm, which are glued together by a thin layer of protein not more than 2 nm. White et al. [27–29] highlighted that there were large amount of remaining proteins among the rods, which could permit limited movements of the adjacent rods toward different directions so as to prevent the catastrophic damages (Fig. 9). Although the early carious enamel owns a relatively intact surface, minerals underneath the surface dissolved. Because of the low solubility of protein, the structures of proteins still exist. After remineralization, different structures emerged on the surface of the enamel. Three layers structure model of the reminer-
Fig. 8. Micrographs of delamination: (a) before remineralization; (b) after remineralization.
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Fig. 9. Schematic of a tooth section cut perpendicularly to the occlusal surface along the long axis and the response of early carious enamel to the scratching load.
alized enamel via fluoride has been proposed in previous research [30]. From the inner to the outer part, they are respectively fluoride apatite (FAp), CaF2 and Ca(OH)2 respectively [30]. In addition, the rod sheath becomes narrow due to the formation of fluorapitite, and the structure of rod is not as clear as before. 4.3. The effect of proteins and the microstructures on the wear mechanisms After we know the distinction of these structures, it is relatively easy for us to understand the wear mechanism. After remineralization, just because more crystal deposited on the surface, the effect of the protein became slighter. The important effects of protein were that it was a type of biopolymer with viscoelastic properties stemming from the configurational rearrangements, disposition and interaction among the macromolecules in both their short- and long-range interrelations [31]. Under the load, these macromolecular chains in the early carious enamel might deform through changing the angle, unfolding and even moving their positions. From a thermodynamics perspective, the changes of macromolecules were unstable. After unloading, the molecules had a tendency of returning to their initial form and positions. The presence of this minor protein component had been regarded as the reason why enamel showed “metal-like ductility” rather than “brittleness”. The response of the remineralized enamel to nanoscratch was closely related with the protein. After remineralization, the proportion of protein decreased, and “metal-like ductility” became not so obvious. Owing to the effect of scratching, the loose minerals deposited on the surface mainly manifested the characteristic of brittleness. In the natural enamel, the arrangement of crystal was regulated by the protein. Without the protein, the arrangements were not as regular as in the natural enamel. Meanwhile, for lack of the protein combination, the bond between crystals decreased. The feature of being more brittle could be observed. And cracks were apt to form on the interface of crystals as well (Fig. 10). The main differences in wear behaviors of the early carious enamel before and after reminearlization were the dislocation of enamel rods, and the delamination occurred on the edge. These distinctions reflected different wear mechanisms. Before remineralization, the minerals content of enamel was low. Loosened crystals owned better elastic-plastic properties due to the effect of proteins. Plastic deformation and adhesive wear were the main damage mechanisms. After remineralization, the minerals content increased, but the crystals were not so compact as the natural enamel. High level
Fig. 10. The response of the remineralized enamel to the scratching load.
of minerals made the enamel brittle. Thus deformation was suppressed in remineralized enamel. The brittle cracks were inclined to grow and might lead to delamination owing to the stress. Cracks and delaminations were the main damage mechanisms [32]. It was suggested that remineralization with fluoride should not increase the wear resistance. Furthermore, it degraded the wear resistance of the early carious enamel. It puts forward a new question to us. It is urgent to explore a new remineralizing technique completely different form the previous ones, which could not only prevent the early enamel caries, but also improve its wear resistance. The evolution of the friction coefficient with the increasing of the normal loads contradicted the classic models. The increase of friction coefficient might be relevant to the damages during the process of scratch. As shown in Figs. 4 and 5, the severer damages occurred on the scratches, microcracks initiated and propagated, the structures under scratches deformed and delaminated. Perhaps these damages made the friction coefficient increase with the loads. The specific reasons still need be explored in the future work. 5. Conclusions The wear resistance tests of the early carious enamel before and after remineralization were carried out in vitro. The main conclusions were summarized as the followings.
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1. The surface microhardness of enamel blocks after fluoride remineralization improved by 42% on average compared with that of the early carious enamel, but the hardness or the contents of minerals had no direct relations with the wear resistance of the early enamel caries. The early carious enamel had a worse wear resistance after remineralization. 2. The main wear mechanisms of the early carious enamel were plastic deformations and adherences. After the fluoride remineralization, the wear mechanisms were dominated by a combination of brittle cracks, plastic deformations and brittle delaminations. Acknowledgement The authors wish to acknowledge the financial support from National Natural Science Foundation of China (No: 30572064) and Program for New Century Excellent Talents in University (NCET-060794). References [1] O. Fejerskov, E.A.M. Kidd, Dental caries: The Disease and its Clinical Management, Blackwell Monksgard, Copenhagen, Denmark, 2003. [2] P. Marsh, M.V. Martin, Oral Microbiology, 4th ed., Wright, Oxford, 1999. [3] G.G. Franklin, M.J. Hicks, Maintaining the integrity of the enamel surface: the role of dental biofilm, saliva and preventive agents in enamel demineralization and remineralization, J. Am. Dent. Assoc. 139 (2008) 25–34. [4] J.D. Featherstone, The continuum of dental caries—evidence for a dynamic disease process, J. Dent. Res. 83 (2004) 39–42. [5] P.W. Caueld, A.L. Grien, Dental caries-an infectious and transmissible disease, Pediatr. Clin. N. Am. 47 (2000) 1001–1019. [6] W.K. Seow, Biological mechanisms of early childhood caries, Commun. Dent. Oral. 26 (1998) S8–S27. [7] R.H. Selwitz, A.L. Ismail, N.B. Pitts, Dent. caries. Lancet. 369 (2007) 51–59. [8] J.P. Chu, J.Y. Li, Y.Q. Hao, et al., Effect of compounds of Galla chinensis on remineralisation of initial enamel carious lesions in vitro, J. Dent. 35 (2007) 383–387. [9] W. Buchalla, T. Attin, J. Schulte-Mönting, et al., Fluoride uptake, retention, and remineralization efficacy of a highly concentrated fluoride solution on enamel lesions in situ, J. Dent. Res. 81 (2002) 329–333. [10] C. Rahiotis, G. Vougiouklakis, Effect of a CPP-ACP agent on the demineralization and remineralization of dentine in vitro, J. Dent. 35 (2007) 695–698. [11] B. Li, J. Wang, Z. Zhao, et al., Mineralizing of nano-hydroxyapatite powers on artificial caries, Rare Metal. Mater. Eng. 36 (S2) (2007) 128–130.
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[12] M. Eisenburger, M. Addy, J.A. Hughes, et al., Effect of time on the remineralisation of enamel by synthetic saliva after citric acid erosion, Caries. Res. 35 (2001) 211–215. [13] R.S. Jones, D. Fried, Remineralization of enamel caries can decrease optical reflectivity, J. Dent. Res. 85 (2006) 804–808. [14] C.P. Turssi, J.J. Faraoni, A.L. Rodrigues Jr., et al., An in situ investigation into the abrasion of eroded dental hard tissue by a whitening dentifrice, Caries. Res. 38 (2003) 473–477. [15] T. Attin, S. Siegel, W. Buchalla, et al., Brushing abrasion of softened and remineralised dentin: an in situ study, Caries. Res. 38 (2004) 62–66. [16] T. Attin, W. Buchalla, M. Gollner, et al., Use of variable remineralization periods to improve the abrasion resistance of previously eroded enamel, Caries. Res. 34 (2000) 48–52. [17] D.J. White, The application of in vitro models to research on demineralization and remineralization of the teeth, Adv. Dent. Res. 9 (1995) 175–193. [18] C. Robinson, R.C. Shore, S.J. Brookes, et al., The chemistry of enamel caries, Crit. Rev. Oral. Biol. Med. 11 (2000) 481–495. [19] J.A. Kaidonis, Tooth wear: the view of the anthropologist, Clin. Oral. Invest. 12 (2008) S21–S26. [20] S.F. Rosenstiel, M.F. Land, J. Fujimoto, Contemporary Fixed Prosthodontics, 3rd ed., Mosby, Inc., USA, 2001. [21] F. Lippert, D.M. Parker, K.D. Jandt, In vitro demnieralization/remineralization cycles at human tooth enamel surfaces investigated by AFM and nanoindentation, J. Colloid. Interf. Sci. 280 (2004) 442–448. [22] J.S. Wefel, J.K. Harless, Comparison of artificial white spots by microradiography and polarized light microscopy, J. Dent. Res. 63 (1984) 1271–1275. [23] J.D. Featherston, Prevention and reversal of dental caries: role of low level fluoride, Commun. Dent. Oral. 27 (1999) 31–40. [24] D.T. Zero, Dental caries process, Dent. Clin. N. Am. 43 (1999) 635–664. [25] J.M. Ten Cate, C. Van Loveren, Fluoride mechanisms, Dent. Clin. N. Am. 43 (1999) 713–742. [26] I.M. Low, N. Duraman, U. Mahmood, Mapping the structure, composition and mechanical properties of human teeth, Mater. Sci. Eng. C 28 (2008) 243–247. [27] S.N. White, W. Luo, M.L. Paine, et al., Biological organization of hydroxyapatite crystallites into a fibrous continuum toughness and controls anisotropy in human enamel, J. Dent. Res. 80 (2001) 321–327. [28] S. Roy, B. Basu, Mechanical and tribological characterization of human tooth, Mater. Charact. 59 (2008) 747–756. [29] Z. Xie, M. Swain, P. Munroe, et al., On the critical parameters that regulate the deformation behaviour of tooth enamel, Biomaterials 29 (2008) 2697–2703. [30] H.U.V. Gerth, T. Dammaschke, E. Schafer, et al., A three layer structure model of fluoridated enamel containing CaF2 , Ca(OH)2 and FAp, Dent. Mater. 23 (2007) 1521–1528. [31] B. Ji, H. Gao, Mechanical properties of nanostructure of biological materials, J. Mech. Phys. Solids 52 (2004) 1963–1990. [32] P. Zioupos, K.D. Rogers, Complementary physical and mechanical techniques to characterise tooth: a boon-like tissue, J. Bionic. Eng. 3 (2006) 19–31.
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Wear journal homepage: www.elsevier.com/locate/wear
Wear behavior of early carious enamel before and after remineralization Shanshan Gao a , Shengbin Huang a , Linmao Qian b , Haiyang Yu a,∗ , Zhongrong Zhou b a b
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, PR China Tribology Research Institute, National Traction Power Laboratory, Southwest Jiaotong University, Chengdu 610031, PR China
a r t i c l e
i n f o
Article history: Received 28 August 2008 Received in revised form 22 November 2008 Accepted 23 November 2008 Keywords: Biotribology Wear mechanism Early carious enamel Remineralization
a b s t r a c t The purpose of the research was to evaluate the effect of remineralization treatment on the wear behavior of human early carious enamel. The degree of remineralization was observed with polarized light microscope and microhardness tester. Then the tribological properties of all the specimens were investigated by a CSEM nano-stratch tester system. The results showed that the hardness and density of carious enamel increased obviously, and the friction coefficient became higher after remineralization. It was easier to form cracks and debris on the remineralized enamel. It was concluded that the most effective remineralization agent (fluoride) in clinic could decrease the wear resistance of early carious enamel. There was no direct relationship between the content of minerals and the wear resistance of early carious enamel. The wear resistance of enamel could not be improved veritably by increasing the hardness. The main damage mechanisms of early carious enamel were the plastic deformation and adhesive wear while those of remineralized one were the combination of brittle cracks, plastic deformation and brittle delamination. New technique of remineralization is urgently needed clinically to improve the wear resistance. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Carious enamel is the susceptible dental enamel destructed locally by acidic by-products of carbohydrates [1,2]. The acid makes local pH values fall below a critical value (about 5.5), which causes the demineralization [3–5]. If the dissipation of calcium, phosphate, and carbonate continues, cavitation will eventually occur [6]. The process of demineralization can be reversed in its early stages by intake of calcium, phosphate, and fluoride [7]. Remineralization is a process of restoring mineral ions into the hydroxyapatite’s latticework structure [3]. Therefore remineralization, especially with fluoride, can repair damage caused by demineralization. It is of crucial importance to the prevention from cavitation [8]. Nowadays the researches on the remineralizing agents are mainly about fluoride, casein-phosphopeptide (CPP) with amorphous calcium phosphate (ACP), calcium carbonate (CaCO3 ), nano-hydroxyapatite, the Chinese herbal medicine such as Galla chinensis (G. chinensis) and so on [3,8–11]. Many of them have proved effective in preventing caries. Apart from new remineralizing agents, various methods have been used to assess the remineralization effect on early enamel caries. And they can be divided into two categories, direct and indirect technique. By direct one researcher detect the mineral intake and loss via microradiography, chemical analysis, and so on. By
∗ Corresponding author. Tel.: +86 28 85502869; fax: +86 28 85502869. E-mail address: [email protected] (H. Yu). 0043-1648/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.wear.2008.11.018
indirect one they determine the physical indexes of the enamel changing with mineral content, such as the microhardness, and the profilometry [12]. All the methods are mainly applied to assess the depth-resolved changes within remineralized enamel according to their mineral content, and histological features, such as their surface texture and appearances [13]. The most important function of teeth is to grind food through friction. Wear of teeth would take place inevitably during the process. Thus it is impossible to evaluate the effect of the remineralization correctly without the tribological researches. There were some researches on the wear resistance of remineralized enamel [14–16]. But most of them focused on the eroded enamel and proved that remineralization could increase the wear resistance of the eroded enamel [14,16]. Early carious enamel is different from the eroded enamel. Erosion had been described as a chronic, pathologic, localized loss of dental hard tissue etched away chemically from the tooth surface [16]. This chemical erosion might be caused by acidic substances and/or chelation without bacterial involvement. But early carious enamel owns apparently a relatively “intact” surface zone, rather than simple erosion [17,18]. And the researches on the wear resistance of the remineralized early carious enamel are rather scarce [14–16]. The early carious enamel after remineralization had better have a similar wear resistance with natural enamel. The inherent anisotropy of human tooth, such as mineral concentration gradient and consequent mechanical properties variation in enamel, influences its tribological behaviors as well. Early carious enamel before
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and after remineralization has different mineral concentration. So the different tribological properties may be embodied. An affective method providing a relatively rapid measure of wear mechanism of slender enamel is scratch test [19]. So the purpose of the research was to investigate the wear behavior of the remineralized enamel by nanoscratching test, to evaluate the wear resistance of the remineralizing agent, and to explore the tribological fundamentals of producing the remineralizing agents.
Table 1 The main compositions of demineralization solution.
2. Materials and methods
CaCl2 (mM)
KH2 PO4 (mM)
KCl (mM)
NaN3 (mM)
HEPES (mmol/L)
1.5
0.9
130
1
20
2.1. Preparation of early carious enamel specimens
Ca(NO3 )2 (mM)
KH2 PO4 (mM)
NaF (ppm)
Acetic acid (mM)
2.2
2.2
0.1
50
The PH of demineralization solution was adjusted to pH 4.5 using KOH.
Table 2 The main compositions of remineralization solution.
The PH of remineralization solution was adjusted to pH 7.0 using KOH.
Forty young’s premolars (11–14 years old) extracted for orthodontic treatment were collected immediately. Eight teeth with cracks, flaws or natural hetroplasia were excluded after investigation via microscope. Thirty two teeth were rinsed under tap water and kept in the water containing 0.05% thymol at four centigrade before they were taken for use [8]. All the teeth were sectioned carefully along the buccal-lingual direction. So there were sixty-four specimens altogether. These specimens were embedded into polymethyl methacrylate (PMMA). Tooth surfaces of the blocks were covered with PMMA except an exposed window about 4 × 4 mm. Then the surfaces were ground flat and polished with silicon carbide abrasive papers of a decreasing grit size, up to 2000 grit (Struers, Copenhagen, Denmark). Later they were ground to a mirror finish, thus the outer enamel layer of about 125 ± 23 m was removed (the thinnest thickness of normal premolar’s enamel is about 1.1 mm [20]). Finally, the specimens were ultrasonically cleaned in the distilled water for 5 min to remove the surface debris. The surface microhardness (SMH) of all the specimens were measured by a microhardness tester (Duramin-1/-2; Struers, Copenhagen, Denmark) with a Knoop indenter set at a load of 10 g for 15 s. Forty two enamel blocks with a baseline SMH between 256.1 KHN and 302.9 KHN were selected for the study [21].
Secondly, early carious enamel specimens were produced basically according to previous methods [8]. Each specimen was immersed in 8 mL of demineralization solution (Table 1) for 72 h at 37 ◦ C. Subsequently the SMH was measured again and recorded as SMH1. 2.2. Remineralization model Then half of each specimen was covered with an acid-resistant varnish to protect the early carious enamel. The cycling schedule was designed to imitate the oral environment’s pH dynamics [8]. The selected treatment solution was 1000 ppm NaF aqueous solution [8]. The main compositions of remineralization solution were listed in Table 2. 2.3. Surface microhardness analysis After the pH-cycling, SMH was measured again and recorded as SMH2. The measurement unit of hardness is kg/mm2 . The mean values at three different phases (baseline, after demineralization and after remineralization) were compared and the percentage SMH
Fig. 1. Design of whole test.
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S. Gao et al. / Wear 267 (2009) 726–733 Table 3 Surface microhardness analysis of enamel blocks at different phases. Treatment
Baseline SMH
Before pH-cycling
After pH-cycling
%SMHR
NaF
257.2 ± 13.0
80.5 ± 10.6
155.2 ± 18.8
42.1 ± 8.8
*
*
SMH values between before and after pH-cycling (p < 0.05). * Surface microhardness analysis of enamel blocks showed different surface microhardness.
were carefully observed via a Field Emission Scanning Electron Microscopy (INSPECT F, Czech Republic) to reveal the deformation and fracture patterns. 2.6. Statistical analysis Data were analyzed by applying SPSS 11.0 software. Student’s paired t-test was adopted to compare Knoop surface microhardness before and after remineralization. The statistical significance was considered only if p-value was less than 0.05 (p < 0.05).
Fig. 2. The selected diamond Brinell indenter with a nominal radius of 2 m.
3. Results 3.1. Surface microhardness
recovery [%SMHR = 100(SMH2 − SMH1)/(SMH1 − SMH)] was calculated. 2.4. Polarized microscopy examination Slices with the thickness of 500 m were cut from the central part of each specimen with a diamond blade, and all the sections were ground till becoming about 100 m thick with a watercooled diamond disc (Struers, Copenhagen, Denmark). Then they were examined under a Polarized Light Microscope (PLM) (ECLIPSE ME600L, Nikon, Tokyo, Japan) (Fig. 1). 2.5. Scratching tests After the treatment, the regions with the acid-resistant varnish were cleaned with acetone. Nanoscratch tests were conducted on the carious enamel and the remineralized enamel at room temperature. All scratch experiments had been performed on a CSEM nanoscratch tester apparatus (CSEM Instruments, Switzerland) in the same environmental conditions. A diamond Brinell indenter with a nominal radius of 2 m was used for scratch test (Fig. 2). The scratch test was performed, with a progressive load from 0.1 mN to 80 mN. The scratch velocity was set at 400 m/min. At least three scratches were conducted in one test region. The friction forces were recorded during the experiments. The remnant depths of the scratch grooves were measured with an AMBIOS XP-2 stylus profilometer (Ambios technology, Inc). All the morphologies
Table 3 shows the values of the SMH of enamel blocks at different stages. Statistically significant (p < 0.05) re-hardness was observed after remineralization. The %SMH of enamel blocks after remineralization improved 42.1 ± 8.6 compared with that of the early carious enamel. The results confirmed the remineralizing effect of fluoride on the early carious enamel. 3.2. PLM examination Polarized light evaluations of enamels are useful in describing the early carious enamel and the alteration of structure after remineralization [22]. Fig. 3 shows how early carious enamel developed before and after remineralization viewed with PLM. It could be easily observed that the intact enamel has a symmetrical higher density than early carious enamel (Fig. 3(a)). However, the early carious enamel has a surface with a relatively high density. Underneath the surface, the dark area represented the effect of demineralization. The mean depth of early carious enamel is 79 ± 14 m. It confirmed that the artificially induced carious enamel adopted in this study were similar to the natural caries enamel [18]. After remineralization, the density of the surface and the subsurface of enamel increased, and the surface became thicker (Fig. 3(b)). The depth (62 ± 11 m) of carious enamel decreased. Negative birefringent bands of the subsurface similar to that of the surface layer appeared.
Fig. 3. Polarized light microscopic pictures of enamel sections before and after the pH-cycling (a) before pH-cycling (b) after pH-cycling.
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Fig. 4. Evolution of friction coefficient versus displacement during scratching.
Fig. 6. Typical acquisition of remnant depth during the scratch experiment with an increasing normal load performed on the enamel before and after remineralization.
3.3. Coefficient of friction
rapidly. With the loads growing, the increasing rate decreased slightly. COF of the remineralized enamel were higher than that of the early carious enamel during the whole scratch process.
Fig. 4 shows the typical curves of friction coefficient versus displacement obtained during the scratching. Coefficient of friction (COF) was obtained from the ratio of the tangential force to the normal one. COF of early carious enamel increased linearly from 0 to 0.38 during the scratch process. As a comparison, COF of the remineralized enamel increased from 0 to 0.61, and the increasing rate was not constant. At the initiation of the scratching, COF increased
3.4. Micrographs The micrographs of the early carious enamel and the ones after remineralization are shown in Fig. 5(a) and (c). There were no obvious differences about the size and density of crystal. It
Fig. 5. Micrographs of enamel. (a) early carious enamel (b) early carious enamel after scratching (c) remineralized enamel (d) remineralized enamel after scratching.
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indicated that the crystals produced on the surface during the remineralization process were similar to those of the early carious enamel. The scratched regions are shown in Fig. 5(b) and (d). The arrangement of crystals became more compact, and the sizes of crystal diminished, which indicated that minerals in the early carious enamel and the new sediment were loose. The scratched surfaces before and after remineralization showed no obvious difference. Fig. 6 is the typical remnant depth curves after scratch experiment performed on two kinds of enamel (the early carious enamel
and the remineralized enamel). With the increase of the imposed load, the remnant depth increased accordingly. And the distinction of the remnant depths between two kinds of enamel became large. The remnant depth increased steadily before remineralization, while it fluctuated slightly after it. The remnant depths in the remineralized enamel were deeper. It indicated that the damages were severer in the remineralized enamel. The maximum damage depth would be less than 16 m in all specimens. The effect depth of remineralization was about 80 and the damage was located in the region of early carious enamel and the remineralized enamel. So the
Fig. 7. Micrographs of scratch before and after remineralization. (a–d) Before the remineralization; (e–h) after the remineralization.
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damage degree in the scratching test reflected the real wear resistance of the early carious enamel and the remineralized enamel. Typical micrographs of scratch conducted under ramping loads from 0.1 mN to 80 mN are presented in Fig. 7. Fig. 7(a)–(d) shows the early carious enamel, and Fig. 7(e)–(h) shows the remineralized enamel. On the surface of the early carious enamel, the width of the scratch increased with the loads (Fig. 7(a)). Traces of the plastically deformed grooves with slight rod dislodgement on the edges along the length were observed on the initial load levels (Fig. 7(b)). At the same time, there were some ripple-type deformations along the trace. The areas with dislodgement increased with the rising load, and some very slight delaminations occurred on the edge of the trace as the loads increased to about 60 mN (Fig. 7(c)). When loads reached about 70 mN, microcracks formed on some interfaces of ripple-type deformation (Fig. 7(d)). All the cracks focused on the trace, and the orientations of cracks were oblique to the scratching direction and formed an angle of 45◦ . Outside the scratching test areas, no cracks originated and propagated. The stress existed outside the trace would only produce the rod dislodgement. The enamel rods were compressed and deformed, which resulted in the dislocation on the interfaces of enamel rods. Severer damages occurred on the rematerialized surface (Fig. 7(e)–(h)). At the beginning of the scratching, only plastic deformation formed on the surface. Some closely spaced parallel lines (the white dash labeled in Fig. 7(f)) could be found in the trace, which also indicated the plastic deformation during the scratch process. The widths of the scratches became wider, and the interfaces of the enamel rods were not as evident as the cases before remineralization, which indicated that the dislocation deformation on the edge of the trace was not obvious. Cracks initiated on the lower level of load. When the loads increased to approximately 40 mN, cracks came into being at the two sides of the trace (Fig. 7(g)). Meanwhile, abundant delaminations occurred on the edge of the scratch. With the further increase of the loads, the distances between cracks shortened and the cracks lengthened. More brittle delaminations of the enamel tissue emerged around the scratches (Fig. 7(h)). The whole damage presented the character of a brittle damage. The distinct characteristics about wear resistance of the early carious enamel and of the enamel after remineralization would be explained in the part of discussion. Fig. 8 shows the delamination feature developed inside the scratch groove. After delamination, fresh tissues were exposed. Compared with the scratched area, the delamination in the early carious enamel was slight. Only part of enamel on the edge of trace was peeled off (Fig. 8(a)). Moreover the exposed fresh crystals seem to be looser and larger than those on the surface layer. It indicated
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that the crushed crystals only occurred on the surface. The crystals underneath were not affected by the scratching loads. The slip lines in the trace were still intact. While massive enamel tissues were delaminated under the same load after remineralization, the slip lines became the origins of the cracks, and formed the cracks in the trace (Fig. 8(b)). In addition, after remineralization, the size of the exposed crystal was smaller. 4. Discussion 4.1. Selection of remineralization agent In general, the early carious enamel is observed clinically as the white spot lesion, which is a small area of subsurface demineralization beneath the dental plaque [7]. The lesion is covered with a relatively intact, mineral-rich, porous surface layer, under which the mineral content is quite low [23]. Fluoride has been recommended and widely adopted as a preventive measure for caries, which proved to be the most effective remineralization agent in caries prevention to inhibit demineralization on the crystal surfaces and enhance remineralization on the crystal surfaces [24,25]. It is usually used as the positive control to evaluate other remineralization agents [8]. So fluoride was selected as the remineralization agent in this research. 4.2. The difference among the normal enamel, and the early carious enamel before and after remineralization The differences of wear between the early carious enamel and the remineralized enamel might lie in the effect of the minerals and protein. To understand it better, it was necessary to introduce the structures of the intact enamel, the early carious enamel and the remineralized enamel. Natural enamel is the highest mineralized tissue in the human body [26]. Crystallites of enamel are roughly rectangular in cross-section with an average width of 68 nm and an average thickness of 25 nm, which are glued together by a thin layer of protein not more than 2 nm. White et al. [27–29] highlighted that there were large amount of remaining proteins among the rods, which could permit limited movements of the adjacent rods toward different directions so as to prevent the catastrophic damages (Fig. 9). Although the early carious enamel owns a relatively intact surface, minerals underneath the surface dissolved. Because of the low solubility of protein, the structures of proteins still exist. After remineralization, different structures emerged on the surface of the enamel. Three layers structure model of the reminer-
Fig. 8. Micrographs of delamination: (a) before remineralization; (b) after remineralization.
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Fig. 9. Schematic of a tooth section cut perpendicularly to the occlusal surface along the long axis and the response of early carious enamel to the scratching load.
alized enamel via fluoride has been proposed in previous research [30]. From the inner to the outer part, they are respectively fluoride apatite (FAp), CaF2 and Ca(OH)2 respectively [30]. In addition, the rod sheath becomes narrow due to the formation of fluorapitite, and the structure of rod is not as clear as before. 4.3. The effect of proteins and the microstructures on the wear mechanisms After we know the distinction of these structures, it is relatively easy for us to understand the wear mechanism. After remineralization, just because more crystal deposited on the surface, the effect of the protein became slighter. The important effects of protein were that it was a type of biopolymer with viscoelastic properties stemming from the configurational rearrangements, disposition and interaction among the macromolecules in both their short- and long-range interrelations [31]. Under the load, these macromolecular chains in the early carious enamel might deform through changing the angle, unfolding and even moving their positions. From a thermodynamics perspective, the changes of macromolecules were unstable. After unloading, the molecules had a tendency of returning to their initial form and positions. The presence of this minor protein component had been regarded as the reason why enamel showed “metal-like ductility” rather than “brittleness”. The response of the remineralized enamel to nanoscratch was closely related with the protein. After remineralization, the proportion of protein decreased, and “metal-like ductility” became not so obvious. Owing to the effect of scratching, the loose minerals deposited on the surface mainly manifested the characteristic of brittleness. In the natural enamel, the arrangement of crystal was regulated by the protein. Without the protein, the arrangements were not as regular as in the natural enamel. Meanwhile, for lack of the protein combination, the bond between crystals decreased. The feature of being more brittle could be observed. And cracks were apt to form on the interface of crystals as well (Fig. 10). The main differences in wear behaviors of the early carious enamel before and after reminearlization were the dislocation of enamel rods, and the delamination occurred on the edge. These distinctions reflected different wear mechanisms. Before remineralization, the minerals content of enamel was low. Loosened crystals owned better elastic-plastic properties due to the effect of proteins. Plastic deformation and adhesive wear were the main damage mechanisms. After remineralization, the minerals content increased, but the crystals were not so compact as the natural enamel. High level
Fig. 10. The response of the remineralized enamel to the scratching load.
of minerals made the enamel brittle. Thus deformation was suppressed in remineralized enamel. The brittle cracks were inclined to grow and might lead to delamination owing to the stress. Cracks and delaminations were the main damage mechanisms [32]. It was suggested that remineralization with fluoride should not increase the wear resistance. Furthermore, it degraded the wear resistance of the early carious enamel. It puts forward a new question to us. It is urgent to explore a new remineralizing technique completely different form the previous ones, which could not only prevent the early enamel caries, but also improve its wear resistance. The evolution of the friction coefficient with the increasing of the normal loads contradicted the classic models. The increase of friction coefficient might be relevant to the damages during the process of scratch. As shown in Figs. 4 and 5, the severer damages occurred on the scratches, microcracks initiated and propagated, the structures under scratches deformed and delaminated. Perhaps these damages made the friction coefficient increase with the loads. The specific reasons still need be explored in the future work. 5. Conclusions The wear resistance tests of the early carious enamel before and after remineralization were carried out in vitro. The main conclusions were summarized as the followings.
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1. The surface microhardness of enamel blocks after fluoride remineralization improved by 42% on average compared with that of the early carious enamel, but the hardness or the contents of minerals had no direct relations with the wear resistance of the early enamel caries. The early carious enamel had a worse wear resistance after remineralization. 2. The main wear mechanisms of the early carious enamel were plastic deformations and adherences. After the fluoride remineralization, the wear mechanisms were dominated by a combination of brittle cracks, plastic deformations and brittle delaminations. Acknowledgement The authors wish to acknowledge the financial support from National Natural Science Foundation of China (No: 30572064) and Program for New Century Excellent Talents in University (NCET-060794). References [1] O. Fejerskov, E.A.M. Kidd, Dental caries: The Disease and its Clinical Management, Blackwell Monksgard, Copenhagen, Denmark, 2003. [2] P. Marsh, M.V. Martin, Oral Microbiology, 4th ed., Wright, Oxford, 1999. [3] G.G. Franklin, M.J. Hicks, Maintaining the integrity of the enamel surface: the role of dental biofilm, saliva and preventive agents in enamel demineralization and remineralization, J. Am. Dent. Assoc. 139 (2008) 25–34. [4] J.D. Featherstone, The continuum of dental caries—evidence for a dynamic disease process, J. Dent. Res. 83 (2004) 39–42. [5] P.W. Caueld, A.L. Grien, Dental caries-an infectious and transmissible disease, Pediatr. Clin. N. Am. 47 (2000) 1001–1019. [6] W.K. Seow, Biological mechanisms of early childhood caries, Commun. Dent. Oral. 26 (1998) S8–S27. [7] R.H. Selwitz, A.L. Ismail, N.B. Pitts, Dent. caries. Lancet. 369 (2007) 51–59. [8] J.P. Chu, J.Y. Li, Y.Q. Hao, et al., Effect of compounds of Galla chinensis on remineralisation of initial enamel carious lesions in vitro, J. Dent. 35 (2007) 383–387. [9] W. Buchalla, T. Attin, J. Schulte-Mönting, et al., Fluoride uptake, retention, and remineralization efficacy of a highly concentrated fluoride solution on enamel lesions in situ, J. Dent. Res. 81 (2002) 329–333. [10] C. Rahiotis, G. Vougiouklakis, Effect of a CPP-ACP agent on the demineralization and remineralization of dentine in vitro, J. Dent. 35 (2007) 695–698. [11] B. Li, J. Wang, Z. Zhao, et al., Mineralizing of nano-hydroxyapatite powers on artificial caries, Rare Metal. Mater. Eng. 36 (S2) (2007) 128–130.
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[12] M. Eisenburger, M. Addy, J.A. Hughes, et al., Effect of time on the remineralisation of enamel by synthetic saliva after citric acid erosion, Caries. Res. 35 (2001) 211–215. [13] R.S. Jones, D. Fried, Remineralization of enamel caries can decrease optical reflectivity, J. Dent. Res. 85 (2006) 804–808. [14] C.P. Turssi, J.J. Faraoni, A.L. Rodrigues Jr., et al., An in situ investigation into the abrasion of eroded dental hard tissue by a whitening dentifrice, Caries. Res. 38 (2003) 473–477. [15] T. Attin, S. Siegel, W. Buchalla, et al., Brushing abrasion of softened and remineralised dentin: an in situ study, Caries. Res. 38 (2004) 62–66. [16] T. Attin, W. Buchalla, M. Gollner, et al., Use of variable remineralization periods to improve the abrasion resistance of previously eroded enamel, Caries. Res. 34 (2000) 48–52. [17] D.J. White, The application of in vitro models to research on demineralization and remineralization of the teeth, Adv. Dent. Res. 9 (1995) 175–193. [18] C. Robinson, R.C. Shore, S.J. Brookes, et al., The chemistry of enamel caries, Crit. Rev. Oral. Biol. Med. 11 (2000) 481–495. [19] J.A. Kaidonis, Tooth wear: the view of the anthropologist, Clin. Oral. Invest. 12 (2008) S21–S26. [20] S.F. Rosenstiel, M.F. Land, J. Fujimoto, Contemporary Fixed Prosthodontics, 3rd ed., Mosby, Inc., USA, 2001. [21] F. Lippert, D.M. Parker, K.D. Jandt, In vitro demnieralization/remineralization cycles at human tooth enamel surfaces investigated by AFM and nanoindentation, J. Colloid. Interf. Sci. 280 (2004) 442–448. [22] J.S. Wefel, J.K. Harless, Comparison of artificial white spots by microradiography and polarized light microscopy, J. Dent. Res. 63 (1984) 1271–1275. [23] J.D. Featherston, Prevention and reversal of dental caries: role of low level fluoride, Commun. Dent. Oral. 27 (1999) 31–40. [24] D.T. Zero, Dental caries process, Dent. Clin. N. Am. 43 (1999) 635–664. [25] J.M. Ten Cate, C. Van Loveren, Fluoride mechanisms, Dent. Clin. N. Am. 43 (1999) 713–742. [26] I.M. Low, N. Duraman, U. Mahmood, Mapping the structure, composition and mechanical properties of human teeth, Mater. Sci. Eng. C 28 (2008) 243–247. [27] S.N. White, W. Luo, M.L. Paine, et al., Biological organization of hydroxyapatite crystallites into a fibrous continuum toughness and controls anisotropy in human enamel, J. Dent. Res. 80 (2001) 321–327. [28] S. Roy, B. Basu, Mechanical and tribological characterization of human tooth, Mater. Charact. 59 (2008) 747–756. [29] Z. Xie, M. Swain, P. Munroe, et al., On the critical parameters that regulate the deformation behaviour of tooth enamel, Biomaterials 29 (2008) 2697–2703. [30] H.U.V. Gerth, T. Dammaschke, E. Schafer, et al., A three layer structure model of fluoridated enamel containing CaF2 , Ca(OH)2 and FAp, Dent. Mater. 23 (2007) 1521–1528. [31] B. Ji, H. Gao, Mechanical properties of nanostructure of biological materials, J. Mech. Phys. Solids 52 (2004) 1963–1990. [32] P. Zioupos, K.D. Rogers, Complementary physical and mechanical techniques to characterise tooth: a boon-like tissue, J. Bionic. Eng. 3 (2006) 19–31.