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Wear 267 (2009) 726–733

Contents lists available at ScienceDirect

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ear behavior of early carious enamel before and after remineralization

hanshan Gaoa, Shengbin Huanga, Linmao Qianb, Haiyang Yua,∗, Zhongrong Zhoub

State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, PR ChinaTribology Research Institute, National Traction Power Laboratory, Southwest Jiaotong University, Chengdu 610031, PR China

r t i c l e i n f o

rticle history:eceived 28 August 2008eceived in revised form2 November 2008ccepted 23 November 2008

a b s t r a c t

The purpose of the research was to evaluate the effect of remineralization treatment on the wear behaviorof human early carious enamel. The degree of remineralization was observed with polarized light micro-scope and microhardness tester. Then the tribological properties of all the specimens were investigated bya CSEM nano-stratch tester system. The results showed that the hardness and density of carious enamelincreased obviously, and the friction coefficient became higher after remineralization. It was easier to

eywords:iotribologyear mechanism

arly carious enamelemineralization

form cracks and debris on the remineralized enamel. It was concluded that the most effective remineral-ization agent (fluoride) in clinic could decrease the wear resistance of early carious enamel. There was nodirect relationship between the content of minerals and the wear resistance of early carious enamel. Thewear resistance of enamel could not be improved veritably by increasing the hardness. The main dam-age mechanisms of early carious enamel were the plastic deformation and adhesive wear while those ofremineralized one were the combination of brittle cracks, plastic deformation and brittle delamination.

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New technique of remine

. Introduction

Carious enamel is the susceptible dental enamel destructedocally by acidic by-products of carbohydrates [1,2]. The acid

akes local pH values fall below a critical value (about 5.5), whichauses the demineralization [3–5]. If the dissipation of calcium,hosphate, and carbonate continues, cavitation will eventuallyccur [6]. The process of demineralization can be reversed in itsarly stages by intake of calcium, phosphate, and fluoride [7].emineralization is a process of restoring mineral ions into theydroxyapatite’s latticework structure [3]. Therefore remineral-

zation, especially with fluoride, can repair damage caused byemineralization. It is of crucial importance to the prevention fromavitation [8]. Nowadays the researches on the remineralizinggents are mainly about fluoride, casein-phosphopeptide (CPP)ith amorphous calcium phosphate (ACP), calcium carbonate

CaCO3), nano-hydroxyapatite, the Chinese herbal medicine suchs Galla chinensis (G. chinensis) and so on [3,8–11]. Many of themave proved effective in preventing caries.

Apart from new remineralizing agents, various methods have

een used to assess the remineralization effect on early enamelaries. And they can be divided into two categories, direct and indi-ect technique. By direct one researcher detect the mineral intakend loss via microradiography, chemical analysis, and so on. By

∗ Corresponding author. Tel.: +86 28 85502869; fax: +86 28 85502869.E-mail address: yhyang6812@scu.edu.cn (H. Yu).

043-1648/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.wear.2008.11.018

tion is urgently needed clinically to improve the wear resistance.© 2009 Elsevier B.V. All rights reserved.

indirect one they determine the physical indexes of the enamelchanging with mineral content, such as the microhardness, and theprofilometry [12]. All the methods are mainly applied to assess thedepth-resolved changes within remineralized enamel according totheir mineral content, and histological features, such as their sur-face texture and appearances [13]. The most important functionof teeth is to grind food through friction. Wear of teeth would takeplace inevitably during the process. Thus it is impossible to evaluatethe effect of the remineralization correctly without the tribologicalresearches.

There were some researches on the wear resistance of rem-ineralized enamel [14–16]. But most of them focused on theeroded enamel and proved that remineralization could increasethe wear resistance of the eroded enamel [14,16]. Early cariousenamel is different from the eroded enamel. Erosion had beendescribed as a chronic, pathologic, localized loss of dental hardtissue etched away chemically from the tooth surface [16]. Thischemical erosion might be caused by acidic substances and/orchelation without bacterial involvement. But early carious enamelowns apparently a relatively “intact” surface zone, rather thansimple erosion [17,18]. And the researches on the wear resis-tance of the remineralized early carious enamel are rather scarce[14–16].

The early carious enamel after remineralization had betterhave a similar wear resistance with natural enamel. The inherentanisotropy of human tooth, such as mineral concentration gradientand consequent mechanical properties variation in enamel, influ-ences its tribological behaviors as well. Early carious enamel before

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Table 1The main compositions of demineralization solution.

Ca(NO3)2 (mM) KH2PO4 (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 2The main compositions of remineralization solution.

S. Gao et al. / Wea

nd after remineralization has different mineral concentration. Sohe different tribological properties may be embodied.

An affective method providing a relatively rapid measure of wearechanism of slender enamel is scratch test [19]. So the purpose of

he research was to investigate the wear behavior of the remineral-zed enamel by nanoscratching test, to evaluate the wear resistancef the remineralizing agent, and to explore the tribological funda-entals of producing the remineralizing agents.

. Materials and methods

.1. Preparation of early carious enamel specimens

Forty young’s premolars (11–14 years old) extracted forrthodontic treatment were collected immediately. Eight teethith cracks, flaws or natural hetroplasia were excluded after inves-

igation via microscope. Thirty two teeth were rinsed under tapater and kept in the water containing 0.05% thymol at four

entigrade before they were taken for use [8]. All the teeth were sec-ioned carefully along the buccal-lingual direction. So there wereixty-four specimens altogether. These specimens were embed-ed into polymethyl methacrylate (PMMA). Tooth surfaces of thelocks were covered with PMMA except an exposed window about× 4 mm. Then the surfaces were ground flat and polished with

ilicon carbide abrasive papers of a decreasing grit size, up to 2000rit (Struers, Copenhagen, Denmark). Later they were ground to airror finish, thus the outer enamel layer of about 125 ± 23 �mas removed (the thinnest thickness of normal premolar’s enamel

s about 1.1 mm [20]). Finally, the specimens were ultrasonicallyleaned in the distilled water for 5 min to remove the surface debris.

The surface microhardness (SMH) of all the specimens wereeasured by a microhardness tester (Duramin-1/-2; Struers,

openhagen, Denmark) with a Knoop indenter set at a load of 10 gor 15 s. Forty two enamel blocks with a baseline SMH between56.1 KHN and 302.9 KHN were selected for the study [21].

Fig. 1. Design of

CaCl2 (mM) KH2 PO4 (mM) KCl (mM) NaN3 (mM) HEPES (mmol/L)

1.5 0.9 130 1 20

The PH of remineralization solution was adjusted to pH 7.0 using KOH.

Secondly, early carious enamel specimens were produced basi-cally according to previous methods [8]. Each specimen wasimmersed in 8 mL of demineralization solution (Table 1) for 72 hat 37 ◦C. Subsequently the SMH was measured again and recordedas SMH1.

2.2. Remineralization model

Then half of each specimen was covered with an acid-resistantvarnish to protect the early carious enamel. The cycling schedulewas designed to imitate the oral environment’s pH dynamics [8].The selected treatment solution was 1000 ppm NaF aqueous solu-tion [8]. The main compositions of remineralization solution werelisted in Table 2.

2.3. Surface microhardness analysis

After the pH-cycling, SMH was measured again and recorded asSMH2. The measurement unit of hardness is kg/mm2. The mean val-ues at three different phases (baseline, after demineralization andafter remineralization) were compared and the percentage SMH

whole test.

728 S. Gao et al. / Wear 267 (2009) 726–733

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Table 3Surface 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

[18].

Fig. 2. The selected diamond Brinell indenter with a nominal radius of 2 �m.

ecovery [%SMHR = 100(SMH2 − SMH1)/(SMH1 − SMH)] was calcu-ated.

.4. Polarized microscopy examination

Slices with the thickness of 500 �m were cut from the centralart of each specimen with a diamond blade, and all the sectionsere ground till becoming about 100 �m thick with a water-

ooled diamond disc (Struers, Copenhagen, Denmark). Then theyere examined under a Polarized Light Microscope (PLM) (ECLIPSEE600L, Nikon, Tokyo, Japan) (Fig. 1).

.5. Scratching tests

After the treatment, the regions with the acid-resistant varnishere cleaned with acetone. Nanoscratch tests were conducted on

he carious enamel and the remineralized enamel at room tem-erature. All scratch experiments had been performed on a CSEManoscratch tester apparatus (CSEM Instruments, Switzerland) inhe same environmental conditions. A diamond Brinell indenterith a nominal radius of 2 �m was used for scratch test (Fig. 2). The

cratch test was performed, with a progressive load from 0.1 mN

o 80 mN. The scratch velocity was set at 400 �m/min. At leasthree scratches were conducted in one test region. The frictionorces were recorded during the experiments. The remnant depthsf the scratch grooves were measured with an AMBIOS XP-2 sty-us profilometer (Ambios technology, Inc). All the morphologies

Fig. 3. Polarized light microscopic pictures of enamel sections before an

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 ElectronMicroscopy (INSPECT F, Czech Republic) to reveal the deformationand fracture patterns.

2.6. Statistical analysis

Data were analyzed by applying SPSS 11.0 software. Student’spaired t-test was adopted to compare Knoop surface microhardnessbefore and after remineralization. The statistical significance wasconsidered only if p-value was less than 0.05 (p < 0.05).

3. Results

3.1. Surface microhardness

Table 3 shows the values of the SMH of enamel blocks at differentstages. Statistically significant (p < 0.05) re-hardness was observedafter remineralization. The %SMH of enamel blocks after remineral-ization improved 42.1 ± 8.6 compared with that of the early cariousenamel. The results confirmed the remineralizing effect of fluorideon the early carious enamel.

3.2. PLM examination

Polarized light evaluations of enamels are useful in describingthe early carious enamel and the alteration of structure after rem-ineralization [22]. Fig. 3 shows how early carious enamel developedbefore and after remineralization viewed with PLM. It could beeasily observed that the intact enamel has a symmetrical higherdensity than early carious enamel (Fig. 3(a)). However, the early car-ious enamel has a surface with a relatively high density. Underneaththe surface, the dark area represented the effect of demineral-ization. The mean depth of early carious enamel is 79 ± 14 �m.It confirmed that the artificially induced carious enamel adoptedin this study were similar to the natural caries enamel

After remineralization, the density of the surface and the subsur-face of enamel increased, and the surface became thicker (Fig. 3(b)).The depth (62 ± 11 �m) of carious enamel decreased. Negative bire-fringent bands of the subsurface similar to that of the surface layerappeared.

d after the pH-cycling (a) before pH-cycling (b) after pH-cycling.

S. Gao et al. / Wear 267 (2009) 726–733 729

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3.4. Micrographs

ig. 4. Evolution of friction coefficient versus displacement during scratching.

.3. Coefficient of friction

Fig. 4 shows the typical curves of friction coefficient versus dis-lacement obtained during the scratching. Coefficient of frictionCOF) was obtained from the ratio of the tangential force to the

ormal one. COF of early carious enamel increased linearly from 0o 0.38 during the scratch process. As a comparison, COF of the rem-neralized enamel increased from 0 to 0.61, and the increasing rateas not constant. At the initiation of the scratching, COF increased

Fig. 5. Micrographs of enamel. (a) early carious enamel (b) early carious enamel after

Fig. 6. Typical acquisition of remnant depth during the scratch experiment with anincreasing normal load performed on the enamel before and after remineralization.

rapidly. With the loads growing, the increasing rate decreasedslightly. COF of the remineralized enamel were higher than thatof the early carious enamel during the whole scratch process.

The micrographs of the early carious enamel and the onesafter remineralization are shown in Fig. 5(a) and (c). There wereno obvious differences about the size and density of crystal. It

scratching (c) remineralized enamel (d) remineralized enamel after scratching.

7 r 267

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ndicated that the crystals produced on the surface during theemineralization process were similar to those of the early cari-us enamel. The scratched regions are shown in Fig. 5(b) and (d).he arrangement of crystals became more compact, and the sizesf crystal diminished, which indicated that minerals in the early

arious enamel and the new sediment were loose. The scratchedurfaces before and after remineralization showed no obviousifference.

Fig. 6 is the typical remnant depth curves after scratch experi-ent performed on two kinds of enamel (the early carious enamel

Fig. 7. Micrographs of scratch before and after remineralization. (a–d

(2009) 726–733

and the remineralized enamel). With the increase of the imposedload, the remnant depth increased accordingly. And the distinc-tion of the remnant depths between two kinds of enamel becamelarge. The remnant depth increased steadily before remineraliza-tion, while it fluctuated slightly after it. The remnant depths in the

remineralized enamel were deeper. It indicated that the damageswere severer in the remineralized enamel. The maximum damagedepth would be less than 16 �m in all specimens. The effect depthof remineralization was about 80 and the damage was located in theregion of early carious enamel and the remineralized enamel. So the

) Before the remineralization; (e–h) after the remineralization.

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amage degree in the scratching test reflected the real wear resis-ance of the early carious enamel and the remineralized enamel.

Typical micrographs of scratch conducted under ramping loadsrom 0.1 mN to 80 mN are presented in Fig. 7. Fig. 7(a)–(d) showshe early carious enamel, and Fig. 7(e)–(h) shows the remineralizednamel. On the surface of the early carious enamel, the width of thecratch increased with the loads (Fig. 7(a)). Traces of the plasticallyeformed grooves with slight rod dislodgement on the edges alonghe length were observed on the initial load levels (Fig. 7(b)). Athe same time, there were some ripple-type deformations alonghe trace. The areas with dislodgement increased with the risingoad, and some very slight delaminations occurred on the edge ofhe trace as the loads increased to about 60 mN (Fig. 7(c)). Whenoads reached about 70 mN, microcracks formed on some interfacesf ripple-type deformation (Fig. 7(d)). All the cracks focused on therace, and the orientations of cracks were oblique to the scratch-ng direction and formed an angle of 45◦. Outside the scratchingest areas, no cracks originated and propagated. The stress existedutside the trace would only produce the rod dislodgement. Thenamel rods were compressed and deformed, which resulted inhe dislocation on the interfaces of enamel rods.

Severer damages occurred on the rematerialized surfaceFig. 7(e)–(h)). At the beginning of the scratching, only plastic defor-

ation formed on the surface. Some closely spaced parallel linesthe white dash labeled in Fig. 7(f)) could be found in the trace,hich also indicated the plastic deformation during the scratch pro-

ess. The widths of the scratches became wider, and the interfacesf the enamel rods were not as evident as the cases before rem-neralization, which indicated that the dislocation deformation onhe edge of the trace was not obvious. Cracks initiated on the lowerevel of load. When the loads increased to approximately 40 mN,racks came into being at the two sides of the trace (Fig. 7(g)). Mean-hile, abundant delaminations occurred on the edge of the scratch.ith the further increase of the loads, the distances between cracks

hortened and the cracks lengthened. More brittle delaminationsf the enamel tissue emerged around the scratches (Fig. 7(h)).he whole damage presented the character of a brittle damage.he distinct characteristics about wear resistance of the early car-ous enamel and of the enamel after remineralization would bexplained in the part of discussion.

Fig. 8 shows the delamination feature developed inside the

cratch groove. After delamination, fresh tissues were exposed.ompared with the scratched area, the delamination in the earlyarious enamel was slight. Only part of enamel on the edge of traceas peeled off (Fig. 8(a)). Moreover the exposed fresh crystals seem

o be looser and larger than those on the surface layer. It indicated

Fig. 8. Micrographs of delamination: (a) before re

(2009) 726–733 731

that the crushed crystals only occurred on the surface. The crys-tals underneath were not affected by the scratching loads. The sliplines in the trace were still intact. While massive enamel tissueswere delaminated under the same load after remineralization, theslip lines became the origins of the cracks, and formed the cracksin the trace (Fig. 8(b)). In addition, after remineralization, the sizeof the exposed crystal was smaller.

4. Discussion

4.1. Selection of remineralization agent

In general, the early carious enamel is observed clinically as thewhite spot lesion, which is a small area of subsurface demineral-ization beneath the dental plaque [7]. The lesion is covered witha relatively intact, mineral-rich, porous surface layer, under whichthe mineral content is quite low [23]. Fluoride has been recom-mended and widely adopted as a preventive measure for caries,which proved to be the most effective remineralization agent incaries prevention to inhibit demineralization on the crystal surfacesand enhance remineralization on the crystal surfaces [24,25]. It isusually used as the positive control to evaluate other remineral-ization agents [8]. So fluoride was selected as the remineralizationagent in this research.

4.2. The difference among the normal enamel, and the earlycarious enamel before and after remineralization

The differences of wear between the early carious enamel andthe remineralized enamel might lie in the effect of the minerals andprotein. To understand it better, it was necessary to introduce thestructures of the intact enamel, the early carious enamel and theremineralized enamel. Natural enamel is the highest mineralizedtissue in the human body [26]. Crystallites of enamel are roughlyrectangular in cross-section with an average width of 68 nm andan average thickness of 25 nm, which are glued together by a thinlayer of protein not more than 2 nm. White et al. [27–29] high-lighted that there were large amount of remaining proteins amongthe rods, which could permit limited movements of the adjacentrods toward different directions so as to prevent the catastrophicdamages (Fig. 9).

Although the early carious enamel owns a relatively intact sur-face, minerals underneath the surface dissolved. Because of the lowsolubility of protein, the structures of proteins still exist.

After remineralization, different structures emerged on thesurface of the enamel. Three layers structure model of the reminer-

mineralization; (b) after remineralization.

732 S. Gao et al. / Wear 267 (2009) 726–733

along the long axis and the response of early carious enamel to the scratching load.

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lized enamel via fluoride has been proposed in previous research30]. From the inner to the outer part, they are respectively fluoridepatite (FAp), CaF2 and Ca(OH)2 respectively [30]. In addition, theod sheath becomes narrow due to the formation of fluorapitite,nd the structure of rod is not as clear as before.

.3. The effect of proteins and the microstructures on the wearechanisms

After we know the distinction of these structures, it is relativelyasy for us to understand the wear mechanism. After remineral-zation, just because more crystal deposited on the surface, theffect of the protein became slighter. The important effects ofrotein were that it was a type of biopolymer with viscoelas-ic properties stemming from the configurational rearrangements,isposition and interaction among the macromolecules in bothheir short- and long-range interrelations [31]. Under the load,hese macromolecular chains in the early carious enamel mighteform through changing the angle, unfolding and even movingheir positions. From a thermodynamics perspective, the changesf macromolecules were unstable. After unloading, the moleculesad a tendency of returning to their initial form and positions. Theresence of this minor protein component had been regarded ashe reason why enamel showed “metal-like ductility” rather thanbrittleness”.

The response of the remineralized enamel to nanoscratch waslosely related with the protein. After remineralization, the pro-ortion of protein decreased, and “metal-like ductility” becameot so obvious. Owing to the effect of scratching, the loose min-rals deposited on the surface mainly manifested the characteristicf brittleness. In the natural enamel, the arrangement of crystalas regulated by the protein. Without the protein, the arrange-ents were not as regular as in the natural enamel. Meanwhile,

or lack of the protein combination, the bond between crystalsecreased. The feature of being more brittle could be observed.nd cracks were apt to form on the interface of crystals as well

Fig. 10).The main differences in wear behaviors of the early carious

namel before and after reminearlization were the dislocation ofnamel rods, and the delamination occurred on the edge. Theseistinctions reflected different wear mechanisms. Before rem-

neralization, the minerals content of enamel was low. Loosened

rystals owned better elastic-plastic properties due to the effectf proteins. Plastic deformation and adhesive wear were the mainamage mechanisms.

After remineralization, the minerals content increased, but therystals 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 sup-pressed in remineralized enamel. The brittle cracks were inclinedto grow and might lead to delamination owing to the stress. Cracksand delaminations were the main damage mechanisms [32].

It was suggested that remineralization with fluoride should notincrease the wear resistance. Furthermore, it degraded the wearresistance of the early carious enamel. It puts forward a new ques-tion to us. It is urgent to explore a new remineralizing techniquecompletely different form the previous ones, which could not onlyprevent the early enamel caries, but also improve its wear resis-tance.

The evolution of the friction coefficient with the increasing ofthe normal loads contradicted the classic models. The increase offriction coefficient might be relevant to the damages during theprocess of scratch. As shown in Figs. 4 and 5, the severer damagesoccurred on the scratches, microcracks initiated and propagated,the structures under scratches deformed and delaminated. Perhapsthese 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 andafter remineralization were carried out in vitro. The main conclu-sions were summarized as the followings.

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1. The surface microhardness of enamel blocks after fluoride rem-ineralization improved by 42% on average compared with thatof the early carious enamel, but the hardness or the contents ofminerals had no direct relations with the wear resistance of theearly enamel caries. The early carious enamel had a worse wearresistance after remineralization.

. The main wear mechanisms of the early carious enamel wereplastic deformations and adherences. After the fluoride rem-ineralization, the wear mechanisms were dominated by acombination of brittle cracks, plastic deformations and brittledelaminations.

cknowledgement

The authors wish to acknowledge the financial support fromational Natural Science Foundation of China (No: 30572064) androgram for New Century Excellent Talents in University (NCET-06-794).

eferences

[1] O. Fejerskov, E.A.M. Kidd, Dental caries: The Disease and its Clinical Manage-ment, 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 demineralizationand remineralization, J. Am. Dent. Assoc. 139 (2008) 25–34.

[4] J.D. Featherstone, The continuum of dental caries—evidence for a dynamic dis-ease 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 reminer-

alisation 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 enamellesions in situ, J. Dent. Res. 81 (2002) 329–333.

10] C. Rahiotis, G. Vougiouklakis, Effect of a CPP-ACP agent on the demineralizationand 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 onartificial caries, Rare Metal. Mater. Eng. 36 (S2) (2007) 128–130.

[

[

(2009) 726–733 733

12] M. Eisenburger, M. Addy, J.A. Hughes, et al., Effect of time on the remineralisa-tion 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 opticalreflectivity, 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 theabrasion 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 rem-ineralised dentin: an in situ study, Caries. Res. 38 (2004) 62–66.

16] T. Attin, W. Buchalla, M. Gollner, et al., Use of variable remineralization periodsto 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 demineralizationand 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, 3rded., Mosby, Inc., USA, 2001.

21] F. Lippert, D.M. Parker, K.D. Jandt, In vitro demnieralization/remineralizationcycles at human tooth enamel surfaces investigated by AFM and nanoindenta-tion, J. Colloid. Interf. Sci. 280 (2004) 442–448.

22] J.S. Wefel, J.K. Harless, Comparison of artificial white spots by microradiographyand polarized light microscopy, J. Dent. Res. 63 (1984) 1271–1275.

23] J.D. Featherston, Prevention and reversal of dental caries: role of low levelfluoride, 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 hydroxyap-atite crystallites into a fibrous continuum toughness and controls anisotropy inhuman 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 reg-ulate 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 tocharacterise tooth: a boon-like tissue, J. Bionic. Eng. 3 (2006) 19–31.