Change in surface hardness of enamel by a cola drinkand a CPP–ACP paste

D. Tantbirojn a,*, A. Huang b, M.D. Ericson c, S. Poolthong d

aDepartment of Restorative Sciences, University of Minnesota, Minneapolis, USAb Summer Research Fellow, School of Dentistry, University of Minnesota, Minneapolis, USAcMinnesota Dental Research Center for Biomaterials and Biomechanics, University of Minnesota, Minneapolis, USAdDepartment of Operative Dentistry, Chulalongkorn University, Bangkok, Thailand

j o u r n a l o f d e n t i s t r y 3 6 ( 2 0 0 8 ) 7 4 – 7 9

a r t i c l e i n f o

Article history:

Received 14 September 2007

Received in revised form

15 October 2007

Accepted 17 October 2007

Keywords:

Surface hardness

Enamel

Cola

CPP–ACP

Saliva-substitute solution

Biotene mouthwash

a b s t r a c t

Objectives: This in vitro study used surface microhardness to evaluate whether a paste

containing casein phosphopeptide amorphous calcium phosphate (CPP–ACP) can reharden

tooth enamel softened by a cola drink, and how different saliva-substitute solutions affect

the enamel hardness.

Methods: Twenty-four bovine incisors, each tooth consisting of treatment and control

halves, were immersed in a cola drink (Coke1) for 8 min, then placed under a 0.4 mL/

min drip with various saliva-substitute solutions. The saliva-substitute solutions were:

saliva-like solution (SLS) with 1 ppm fluoride, SLS without fluoride, and Biotene1

mouthwash. CPP–ACP paste was applied to the treatment halves for 3 min at 0, 8, 24,

and 36 h. Knoop microhardness measurements were performed at baseline, after the cola

drink immersion, and after 24 and 48 h contact with saliva-substitute solution.

Results: Enamel hardness significantly decreased after immersion in cola drink (ANOVA,

p < 0.05). After contact with saliva-like solutions for 48 h, those treated with CPP–ACP paste

were significantly harder than those untreated regardless of the presence of 1 ppm fluoride

in the saliva-like solution (ANOVA, p < 0.05). Biotene1 mouthwash significantly softened

the enamel surface (ANOVA, p < 0.05). Two-way ANOVA showed significant effects of the

CPP–ACP paste application and types of saliva-substitute solutions on the changes in surface

hardness of the softened enamel at a significance level of 0.05.

Conclusion: The application of CPP–ACP paste with continuous replenishment of saliva-like

solution for 48 h significantly hardened enamel softened by a cola drink. Biotene1

mouthwash softened enamel surface after 48 h contact.

# 2007 Elsevier Ltd. All rights reserved.

avai lable at www.sc iencedi rec t .com

journal homepage: www. int l .e lsev ierhea l th .com/ journals / jden

1. Introduction

Erosive tooth wear or dental erosion has gained more

attention from the dental profession since the decline in

dental caries in many industrialized countries. Dental erosion

is a localized loss of the tooth surface by a chemical process of

* Corresponding author at: 16-212 Moos Tower, 515 Delaware Street Sfax: +1 612 626 1484.

E-mail address: tant0002@umn.edu (D. Tantbirojn).

0300-5712/$ – see front matter # 2007 Elsevier Ltd. All rights reservedoi:10.1016/j.jdent.2007.10.008

acidic dissolution of nonbacterial origin.1 An epidemiological

study indicated that 16% of adults had erosion lesion(s) on

facial surfaces, and at least one occlusal erosion lesion with

dentin involvement were observed in 30% and 43% of adults

aged 26–30 and 46–50, respectively.2 Excessive consumption of

acidic food and beverages are one of the most common

extrinsic factors that cause dental erosion.3,4

E, Minneapolis, MN 55455, USA. Tel.: +1 612 625 0950;

d.

j o u r n a l o f d e n t i s t r y 3 6 ( 2 0 0 8 ) 7 4 – 7 9 75

Biological and chemical factors in the oral environment

influence the progress of dental erosion. Saliva provides

protective effects by neutralizing and clearing the acid. Saliva

is also a source of inorganic ions necessary for the

remineralization process.5 Enamel softened by acidic bev-

erages was rehardened following exposure to saliva or

artificial saliva.6,7 Patients with low or diminished salivary

flow are thus more susceptible to erosive tooth damage.4,8 In

addition to the buffering and remineralizing capacity, other

biological functions of saliva include lubrication and anti-

bacterial effects. For patients experiencing dry mouth, saliva

substitutes are often prescribed. One of the saliva substitutes

available as an over-the-counter product is Biotene1

Mouthwash (Laclede Inc., CA, USA). According to the

manufacturer, Biotene1 Mouthwash contains calcium, xyli-

tol, lactoferrin, and enzymes naturally found in human saliva

to provide the antibacterial function.

Calcium and phosphate ions are building blocks for the

remineralization process, and are found in saliva. The degree

of saturation of these ions differs from person to person,

among various salivary glands, and with secretion rate. The

dissolution and precipitation of tooth minerals depends on the

pH and the concentrations of ions in the fluid phase

surrounding the tooth structures.9 Recently, casein phospho-

peptide amorphous calcium phosphate complex (CPP–ACP)

has been introduced as a supplemental source of calcium and

phosphate ions in the oral environment. Casein phosphopep-

tides bind to calcium and phosphates in nano-particles,

preventing the crystals from growing to critical size and

precipitating out of solution.10 The amorphous calcium

phosphate is biologically active, and is able to release calcium

and phosphate ions to maintain the supersaturated state, thus

enhancing the remineralization process. Previous studies

have shown that CPP–ACP was effective in the remineraliza-

tion of carious lesions.11–13 A topical paste containing CPP–ACP

(ProspecTM MI Paste, GC Corporation, Tokyo, Japan) is

recommended by the manufacturer to reduce erosion. The

Fig. 1 – Overall experimental design. In Phase I, enamel surface

specimens received MI paste application and were subjected to

was measured at various stages and the changes in hardness w

question remains whether CPP–ACP can enhance reminer-

alization in the early stage of erosion as well.

It is well established that fluoride in the fluid phase

surrounding the tooth structures shifts the equilibrium

towards remineralization. Fluoride enhances remineraliza-

tion of early carious lesions by adsorbing onto the partially

dissolved crystal lattice, which attracts calcium and phos-

phate ions to precipitate.14 Fluoride should similarly enhance

the remineralization process of erosion lesions. In the initial

stage of erosion where a scaffold of mineral crystals still

remains, the lesion could be remineralized.6 When the surface

is completely lost, the erosion process cannot be reversed.6

Softening of the enamel surface is an early manifestation of

the erosion process. Reduced surface hardness which accom-

panies erosion of the enamel surface by acidic beverages can

be assessed using a physical measurement such as the

hardness test.5,15 The purpose of this study was to evaluate

the effect of CPP–ACP treatment and types of saliva-substitute

solutions on changes in enamel surface hardness softened by

a carbonated cola drink.

2. Materials and methods

The overall experimental design, shown in Fig. 1, consisted of

two phases. In Phase I, enamel surfaces were softened by

immersion in a carbonated cola drink. The softened speci-

mens were then subjected to different remineralizing proto-

cols in Phase II to assess the effect of CPP–ACP treatment on

the changes in enamel surface hardness under continuous

replenishment of various saliva-substitute solutions.

2.1. Phase I: surface microhardness measurement andenamel softening

Extracted bovine incisors were cut longitudinally, one half

served as a control and the other half as treatment. The

s were softened by a cola drink. In Phase II, the softened

three remineralizing protocols. Enamel surface hardness

ere calculated.

Fig. 2 – Specimens were under continuous replenishment of saliva-substitute solution by dripping the solution onto the

enamel surface at a rate of 0.4 mL/min.

j o u r n a l o f d e n t i s t r y 3 6 ( 2 0 0 8 ) 7 4 – 7 976

extracted teeth were fully erupted and obtained from animals

slaughtered at an age of 30 months and above. The specimens

were embedded in Orthodontic Resin (L.D. Caulk, Milford, DE).

The labial surfaces were ground wet using 240, 400, and

600 grit silicon carbide paper (Buehler, Lake Bluff, IL) and

polished with 1.0 and 0.05 mm alumina suspension (Buehler)

using an Ecomet 3 Grinder-Polisher (Buehler) to expose a flat

enamel, ca 3 mm � 6 mm. Baseline surface hardness of the

sound enamel was measured with a microhardness tester

(Micromet, 2004, Buehler) using a Knoop indenter at 50 g load

for 10 s. Four indentations per test were performed on each

specimen at every experimental stage.

The specimens were immersed in 6 mL of a cola drink

(Classic Coke#, Coca-Cola, Atlanta, GA) for 2 min at room

temperature before rinsing with deionized water. The pH of

coke measured with a pH meter (Accumet AB15, Fisher

Scientific, Pittsburgh, PA) was 2.7. Four consecutive intervals

of the immersion procedure were carried out. Knoop hardness

number (KHN) was measured after 4 and 8 min immersion.

The difference in KHNs between the baseline and 8 min

immersion (DKHNsoftening) of each specimen was calculated.

DKHNsoftening was used to assign the specimens to three

experimental groups in a balanced manner, i.e., each group

had a mixture of teeth ranging from low to high enamel

softening. Each experimental group consisted of eight sof-

tened enamel pairs (treatment and control halves).

2.2. Phase II: remineralization of softened enamel

Each softened enamel specimen was subjected to two inde-

pendent variables within the remineralizing protocol, namely, a

CPP–ACP treatment (ProspecTM MI Paste, GC Corporation,

Tokyo, Japan), and one of the saliva-substitute solutions. MI

paste was applied onto the surface of the specimens in the

treatment group for 3 min and wiped off, whereas the matching

control specimens received no treatment. The specimens were

placed under variable-speed low flow pumps (Control Com-

pany, Texas) for continuous replenishment of the saliva-

substitute solutions (Fig. 2) at room temperature. The pumps

were adjusted so that saliva-substitute solutions dripped at a

rate of 0.4 mL/min to mimic unstimulated saliva flow rate.16

Saliva-substitute solutions used in the three experimental

groups were: (1) saliva-like solution (SLS) containing 1.5 mM

CaCl2, 0.9 mM KH2PO4, 130 mM KCl, 20 mM HEPES, adjusted to

pH 7.0 with 1 M KOH, modified from Mukai et al.17; (2) SLS with

1 ppm NaF; (3) Biotene1 mouthwash (Laclede Inc., CA, USA).

The MI paste was applied at 0, 8, 24, and 32 h. The experiment

was completed after48 h of continuous dripping. Knoop surface

hardness was obtained at 24 and 48 h. The differences in KHNs

of enamel surface between the 8-min cola immersion and after

remineralization for 24 h (DKHNR24 h) and 48 h (DKHNR48 h) were

calculated.

2.3. Statistical analysis

Surface KHNs at various time intervals within each group were

compared using one-way ANOVA at a significance level 0.05,

followed by a Student–Newman–Keuls post hoc test. The

effects of MI paste treatment and various saliva-substitute

solutions on the changes in enamel surface hardness

(DKHNR24 h and DKHNR48 h) were analyzed using two-way

ANOVA and least squares means at a significance level 0.05.

3. Results

The average values of Knoop hardness number (KHN) of surface

enamel in each group measured at different time intervals

Fig. 3 – Enamel surface hardness at different time intervals

during the experiment. C4 min and C8 min are 4 and 8 min

immersion in a cola drink. R24 h and R48 h are 24 and 48 h

remineralization.

j o u r n a l o f d e n t i s t r y 3 6 ( 2 0 0 8 ) 7 4 – 7 9 77

during the course of the experiment are shown in Fig. 3. Surface

hardness values of sound enamel (baseline) were not signifi-

cantly different among the experimental groups (ANOVA,

p = 0.30). Immersion in a cola beverage for 4 and 8 min

significantly reduced enamel surface hardness (ANOVA and

Student–Newman–Keuls post hoc, p < 0.05). Surface hardness

of the softened enamel increased when the specimens were

placed under a continuous drip of saliva-like solution. However,

the hardness increased significantly only when the specimens

Table 1 – Mean (S.D.) of change in surface hardness (DKHNR24 h

remineralization protocols for 24 and 48 h

Time Treatment SLS

24 h MI paste 31.8 (8.0)

(DKHNR24 h) No treatment 2.9 (16.3

48 h MI paste 42.8 (12.4

(DKHNR48 h) No treatment 10.6 (21.0

SLS is saliva-like solution without fluoride, SLS/F is saliva-like soluti

significantly different ( p > 0.05) within the same time period.

Fig. 4 – Change in surface hardness of the softened enamel afte

letters (a–c) above each bar denote values that are not significan

saliva-like solution without fluoride, SLS/F is saliva-like solutio

were treated with MI paste, regardless of the presence of

fluoride in the saliva-like solution (ANOVA and Student–

Newman–Keuls post hoc, p < 0.05). Biotene1 mouthwash

significantly reduced surface hardness of the specimens

(ANOVA and Student–Newman–Keuls post hoc, p < 0.05).

Changes in enamel surface hardness after 8 min immer-

sion in cola beverage and after 24 h (DKHNR24 h) as well as 48 h

(DKHNR48 h) of remineralization are shown in Table 1 and

Fig. 4. Two-way ANOVA showed a significant effect of MI paste

application and various types of saliva-substitute solutions on

the changes in surface hardness of the softened enamel, but

there was no interaction between the two variables at the

significance level of 0.05. There was no difference in hardness

change between the saliva-like solutions with or without

1 ppm fluoride. The application of MI paste increased surface

hardness when the specimens were dripped with saliva-like

solutions. When the specimens were softened from the

Biotene1 drip, those that received MI paste exhibited less

hardness decrease than the untreated controls.

4. Discussion

Baseline microhardness values for enamel in this study

ranged from 244 to 337 KHN. These values are similar to

previous studies.18,19 Our study design required a sufficiently

flat area to allow microhardness measurements, thus the area

subjected to erosion was not the original surface enamel.

Microhardness decreases from the outer enamel surface

toward the dentinoenamel junction,18 which may explain

the range of baseline values. The average baseline hardness

and DKHNR48 h) of the softened enamel subjected to various

SLS/F Biotene1

a 28.2 (20.4) a b �81.1 (20.6)

) c 13.8 (13.2) b c �121.3 (18.2)

) a 38.8 (24.8) a �111.6 (14.9)

) b 18.0 (18.5) b �149.8 (13.9)

on with 1 ppm fluoride. Letters (a–c) denote values that are not

r 24 and 48 h of the remineralizing protocol. Lower case

tly different ( p > 0.05) within the same time period. SLS is

n with 1 ppm fluoride.

j o u r n a l o f d e n t i s t r y 3 6 ( 2 0 0 8 ) 7 4 – 7 978

values were not significantly different among the experi-

mental groups (ANOVA, p = 0.30).

Indentation hardness testing with either Knoop or Vickers

indenter have been used for the measurement of initial enamel

hardness, enamel softening as an early manifestation of the

erosion process, as well as enamel hardening after reminer-

alization.5,7,15,18–22 Both indenters are suitable for hardness

testing of non-metallic materials. The measurement of Knoop’s

long diagonal is less affected by elastic recovery than its short

diagonal or the equal diagonals of the 1368 diamond pyramid of

Vickers indenter.23 Knoop hardness number is expressed by the

formula: I = L/l2Cp, where I is Knoop hardness number, L is load

(kg), l is measured length of the long diagonal of the indentation

(mm), Cp is a constant equaling 7.028 � 10�2.23 Knoop hardness

number has been correlated with volume percent mineral of

enamel.24 The Knoopindenter with 50 g loadused inthe present

study is similar to a previous study where surface hardness

measurement was used to identify the effect of different factors

on enamel erosion.20 The 50 g load was selected because it

provided the appropriate size of indentations for accurate

measurement with the available equipment and the present

experimental design.

Devlin et al. measured the surface hardness of human

enamel exposed to Coca-Cola and artificial saliva.7 Similar to

the present study, they found that the hardness decreased

with the Coca-Cola exposure, and a partial recovery of

hardness when samples were exposed to artificial saliva.

However, the decrease was quite different between these two

experiments. After 8 min of exposure to the cola drink, we

found the surface hardness to be approximately 70% of the

baseline. Devlin et al. reported a reduction to 92.6% of the

baseline hardness after 1 h exposure, and 85.7% after 15 h.7

The difference could result from the substrates. In our

experiment we used bovine teeth while Devlin et al. used

human teeth. Artificial caries lesions formed in bovine tooth

enamel were twice as deep as those formed in human teeth.25

Another study using human enamel found 63% reduction in

Vickers hardness after the teeth were alternately immersed in

a cola drink and in artificial saliva for 5 s each and 10 cycles.15

In the present study, the cola drink was replenished every

2 min to ensure that it was carbonated and to reduce the

buffering effect from ions dissolved from the enamel surface.

This maintained the tooth in the initial stages of the erosion

process, without reaching the level of visible surface loss.

An agent applied onto a sound tooth surface may be

washed away by saliva or bind to biofilm. To simulate the

washing effect of saliva, we designed a technique to replenish

the saliva-substitute solutions by dripping them at 0.4 mL/

min. This prevents the accumulation of agents such as the MI

paste in a static fluid phase surrounding the tooth. The MI

paste was applied with a cotton tip applicator and wiped off

with Kimwipes (Kimberly-Clark, Roswell, GA) after 3 min.

Neither action should physically change the tooth surface. The

control group did not receive any treatment. The specimens

were subjected to continuous dripping with saliva-substitute

solutions. Despite this vigorous condition of continuous

dripping, the specimens treated with MI paste showed a

significant increase in hardness. It is speculated that ions from

the fluid phase readily diffused through the porous lesion and

deposited onto the partially demineralized enamel crystals.

A clinical study found that Biotene1 (mouthwash, tooth-

paste, and chewing gum) improved symptoms of radiation-

induced xerostomia in head and neck cancer patients.26

Biotene1 was chosen in this experiment because it contains

protein components that are not available in the saliva-like

solutions. Casein phosphopeptide not only stabilizes amor-

phous calcium phosphate, it also binds onto adsorbed macro-

molecules of biofilm on the tooth surface and serves as a

reservoir for calcium and phosphate ions.27 We expected that

the protein components of Biotene1 would enhance the

retention of the MI paste on the enamel surface. On the

contrary, prolonged contact with Biotene1 lowered the hard-

ness of enamel. We measured pH of Biotene1, and found it to be

5.24 which is far more acidic than human saliva. Indeed,

Biotene1 has previously been shown to cause significant

erosion of enamel of sound and predemineralized bovine

incisors, as well as predemineralized bovine dentin.28,29 It

should be noted that some conditions used in this experiment

were not the same as in a clinical setting. When Biotene1 was

used in this study, no saliva was present to buffer and supply

calcium and phosphate, while the hardest outer layer of enamel

was removed during polishing. The mouthwash was also

continuously dripped on the specimens for 2 days with no

intermission. Nevertheless, patients with extreme xerostomia

who display very limited salivary function should be cautioned

about overusing Biotene1 mouthwash.

Contrary to our study where no additional benefit was

found using the fluorinated SLS with CPP–ACP, Reynolds et al.

found an additive effect between CPP–ACP and fluoride to

prevent dental caries.30 This may be explained by differences

in the experimental designs. Reynolds et al. focussed on the

anticariogenicity of CPP–ACP in an in vivo rat model and caries

incidence. They also employed a much higher concentration

of fluoride (500 ppm) which was combined with CPP–ACP into

a solution before application.30 We attempted to remineralize

softened enamel, and monitored the change in hardness of the

surface enamel. Without the MI paste treatment, the hardness

of the softened enamel specimens increased more when the

saliva-like solution contained 1 ppm fluoride (Table 1 and

Fig. 4). However, with the MI paste treatment, no difference in

surface hardening was seen between saliva-like solution with

or without 1 ppm fluoride. The present study did not show an

additive effect of low fluoride level in saliva-like solution and

MI paste on the early stage of erosion. The remineralization

effect of MI paste may be enhanced by the application of

fluoridated toothpaste, which could be the subject for a future

study.

Artificial saliva was not used during the erosion phase in

the present study. Artificial saliva introduced during the

erosion stage would buffer the acidity from the cola drink and

limit the softening of the enamel surface. It was not the scope

of this study to examine the course of erosion, but rather the

remineralization process in which minerals precipitate onto

the enamel. For the remineralization phase, natural saliva and

acquired pellicle might influence the results provided that MI

paste is better retained on the enamel surface. It is speculated

that the effect of MI paste will be enhanced under oral

conditions in the presence of biofilm which can bind to casein

phosphopeptide and act as a reservoir for calcium and

phosphate ions. Although this study could not completely

j o u r n a l o f d e n t i s t r y 3 6 ( 2 0 0 8 ) 7 4 – 7 9 79

simulate the complex oral environment, it showed the

potential of CPP–ACP paste for reversing the harmful effect

of a cola drink on tooth surfaces.

5. Conclusions

The hardness of enamel significantly decreased after 8 min

immersion in a cola drink. This softened enamel, which

represented the early stage of erosion, became hardened after

four applications of a CPP–ACP paste along with continuous

replenishment of saliva-like solution for 48 h. The presence of

1 ppm fluoride in the saliva-like solution did not enhance the

hardness of softened enamel. Biotene1 mouthwash softened

enamel surface after 48 h contact.

Acknowledgements

This study is supported in part by a Summer Research Fellow

Program, University of Minnesota School of Dentistry, Non-

tenured Faculty Award, 3M Foundation, and the Minnesota

Dental Research Center for Biomaterials and Biomechanics.

The authors would like to thank Dr. A. Versluis for proof

reading the manuscript.

r e f e r e n c e s

1. Imfeld T. Dental erosion. Definition, classification and links.European Journal of Oral Sciences 1996;104:151–5.

2. Lussi A, Schaffner M, Hots P, Suter P. Dental erosion in apopulation of Swiss adults. Community Dentistry and OralEpidemiology 1991;19:286–90.

3. Lussi A, Jaeggi T, Schaffner M. Diet and dental erosion.Nutrition 2002;18:780–1.

4. Jarvinen VK, Rytomaa II, Heinonen OP. Risk factors in dentalerosion. Journal of Dental Research 1991;70:942–7.

5. Zero DT, Lussi A. Erosion—chemical and biological factor ofimportance to the dental practitioner. International DentalJournal 2005;55:285–90.

6. Amaechi BT, Higham SM. Dental erosion: possibleapproaches to prevention and control. Journal of Dentistry2005;33:243–52.

7. Devlin H, Bassiouny A, Boston D. Hardness of enamelexposed to Coca-Cola and artificial saliva. Journal of OralRehabilitation 2006;33:26–30.

8. Bevenius J, L’Estrange P. Chairside evaluation of salivaryparameters in patient with tooth surface loss: a pilot study.Australian Dental Journal 1990;35:219–21.

9. ten Cate B. The role of saliva in mineral equilibria—caries,erosion and calculus formation. In: Edgar M, Dawes C,O’Mullane D, editors. Saliva and Oral Health. 3rd ed. London:BDJ Books; 2004. p. 120–35.

10. Reynolds EC. Anticariogenic complexes of amorphouscalcium phosphate stabilized by casein phosphopeptides: areview. Special Care in Dentistry 1998;18:8–16.

11. Reynolds EC. Remineralization of enamel subsurfacelesions by casein phosphopeptide-stabilized calciumphosphate solutions. Journal of Dental Research 1997;76:1587–95.

12. Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC.Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopeptide-

amorphous calcium phosphate. Journal of Dental Research2001;80:2066–70.

13. Walker G, Cai F, Shen P, Reynolds C, Ward B, Fone C, et al.Increased remineralization of tooth enamel by milkcontaining added casein phosphopeptide-amorphouscalcium phosphate. Journal of Dairy Research 2006;73:74–8.

14. Featherstone JDC. The science and practice of cariesprevention. Journal of the American Dental Association2000;131:887–99.

15. Wongkhantee S, Patanapiradej V, Maneenut C, TantbirojnD. Effect of acidic food and drinks on surface hardness ofenamel, dentin, and tooth-coloured filing materials. Journalof Dentistry 2006;34:214–20.

16. Whelton H. Introduction: the anatomy and physiology ofsalivary glands. In: Edgar M, Dawes C, O’Mullane D, editors.Saliva and Oral Health. 3rd ed. London: BDJ Books; 2004. p. 9–10.

17. Mukai Y, Lagerweij MD, ten Cate JM. Effect of a solution withhigh fluoride concentration on remineralization of shallowand deep root surface caries in vitro. Caries Research2001;35:317–24.

18. Meredith N, Sherriff M, Setchell DJ, Swanson SA.Measurement of the microhardness and Young’s modulusof human enamel and dentin using an indentationtechnique. Archives of Oral Biology 1996;41:539–45.

19. Maupome G, Dies-de-Bonilla J, Torres-Villasenor G,Andrade-Delgado LDC, Castano VM. In vitro quantitativeassessment of enamel microhardness after exposure toeroding immersion in a cola drink. Caries Research1998;32:148–53.

20. Lussi A, Jaggi T, Scharer S. The influence of differentfactors on in vitro enamel erosion. Caries Research1993;27:387–93.

21. Schemehorn BR, Orban JC, Wood GD, Fischer GM.Remineralization by fluoride enhanced with calcium andphosphate ingredients. Journal of Clinical Dentistry 1999;10:13–6.

22. Munoz CA, Feller R, Haglund A, Triol CW, Winston AE.Strengthening of tooth enamel by a remineralizingtoothpaste after exposure to an acidic soft drink. Journal ofClinical Dentistry 1999;10:17–21.

23. Lysaght VE. Indentation Hardness Testing. New York:Reinhold Publishing Corp; 1949. p. 222–49.

24. Featherstone JDB, ten Cate JM, Shariati M, Arends J.Comparison of artificial caries-like lesions by quantitativemicroradiography and microhardness profiles. CariesResearch 1983;17:385–91.

25. Edmunds DH, Whittaker DK, Green RM. Suitability ofhuman, bovine, equine, and ovine tooth enamel for studiesof artificial bacterial carious lesions. Caries Research1988;22:327–36.

26. Warde P, Kroll B, O’Sullivan B, Aslandidis J, Tew-George E,Waldron J, et al. A phase II study of Biotene in the treatmentof postradiation xerostomia in patients with head and neckcancer. Support Care Cancer 2000;8:203–8.

27. Reynolds EC, Cai F, Shen P, Walker GD. Retention in plaqueand remineralization of enamel lesions by various forms ofcalcium in a mouthrinse or sugar-free chewing gum. Journalof Dental Research 2003;82:206–11.

28. Kielbassa A, Shohadai S, Schulte-Monting J. Effects of salivasubstitutes on mineral content of demineralized and soundenamel. Support Care Cancer 2000;9:40–7.

29. Meyer-Lueckel H, Schulte-Monting J, Kielbassa A. The effectof commercially available saliva substitutes onpredemineralized bovine dentin in vitro. Oral Diseases2002;8:192–8.

30. Reynolds EC, Cain CJ, Webber FL, Riley PF, Johnson IH, PerichJW. Anticariogenicity of calcium phosphate complexes oftryptic casein phosphopeptides in the rat. Journal of DentalResearch 1995;74:1272–9.