See

discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/44890310

Comparison of remineralization potential of

CPP-ACP and CPP-ACP with 900 ppm fluoride
on eroded human enamel: An In-situ study
ARTICLE in ARCHIVES OF ORAL BIOLOGY JULY 2010
Impact Factor: 1.74 DOI: 10.1016/j.archoralbio.2010.05.002 Source: PubMed

CITATIONS

READS

38

155

3 AUTHORS, INCLUDING:
Narasimhan Srinivasan
Mahatma Gandhi Postgraduate Institute of
7 PUBLICATIONS 79 CITATIONS
SEE PROFILE

Available from: Narasimhan Srinivasan

Retrieved on: 06 October 2015

archives of oral biology 55 (2010) 541544

available at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/aob

Comparison of the remineralization potential of CPPACP and

CPPACP with 900 ppm fluoride on eroded human enamel:
An in situ study
N. Srinivasan *, M. Kavitha, S.C. Loganathan
Department of Conservative Dentistry and Endodontics, Tamilnadu Government Dental College and Hospital, Chennai, Tamilnadu 600003, India

article info

abstract

Article history:

Objective: The aim of this in situ study was to compare the remineralization potential of

Accepted 10 May 2010

pastes containing CPPACP and CPPACP with 900 ppm fluoride on human enamel softened
by a cola drink.

Keywords:

Design: Forty-five enamel specimens obtained from human third molar teeth were eroded in

CPPACP

a cola drink for 8 min and then attached to intra-oral devices worn by five volunteers. The

CPPACFP

specimens were subjected to three different in situ remineralization protocols using: (1)

Erosion

CPPACP (Group I), (2) CPPACP with 900 ppm fluoride (Group II), and (3) saliva (Group III,

Demineralization

control). Vickers microhardness measurements were obtained at baseline followed by

Remineralization

demineralization and remineralization stages.

Results: The CPPACP, CPPACP with 900 ppm fluoride and saliva controls resulted in
46.24%, 64.25% and 2.98% increase in post-erosion microhardness values, respectively.
One-way ANOVA revealed statistically significant differences in the mean microhardness
values between pastes containing CPPACP and CPPACP with 900 ppm fluoride.
Conclusions: Both CPPACP and CPPACP with 900 ppm fluoride substantially remineralized
the softened enamel, with the CPPACP and fluoride combination showing higher remineralization potential than CPPACP. This study confirmed the synergistic effect of fluoride
with CPPACP on remineralization of eroded enamel.
# 2010 Elsevier Ltd. All rights reserved.

1.

Introduction

The prevalence of dental caries has declined in developed

nations in recent decades,1 but erosive tooth wear has gained
wider interest from dental practitioners, researchers and the
community at large.2 There is some evidence that many
children and young adults are presenting with erosive tooth
wear.3 This has been associated with an increasing trend in
the consumption of acidic beverages4 which cause irreversible
erosive demineralization of dental hard tissues.5 Such eroded
tooth structure has a shallow, softened sub-surface zone that

is more liable to abrasive wear by mechanical forces,6,7 but this

softened zone can be hardened (remineralized) by fluoride.8 In
this context, other anticariogenic agents involving calcium
and phosphate ions may have an important role in the
prevention and management of dental erosion.
Casein phosphopeptide amorphous calcium phosphate
(CPPACP) nanocomplexes are casein-derived peptides in
which ACP is stabilized by CPP, and these nanocomplexes
act as a calcium and phosphate reservoir when incorporated
into the dental plaque and on the tooth surface.9 CPPACP
has been shown to reduce demineralization and promote

* Corresponding author at: Department of Conservative Dentistry and Endodontics, Tamilnadu Government Dental College and Hospital,
Chennai, Tamilnadu 600003, India. Tel.: +91 98404 79056.
E-mail address: nbalaji_82@yahoo.com (N. Srinivasan).
00039969/$ see front matter # 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2010.05.002

542

archives of oral biology 55 (2010) 541544

remineralization of carious lesions both in vitro and in situ914

and to reduce erosive tooth wear in vitro.1517 CPP also
stabilizes amorphous calcium fluoride phosphate18 (ACFP)
that has a greater remineralizing effect on carious lesions
compared with those of fluoride or CPPACP individually.19,20
To our knowledge there are no previous studies investigating
the combined effect of CPPACP and fluoride in remineralizing
erosion lesions.
Although in vitro studies have greatly improved our
understanding of demineralization and remineralization process, it cannot simulate the complex nature of oral environment. On the other hand, in situ studies serve as a bridge
between the natural uncontrolled clinical situation and highly
controlled laboratory situation. Hence they are more desirable
to obtain clinically relevant information on remineralization of
eroded tooth structure. Thus the aim of this in situ study was to
assess the remineralization effect of eroded human enamel by
CPPACP, CPPACP with 900 ppm fluoride and natural human
saliva using surface microhardness (SMH) analysis.

2.

Materials and methods

2.1.

Ethical approval and study design

The Institutional Ethical Committee of the Tamilnadu Government Dental College and Hospital, Chennai approved this
study (0421/DE/08). All subjects provided informed written
consent. A single blind study with two different formulations
of remineralization pastes (MI pastes) was conducted. Five
healthy adult volunteers (including two men and three
women) with a mean age of 23 years (range 1928 years) took
part in a four-day study. They wore acrylic palatal appliances,
with each appliance containing two rows of three eroded
(softened) enamel specimens obtained from extracted human
third molar teeth. The left row corresponded to the control
group and the right row corresponded to one of the treatment
groups. Changes in the enamel microhardness were assessed
using SMH analysis.

2.2.
Subject recruitment and assessment of salivary
parameters
The inclusion criteria for this study included the presence of at
least 22 natural teeth with no active caries, periodontal
disease, or other oral pathology. None of the subjects were
using antimicrobials or medications that could affect saliva
quality and flow. Saliva test conducted for each subject 1 h
before breakfast at the beginning of the study showed normal
saliva flow rates and pH values. The unstimulated salivary
flow rate exceeded 0.2 ml/min, and the stimulated flow rate
measured after chewing a pellet of sugar-free chewing gum
exceeded 1.0 ml/min. The salivary pH values were found to be
in the range 6.87.1.

2.3.
Fabrication of intra-oral appliances and enamel
specimen preparation
Removable acrylic mid-palatal appliances were fabricated,
extending from the first premolar to the second molar teeth.

Two 19 gauge stainless steel circumferential clasps were

placed on first premolar and second molar teeth on each side
to provide retention for the appliances. Two bilateral troughs
(20 mm long  10 mm wide  2 mm deep) were cut into the
acrylic base parallel to the alignment of posterior teeth in
the dental arch. Forty-five enamel sections of dimensions
8 mm  4 mm  2 mm were obtained from the buccal and
lingual aspects of 28 extracted, unerupted human third molar
teeth using water-cooled diamond disk. The teeth had been
stored in 10% (v/v) of formalin (pH 7.0) for two weeks at room
temperature (25.0 8C).

2.4.
Surface microhardness assessment and erosion
experiments
Enamel specimens were mounted on an acrylic resin block,
and their outer surfaces were polished flat with water-cooled
carborundum disks (320, 600, 1000 and 1200 grades of Al2O3
papers) followed by a metallographic polisher (up to 1 mm size
of diamond spray) at low speed before baseline SMH values
were obtained. This polishing procedure removed approximately 200 mm of the tooth surface and the polished surface
area of the enamel specimens was nearly 6 mm  3 mm.
Vickers hardness number (VHN) was determined by making
five indentations in different regions of each specimen using a
square based diamond pyramid Vickers indenter under a load
of 70 g for 15 s. The indentations were made 100 mm apart from
each other to avoid residual stress. The mean (SD) baseline
SMH value was noted to be 324 (8.91). All specimens were
then immersed in 5 ml of fresh cola drink (Coca-Cola, India)
(pH 2.3) for 8 min at room temperature (25.0 8C) followed by a
second SMH assessment.

2.5.

In situ remineralization of eroded enamel specimens

The eroded (softened) enamel specimens were randomly

divided into three groups and were subjected to different
remineralization protocols involving treatment with (1) CPP
ACP (Tooth Mousse, GC Corporation, Japan) in Group I, (2) CPP
ACP with 900 ppm fluoride (Tooth Mousse Plus, GC Corporation, Japan) in Group II, and (3) saliva in Group III (control
group) (Table 1).
During the first two days, enamel specimens assigned to
Group I were inset on the right trough of the appliances and
they received a single intra-oral treatment of the CPPACP
paste in the beginning of each day for 3 min as per the
manufacturers instructions. After the CPPACP treatment the
subjects rinsed their mouths and washed the appliances with
running water for 1 min. Control enamel specimens (Group III)
were then inset on the left trough of the appliances. These
specimens were not subjected to treatment with CPPACP or
CPPACP with 900 ppm fluoride. The appliances were then
worn for two days except during periods described below.
After two days the Group III specimens were removed and the
Group I enamel specimens were replaced by Group II enamel
specimens. The Group II enamel specimens received a single
intra-oral treatment of CPPACP with 900 ppm fluoride for
3 min using protocols described for CPPACP. The appliances
with only the Group II enamel specimens inset were then worn
for a further two days except during periods described below.

543

archives of oral biology 55 (2010) 541544

Table 1 In situ remineralization of eroded enamel specimens.

Treatment groups

Treatment received

Group I

Description

In situ CPPACP treatment

for first two days
In situ CPPACP + 900 ppm
F treatment for next two days
Control group received no treatment.
Kept in situ for first two days

Group II
Group III

Three enamel specimens were placed on the right side trough

of appliance in five subjects
Group I specimens were replaced by Group II specimens on
the right side trough
Three enamel specimens kept on the left side of appliance

Table 2 Mean W SD of SMH values and percentage changes in mean SMH values (%SMHC) at various experimental stages.

SMH mean  SD
(%SMHC)

Baseline

Post-erosion
stage

Group I
(CPPACP)

Group II
(CPPACP + 900 ppm F)

324.09  8.91 a

244.54  5.85 b
# 24.54%

281.33  3.56 c
" 46.24%

295.65  6.20 d
" 64.25%

Group III
(saliva control)
246.91  5.29 b
" 2.98%

Different superscripts indicate significant differences in the mean SMH values among the various experimental stages ( p < 0.05).
(#) Indicates decrease in %SMHC from the mean baseline SMH value.
(") Indicates increase in %SMHC from the mean softened SMH value.

During the experimental period, the appliances were worn

for at least 10 h during daytime. The subjects removed their
appliances while performing oral hygiene procedures, eating,
drinking and sleeping. After removing the appliances, each
subject rinsed it briefly with distilled water for few seconds
and then stored it in a sealed, humidified container (zip-lock
bag with a small amount of distilled water) until reinsertion.
The subjects rinsed their mouths with water after eating or
drinking and waited for 10 min before reinserting the
appliances. None of the subjects consumed acidic beverages
and foods during the experimental period. At the completion
of remineralization stages, the enamel specimens were stored
in distilled water for further SMH analysis.

2.6.

Statistical analysis

The microhardness values of different groups were compared

using one-way analysis of variance (ANOVA) followed by
Tukey-HSD post hoc test (GraphPad Instat computer software
program, Version 3.06, GraphPad Software Inc., CA, USA). The
significance was set at the 5% probability level.

3.

Results

Microhardness assessment
The mean (SD) SMH values and percentage changes in
surface microhardness values (%SMHC) at various stages are
presented in Table 2. The mean SMH value of eroded enamel
specimens was 244.54 and those after treatment with
remineralizing agents (CPPACP, CPPACP with 900 ppm
fluoride and saliva control) were 281.33, 295.65 and 246.91,
respectively. There were significant increases in SMH values
when the eroded enamel specimens were treated with CPP
ACP (Group I) and CPPACP with 900 ppm fluoride (Group II)
( p < 0.05), but not with saliva ( p > 0.05). Both the pastes
substantially hardened the softened enamel than saliva alone
(Group III). In addition, SMH values in Group II were
significantly greater than those in Group I ( p < 0.05), with

percentage surface microhardness (%SMH) increase from the

post-erosion stage being 64.25% for CPPACP with 900 ppm
fluoride compared with 46.24% for CPPACP alone.

4.

Discussion

Microhardness assessment needs a flat and polished surface

to enable accurate measurements, thus the area subjected to
erosion was not the ideal surface enamel. Moreover during
polishing, scratch lines were developed in few samples and we
encountered difficulty in achieving a smooth enamel surface.
On exposure to a cola drink for 8 min, we found that the
surface microhardness value decreased to 75.4% of the
baseline value. This value is similar to that reported by
Tantbirojn et al.15 after erosion of bovine enamel in a cola
drink for 8 min. Another study using human enamel found a
63% reduction in Vickers hardness after the teeth were
alternately immersed in a cola drink and artificial saliva.21
Both the remineralizing pastes resulted in a significant
increase in post-erosion microhardness in the present study
(Table 2). It has been proposed that the remineralization
mechanism of CPPACP involves localization of ACP at the
tooth surface, which buffers free calcium and phosphate
ions.22 By maintaining a state of supersaturation with respect
to the hydroxyapatite, these ions depress demineralization
and promote remineralization.
In this study, the remineralization effect of CPPACP with
900 ppm of fluoride was found to be superior to that of CPP
ACP alone (Table 2). It is likely that a combination of CPPACP
and fluoride resulted in co-localization of calcium and
phosphate ions with fluoride ions at the enamel surface,
presumably as CPPACFP nanocomplexes.18 Our findings are
in agreement with those of recent in situ20 and in vitro23
studies that have documented the synergistic effect of CPP
ACP and fluoride in remineralization of carious lesions.
However, erosion and carious lesions have different surface
and sub-surface characteristics. Erosion is characterized
predominantly by surface demineralization with a very
shallow sub-surface lesion, whereas incipient carious lesions

544

archives of oral biology 55 (2010) 541544

have a relatively intact enamel surface24 and a much deeper

sub-surface lesion. Given the differences in the depths of subsurface lesions, the nature of remineralization is probably
different in these lesions.
Remineralization of the eroded lesions may occur by
deposition of mineral into the porous zone rather than growth
of the eroded crystals.25 Hydroxyapatite crystals are formed in
the oral environment from supersaturated calcium and
phosphates ions, and the presence of fluoride ions is further
likely to promote the remineralization process by forming
fluorapatite crystals that are more resistant to future
demineralization than hydroxyapatite.26
Saliva resulted in slight remineralization of enamel surface
(2.98%), which was much lower compared with those of MI
pastes (Table 2). Several studies have reported the remineralization potential of stimulated saliva on erosion.27,28 As the
remineralization potential of eroded lesions by saliva seems to
be limited, it is recommended that a remineralizing agent be
applied shortly after an erosive challenge to prevent abrasion
of softened tooth structure from mechanical forces (from
mastication and friction from oral soft tissues).29
Within the limitations of low sample size and short-term
nature of this in situ study, we conclude that CPPACP
exhibited a higher remineralizing potential when combined
with 900 ppm fluoride than used alone. It is recommended
that CPPACFP be used as a regular self-applied topical coating
to prevent erosive tooth wear from acidic beverages.
Funding: None.
Competing interests: The authors declare that they do not
have competing interests.
Ethical approval: This study was approved by Institutional
Ethical Committee, Tamilnadu Government Dental College
and Hospital, Chennai (Reference No.: 0421/DE/08).

references

1. Peterson G, Bratthall D. The caries decline: a review of

reviews. Eur J Oral Sci 1996;104:43643.
2. Lussi A. Erosive tooth wear a multifactorial condition of
growing concern and increasing knowledge. Monogr Oral Sci
2006;20:18.
3. Jaeggi T, Lussi A. Prevalence, incidence and distribution of
erosion. Monogr Oral Sci 2006;20:4465.
4. Lussi A, Jaeggi T, Zero D. The role of diet in the aetiology of
dental erosion. Caries Res 2004;38:3444.
5. Imfeld T. Dental erosion: definition, classification and links.
Eur J Oral Sci 1996;4:1515.
6. Davis WB, Winter PJ. The effect of abrasion on enamel and
dentine and exposure to dietary acid. Br Dent J 1980;148:2536.
7. Jaeggi T, Lussi A. Toothbrush abrasion of erosively altered
enamel after intraoral exposure to saliva: an in situ study.
Caries Res 1999;33:45561.
8. Amaechi BT, Higham SM. Dental erosion: possible
approaches to prevention and control. J Dent 2005;33:24352.
9. Reynolds EC, Cai F, Shen P, Walker GD. Retention in plaque
and remineralization of enamel lesions by various forms of
calcium in a mouthrinse or sugar-free chewing gum. J Dent
Res 2003;82:20611.
10. Reynolds EC. Remineralization of enamel subsurface lesions
by casein phosphopeptide stabilized calcium phosphate
solutions. J Dent Res 1997;76:158795.

11. Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC.

Remineralization of enamel subsurface lesions by sugarfree chewing gum containing casein phosphopeptide
amorphous calcium phosphate. J Dent Res 2001;80:206670.
12. Iijima Y, Cai F, Shen P, Walker G, Reynolds C, Reynolds EC.
Acid resistance of enamel subsurface lesions remineralized
by a sugar-free chewing gum containing casein
phosphopeptideamorphous calcium phosphate. Caries Res
2004;38:5516.
13. Yamaguchi K, Miyazaki M, Takamizawa T, Inage H, Moore
BK. Effects of CPPACP paste on mechanical properties of
bovine enamel as determined by an ultrasonic device. J Dent
2006;34:2306.
14. Kumar VL, Itthagarun A, King NM. The effect of casein
phosphopeptideamorphous calcium phosphate on
remineralization of artificial caries-like lesions: an in vitro
study. Aust Dent J 2008;53:3440.
15. Tantbirojn D, Huang A, Ericson MD, Poolthong S. Change in
surface hardness of enamel by a cola drink and a CPPACP
paste. J Dent 2008;36:749.
16. Ranjitkar S, Narayana T, Kaidonis JA, Hughes TE, Richards
LC, Townsend GC. The effect of casein phosphopeptide
amorphous calcium phosphate on erosive dentine wear.
Aust Dent J 2009;54:1017.
17. Panich M, Poolthong S. The effect of casein
phosphopeptideamorphous calcium phosphate and a cola
soft drink on in vitro enamel hardness. J Am Dent Assoc
2009;140:45560.
18. Cross KJ, Huq NL, Stanton DP, Sum M, Reynolds EC. NMR
studies of novel calcium, phosphate and fluoride delivery
vehicle-alpha (S1) casein (5979) stabilized amorphous
calcium fluoride phosphate nanocomplexes. Biomaterials
2004;25:50619.
19. Reynolds EC, Cain CJ, Webber FL, Riley PF, Johnson IH, Perich
JW, et al. Anticariogenicity of calcium phosphate complexes
of tryptic casein phosphopeptides in the rat. J Dent Res
1995;74:12729.
20. Reynolds EC, Cai F, Cochrane NJ, Shen P, Walker GD, Morgan
MV, et al. Fluoride and casein phosphopeptideamorphous
calcium phosphate. J Dent Res 2008;87:3448.
21. Wongkhantee S, Patanapiradej V, Maneenut C, Tantbirojn
D. Effect of acidic food and drinks on surface hardness of
enamel, dentin, and tooth-coloured filling materials. J Dent
2006;34:21420.
22. Reynolds EC, Black CL. Reduction of chocolates
cariogenicity by supplementation with sodium caseinate.
Caries Res 1987;21:44551.
23. Cochrane NJ, Saranathan S, Cai F, Cross KJ, Reynolds EC.
Enamel subsurface lesion remineralization with casein
phosphopeptide stabilized solutions of calcium, phosphate
and fluoride. Caries Res 2008;42:8897.
24. Attin T. Methods for assessment of dental erosion. Monogr
Oral Sci 2006;20:15272.
25. Eisenburger M, Addy M, Hughes JA, Shellis RP. Effect of time
on the remineralisation of enamel by synthetic saliva after
citric acid erosion. Caries Res 2001;35:2115.
26. Featherstone JD, Lussi A. Understanding the chemistry of
dental erosion. Monogr Oral Sci 2006;20:6676.
27. Hall AF, Buchanan CA, Millett DT, Creanor SL, Strang R, Foye
RH. The effect of saliva on enamel and dentine erosion. J
Dent 1999;27:3339.
28. Gedalia I, Ionat-Bendat D, Ben-Mosheh S, Shapira L. Tooth
enamel softening with a cola type drink and rehardening
with hard cheese or stimulated saliva in situ. J Oral Rehabil
1991;18:5016.
29. Amaechi BT, Higham SM, Edgar WM. Influence of abrasion
on the clinical manifestation of human dental erosion. J Oral
Rehabil 2003;30:40713.