[Downloaded free from [Link] on Wednesday, June 24, 2015, IP: 122.168.21.

197]

Atomic force microscopic comparison of remineralization with

casein‑phosphopeptide amorphous calcium phosphate paste, acidulated
phosphate fluoride gel and iron supplement in primary and permanent
teeth: An in‑vitro study
Nikita Agrawal, Shashikiran N. D., Shilpy Singla, Ravi K. S. 1, Vinaya Kumar Kulkarni2

Abstract
Context: Demineralization of tooth by erosion is caused by frequent contact between the tooth surface and acids present in soft
drinks. Aim: The present study objective was to evaluate the remineralization potential of casein‑phosphopeptide‑amorphous
calcium phosphate (CPP‑ACP) paste, 1.23% acidulated phosphate fluoride (APF) gel and iron supplement on dental erosion by
soft drinks in human primary and permanent enamel using atomic force microscopy (AFM). Materials and Methods: Specimens
were made from extracted 15 primary and 15 permanent teeth which were randomly divided into three treatment groups: CPP‑ACP
paste, APF gel and iron supplement. AFM was used for baseline readings followed by demineralization and remineralization
cycle. Results and Statistics: Almost all group of samples showed remineralization that is a reduction in surface roughness
which was higher with CPP‑ACP paste. Statistical analysis was performed using by one‑way ANOVA and Mann‑Whitney U‑test
with P < 0.05. Conclusions: It can be concluded that the application of CPP‑ACP paste is effective on preventing dental erosion
from soft drinks.

Keywords: Atomic force microscope, dental erosion, remineralization

Introduction The sucralose soft drinks report published annually

suggests that consumption of soft drinks had increased
Dental erosion is a destructive process that renders the tooth consistently over past 15 years.[6] Such drinks causes a drop
surface hypomineralized, leading to an irreversible loss and in plaque pH and on prolonged exposures leads to dental
its progressive softening thereby increases its susceptibility erosion.[7] According to Jaeggi and Lussi distribution of
toward the mechanical forces.[1,2] Epidemiological studies had erosion in the primary dentition is on occlusal surface of
shown the prevalence of dental erosion in children which molars predominantly and scarcely on the facial surface of
varies widely, ranging from 2% to 57% respectively.[3‑5] maxillary incisors although in the permanent dentition it
is seen on palatal surface of maxillary incisors.[8,9] Besides
that the susceptibility of teeth toward erosion also
Department of Pedodontics and Preventive Dentistry, People’s
accounts on its mineral content, porosity and degree of
College of Dental Science and Research Center, Bhanpur, Bhopal,
enamel microcrystal arrangement as primary teeth been
Madhya Pradesh, India, 1Department of Preventive Dental
Sciences, Division of Pedodontics, College of Dentistry, King
the susceptible one.[10] Eventually erosion is complete
Khalid University, Abha 61471, Assir, Kingdom of Saudi Arabia,
dissolution of hydroxy‑apatite (HAP) that occurs layer
2
Department of Pedodontics and Preventive Dentistry, Modern by layer while carious lesion formation occur by partial
Dental College, Indore, Madhya Pradesh, India dissolution of HAP.[11]

Correspondence: Dr. Nikita Agrawal, Department of Pedodontics The substantial reduction in the intake of such beverages is
and Preventive Dentistry, People’s College of Dental Sciences imperative to restrain the process of erosion.[12] Certain bioactive
and Research Center, People’s Campus, Bhanpur, Bhopal, agents for remineralization are also advocated including
Madhya Pradesh, India. E‑mail: [Link]@[Link] non‑fluoridated agents like casein‑phosphopeptide (CPP)
with amorphous calcium phosphate (ACP) which forms a
Access this article online protective layer and inhibits demineralization.[13,14] Recently
Quick Response Code: introduced iron supplement result in precipitation of ferric
Website: phosphate on the enamel, rendering it more resistant to
[Link]
erosion.[15,16] Correspondingly the iron remineralizes eroded
enamel by apatite nucleation and substitution of calcium in
DOI: apatite as well.[17] Moreover most widely used are fluoridated
10.4103/0976-237X.128672 agent like acidulated phosphate fluoride (APF) gel which
acts by deposition of calcium fluoride (CaF2) predominantly

75 Contemporary Clinical Dentistry | Jan-Mar 2014 | Vol 5 | Issue 1

[Downloaded free from [Link] on Wednesday, June 24, 2015, IP: [Link]]

Agrawal, et al.: Atomic force microscope comparison of CPP‑ACP paste, APF gel and iron supplement remineralization

on the enamel surface which successfully transformed into Group I: CPP‑ACP paste‑applied with applicator tip/brush
fluorapatite crystals.[18,19] for 3 min[23]
Group II: APF gel‑applied with applicator tip/brush for
Dental erosion has been widely investigated through a 4 min[26]
number of macro‑microscopic techniques. The novel one is Group III: Iron supplement‑freshly prepared 6 ml 10 mM/L
atomic force microscope having images in three dimensions, ferrous sulfate solution/specimen for 3 min.[27]
at atomic resolution with minimal sample preparation.[20]
On the contrary scanning electron microscope (SEM) has Atomic force microscope AutoProbe CP 100
the ability to image very rough samples due to its large (Themormicroscopes, Veeco, USA) used for the assessment of
depth of field and lateral field of view. [21] Atomic force surface roughness specimens equipped with a piezoelectric
microscopy (AFM) is widely recommended to study the scanner of 100 mm × 100 mm with a range of 7 mm in the
bio‑minerals and also applied to measure the early stages z‑direction. The root‑mean‑square roughness (Rrms) was obtained
of enamel loss.[22] But contemporary the remineralization of from the AFM investigations with a film area of 30 µm × 30 µm
erosion cavities is not widely investigated using AFM. and resolution of 256 × 256 pixels. The labial surface of each
sample was analyzed for at least 10 different sites.[23] Differences
Thus, this paper aim to evaluate the remineralization potential in the mean values among the groups were analyzed by one way
of CPP‑ACP paste, 1.23% APF gel and iron supplement on ANOVA and Mann‑Whitney U‑test with P < 0.05.
dental erosion produced by soft drinks in human primary
and permanent enamel using AFM. Results

Materials and Methods The pH of soft drink was 2.27 ± 0.04 at 25°C. The mean Rrms
of enamel surface at each phases of treatment were shown
A total of 30 teeth specimens were used in the study, in Table 1. The Rrms values of un‑exposed (baseline) samples
of which 15 were primary teeth and the remaining 15 were in the range of 103.287 ± 5.4‑129.406 ± 64.7 nm
permanent teeth. After extraction, teeth were cleaned and 127.9 ± 61.4‑170.776 ± 80.7 nm in primary and
of soft‑tissue debris by ultrasonic scaler, inspected for permanent teeth respectively. After demineralization, all
cracks, hypoplasia or any white spot lesion under light groups showed an increase in Rrms while on application of
microscope 10X (iNEA Olympus Pvt. Ltd., New Delhi). remineralizing agent a marked reduction in Rrms was present
Moreover disinfected in 5% sodium hypochlorite for 1 h and in Group I (CPP‑ACP paste) with 74.709 ± 19.01 and
stored in de‑ionized water (pH = 7.413). The specimens 125.17 ± 27.4 nm followed by Group II (APF gel) with
were sectioned transversely in the cervical area to separate 66.357 ± 25.1 and 116.259 ± 30.7 and Group III (Iron) with
crown from the root and sliced longitudinally for the labial 146.589 ± 29.6 nm in permanent teeth while an increase
portion using diamond disc (Dentorium, Germany) with a in Rrms was seen with 152.696 ± 98.5 nm in primary teeth.
water spray. The labial segments of tooth specimens were The change in Δ Rrms i.e. Δ UNEXP‑DEMIN, Δ UNEXP‑REMIN,
placed in die stone mold measuring 10 mm × 8mm × 2mm Δ REMIN‑DEMIN was statistically not significant in CPP,
in dimension and embedded in acrylic resin (Dentsply, APF and Iron Group (intra‑group) for primary teeth and
New Delhi). The labial surface of each enamel specimen permanent (P > 0.05 ANOVA) as shown in Table 1.
was grounded using silicon carbide papers (3M products)
with consecutive grades of 600, 1000 and 1200 under When the changes in Rrms were compared between the
water irrigation to remove 50‑100 µm for producing a flat groups (inter‑group: CPP, APF and Iron Group), differences
surface.[23] were statistically not significant for both primary and
permanent teeth as shown in Figure 1. When primary and
The pH of soft drink was determined using digital pH permanent teeth were compared, there were no significant
meter (U‑Tech, Singapore). The primary and permanent differences in Δ R rms for different groups (P > 0.05
teeth specimen were randomly divided into three Mann‑Whitney U‑test).
groups (with n = 5). Then specimens were subjected to a
demineralization cycle by immersing in 6 ml of soft drink In all the groups, unexposed samples was quite smooth
for 2 min stirred at constant speed of 120 rpms[24,25] at room except for some scratches. All the demineralized samples
temperature at four consecutive intervals of 6 h imitating showed “honey‑comb” appearance which on remineralization
the meal time. The application of remineralizing agents undergoes surface deposition as shown in Figures 2 and 3
were carried out at 0, 8, 24 and 36 h intervals[23] according of primary and permanent teeth respectively.
to manufacturer’s instruction and stored for further
assessment. In between the experiment the specimen were Discussion
stored in de‑ionized water. The AFM observations were
made after each phase i.e. baseline, demineralization and The aprisamtic outermost layer of enamel had a dense
remineralization. arrangement, less permeable because of its high mineral

Contemporary Clinical Dentistry | Jan-Mar 2014 | Vol 5 | Issue 1 76

[Downloaded free from [Link] on Wednesday, June 24, 2015, IP: [Link]]

Agrawal, et al.: Atomic force microscope comparison of CPP‑ACP paste, APF gel and iron supplement

S 
S 

S 
S  S 
S 

a b
Figure 1: Change in root mean square roughness after each phase (unexposed, demineralization and remineralization) within
all the treatment groups of (a) promary and (b) permanent teeth

a b c

e f
d

g h i

Figure 2: Primry tyeeth; (a,b,c) unexposed sample of group I, II,III; (d,e,f) demineralized sample of group I,II,III; (g,h,i) remineralized
sample of group I,II,III respectively

content and gradually wears off.[23,28,29] To standardize the the artificial saliva enhances the action of CPP‑ACP paste by
specimens were grounded flat and polished, eliminating interacting with hydrogen ions and forms calcium hydrogen
natural variations from enamel surface. [10,23,30] Further phosphate lead to enamel mineralization.[32] Remineralization
specimens were stored in de‑ionized water which is in cycle was carried out at an interval of 0, 8, 24 and 36 h
accordance to the technical report of the ISO (1991).[31] The so as to stimulate the application of any topically applied
complexity of the oral environment was not stimulate as remineralizing agent.[23]

77 Contemporary Clinical Dentistry | Jan-Mar 2014 | Vol 5 | Issue 1

[Downloaded free from [Link] on Wednesday, June 24, 2015, IP: [Link]]

Agrawal, et al.: Atomic force microscope comparison of CPP‑ACP paste, APF gel and iron supplement remineralization

Table 1: Mean surface roughness values (nm) and within group comparison using one way ANOVA
Mean (VHN)±SD
Group (n=5) ANOVA
Unexposed Demineralization Remineralization
Primary
CPP‑ACP 111.521±14.1 138.916±28.38 74.709±19.01 0.593*
APF gel 103.287±5.4 111.592±23.9 66.357±25.1 0.622*
Iron 129.406±64.7 130.227±42.4 152.696±98.5 0.264*
Permanent
CPP‑ACP 170.776±80.7 175.476±44.8 125.17±27.4 0.802*
APF gel 127.9±61.4 132.712±42.2 116.259±30.7 0.527*
Iron 137.735±20.9 158.582±36.9 146.589±29.6 0.546*
*Not significant P<0.05. VHN: Vickers hardness number; CPP: Casein‑phosphopeptide; ACP: Amorphous calcium phosphate; SD: Standard deviation;
APF: Acidulated phosphate fluoride

a b c

d e f

g h i

Figure 3: Permanent teeth; (a,b,c) unexposed sample of group I, II,III; (d,e,f) demineralized sample of group I,II,III;
(g,h,i)remineralized sample of group I,II,III respectively

The baseline Rrms of the present study was higher than the surface roughness of primary and permanent teeth respectively.
previous studies; with a range of 49‑56 nm by Murakami et al. Previous study had similar mean surface roughness with a range
but was almost similar according to Quartarone et al. with a of 140‑290 nm after demineralization.[23] A marked reduction
range of 50‑120 nm.[20,33] This variation in surface roughness in surface roughness was present in Group I of primary teeth
accounts on anatomical topography of tooth varying with when compared with permanent teeth but was not statistically
geographical location, age, oral environment present. Following significant. The present result elucidated the post‑eruptive
demineralization no significant difference was present in mean enamel maturation advocating that the “older” tooth exposed

Contemporary Clinical Dentistry | Jan-Mar 2014 | Vol 5 | Issue 1 78

[Downloaded free from [Link] on Wednesday, June 24, 2015, IP: [Link]]

Agrawal, et al.: Atomic force microscope comparison of CPP‑ACP paste, APF gel and iron supplement

to the oral environment for longer duration encounter acid and References
fluoride exposure rendering it more resistant. Also increased
porosity and removal of aprismatic layer results primary enamel 1. Mahoney EK, Kilpatrick NM. Dental erosion: Part 1. Aetiology and
prevalence of dental erosion. N Z Dent J 2003;99:33‑41.
more reactive and enhance surface deposition.[28]
2. Kargul B, Bakkal M. Prevalence, etiology, risk factors, diagnosis,
and preventive strategies of dental erosion: Literature review. Acta
A considerable decrease in Rrms was seen after remineralization Stomatol Croat 2009;43:165‑87.
with CPP‑ACP paste (Group I) of both primar y and 3. Deshpande SD, Hugar SM. Dental erosion in children: An
increasing clinical problem. J Indian Soc Pedod Prev Dent
permanent teeth but was not statistically significant
2004;22:118‑27.
which was in accordance to Poggio et al. and Quartarone 4. El Aidi H, Bronkhorst EM, Truin GJ. A longitudinal study of tooth
et al.[20,23] CCP‑ACP paste results in formation of a layer erosion in adolescents. J Dent Res 2008;87:731‑5.
that fills interprism cavities caused by erosion.[19] While 5. Linnett V, Seow WK. Dental erosion in children: A literature review.
Pediatr Dent 2001;23:37‑43.
due to its acidic nature APF gel further results in an 6. May J, Waterhouse PJ. Dental erosion and soft drinks: A qualitative
increase in erosion cavities. Moreover, the presence of iron assessment of knowledge, attitude and behaviour using focus
results in substitution of a phosphate group by carbonate groups of schoolchildren. A preliminary study. Int J Paediatr Dent
thus confronts the negative influence of iron on enamel 2003;13:425‑33.
7. Moazzez R, Smith BG, Bartlett DW. Oral pH and drinking habit
remineralization[34] which is confirmed by Alves et al.[17] Iron during ingestion of a carbonated drink in a group of adolescents
supplement showed increased in surface roughness instead with dental erosion. J Dent 2000;28:395‑7.
which was explained by Weiss et al. as mature enamel had 8. Jaeggi T, Lussi A. Erosion in early school‑age children. Schweiz
a greater affinity toward iron.[35] Monatsschr Zahnmed 2004;114:876‑81.
9. Jaeggi T, Schaffner M, Bürgin W, Lussi A. Erosions and
wedge‑shaped defects in Swiss Army recruits. Schweiz
Present in‑vitro study was designed to assess remineralizing Monatsschr Zahnmed 1999;109:1170‑82.
agent on eroded enamel surface using AFM which is not 10. Lippert F, Parker DM, Jandt KD. Susceptibility of deciduous
and permanent enamel to dietary acid‑induced erosion studied
addressed in literature till now. AFM was used in the present with atomic force microscopy nanoindentation. Eur J Oral Sci
study to verify protective effect of remineralizing agent on 2004;112:61‑6.
eroded enamel surface with images of high contrast and 11. Fejerskov O, Tencate JM, Larsen MJ, Pearce EI. Chemical
resolution.[23] According to Poggio et al., AFM analyzes interactions between the tooth and oral fluids. In: Fejerskov O,
editor. Dental Caries. 2nd ed., Ch. 4. Blackwell Munksgaard,
topographical aspects of the tooth surface qualities like when Garsington Road, Oxford,UK. 2003. p. 49‑60.
enamel was treated with CPP‑ACP paste, a collagen network 12. Yip KH, Smales RJ, Kaidonis JA. The diagnosis and control of
with precipitated crystals was evident which was further extrinsic acid erosion of tooth substance. Gen Dent 2003;51:350‑3.
confirmed by SEM.[36] Similarly Quartarone et al. showed that 13. Wang X, Megert B, Hellwig E, Neuhaus KW, Lussi A. Preventing
erosion with novel agents. J Dent 2011;39:163‑70.
the CPP‑ACP paste significantly reduces the erosion cavities 14. Piekarz C, Ranjitkar S, Hunt D, McIntyre J. An in vitro assessment
depth. This was assessed by AFM data analysis through scaling of the role of tooth mousse in preventing wine erosion. Aust Dent
concept and continuum stochastic equations which revealed J 2008;53:22‑5.
the remineralizing mechanism of calcium phosphate paste.[20] 15. Sales‑Peres SH, Pessan JP, Buzalaf MA. Effect of an iron
mouthrinse on enamel and dentine erosion subjected or
not to abrasion: An in situ/ex vivo study. Arch Oral Biol
Thus, CPP‑ACP paste showed a reduction in surface roughness 2007;52:128‑32.
than APF gel followed by iron supplement both in primary 16. Lussi A, Schaffner M, Bern TJ. Dental erosion – Diagnosis and
and permanent teeth but was not statistically significant. prevention in children and adults. Int Dent J 2007;57:385‑98.
17. Alves KM, Franco KS, Sassaki KT, Buzalaf MA, Delbem AC. Effect
Moreover, CPP‑ACP paste can be self‑applied and expected of iron on enamel demineralization and remineralization in vitro.
to be effective intraorally as saliva will enhance the action of Arch Oral Biol 2011;56:1192‑8.
CPP‑ACP according to the manufacturers. Hence been a very 18. Olmez S, Yüksel B, Çelik H. Scanning electron microscope study
promising remineralizing material. of human enamel surfaces treated with topical fluoride agents.
J Islamic Acad Sci 1993;6:133‑9.
19. Jeng YR, Lin TT, Wong TY, Chang HJ, Shieh DB. Nano‑mechanical
Conclusions properties of fluoride‑treated enamel surfaces. J Dent Res
2008;87:381‑5.
20. Quartarone E, Mustarelli P, Poggio C, Lombardini M. Surface
The following conclusions were derived: kinetic roughening caused by dental erosion: An atomic force
1. All the samples showed an increase in surface roughness microscopy study. J Appl Phys 2008;103:104702‑6.
after demineralization and primary teeth shows greater 21. Russell P, Batchelor D, Thornton J. SEM and AFM: Complementary
demineralization but were not statistically significant Techniques for High Resolution Surface Investigations. Digital
Instruments/Veeco Metrology. [Internet]. Santa Barbara, CA
2. Reduction in surface roughness after remineralization and Raleigh, NC:Veeco Instruments and North Carolina State
seen in all samples except iron supplement group of University;2004 [cited 2011 Sep 24]. Available from: http://
primary teeth [Link]/~appkchu/AP5301/SEM%20and%20
AFM%20-%20Complementary%[Link].
3. Greater remineralization was present in samples of 22. Cheng ZJ, Wang XM, Cui FZ, Ge J, Yan JX. The enamel
CPP‑ACP paste than APF gel and iron supplement but softening and loss during early erosion studied by AFM, SEM and
was not statistically significant. nanoindentation. Biomed Mater 2009;4:015020.

79 Contemporary Clinical Dentistry | Jan-Mar 2014 | Vol 5 | Issue 1

[Downloaded free from [Link] on Wednesday, June 24, 2015, IP: [Link]]

Agrawal, et al.: Atomic force microscope comparison of CPP‑ACP paste, APF gel and iron supplement remineralization

23. Poggio C, Lombardini M, Dagna A, Chiesa M, Bianchi S. Protective of dental composite resin to bovine dentine. Arch Oral Biol
effect on enamel demineralization of a CPP‑ACP paste: An AFM 1998;43:305‑11.
in vitro study. J Dent 2009;37:949‑54. 32. Gedalia I, Dakuar A, Shapira L, Lewinstein I, Goultschin J,
24. Amaechi BT, Higham SM, Edgar WM. Techniques for the Rahamim E. Enamel softening with Coca‑Cola and rehardening
production of dental eroded lesions in vitro. J Oral Rehabil with milk or saliva. Am J Dent 1991;4:120‑2.
1999;26:97‑102. 33. Murakami C, Bönecker M, Corrêa MS, Mendes FM, Rodrigues CR.
25. Amaechi BT, Higham SM. In vitro remineralisation of eroded Effect of fluoride varnish and gel on dental erosion in primary and
enamel lesions by saliva. J Dent 2001;29:371‑6. permanent teeth. Arch Oral Biol 2009;54:997‑1001.
26. Hayacibara MF, Paes Leme AF, Lima YB, Gonçalves NC, 34. Tehrani MH, Ghafournia M, Samimi P, Savabi O, Parisay I,
Queiroz CS, Gomes MJ, et al. Alkali‑soluble fluoride deposition Askari N, et al. Effect of casein phosphopeptide‑amorphous
on enamel after professional application of topical fluoride in vitro. calcium phosphate and acidulated phosphate fluoride gel on
J Appl Oral Sci 2004;12:18‑21. erosive enamel wear. Dent Res J (Isfahan) 2011;8:S64‑70.
27. Kato MT, de Moraes Italiani F, de Araújo JJ, Garcia MD, de Carvalho 35. Weiss G, Stabholz A, Markitziu A, Meyer I, Brayer L, Gedalia I.
Sales‑Peres SH, Buzalaf MA. Preventive effect of an iron varnish The effect of iron on in vitro decalcification of human tooth enamel.
on bovine enamel erosion in vitro. J Dent 2009;37:233‑6. J Oral Rehabil 1985;12:91‑3.
28. Badr SB, Ibrahim MA. Protective effect of three different fluoride 36. Poggio C, Lombardini M, Vigorelli P, Ceci M. Analysis of dentin/
pretreatments on artificially induced dental erosion in primary and enamel remineralization by a CPP‑ACP paste: AFM and SEM
permanent teeth. J Am Sci 2010;6:442‑51. study. Scanning 2013.
29. Gwinnett AJ. The ultrastructure of the “prismless” enamel of
permanent human teeth. Arch Oral Biol 1967;12:381‑8. How to cite this article: Agrawal N, Shashikiran ND, Singla S, Ravi KS,
30. Torres CP, Chinelatti MA, Gomes‑Silva JM, Rizóli FA, Oliveira MA, Kulkarni VK. Atomic force microscopic comparison of remineralization with
casein-phosphopeptide amorphous calcium phosphate paste, acidulated
Palma‑Dibb RG, et al. Surface and subsurface erosion of primary
phosphate fluoride gel and iron supplement in primary and permanent
enamel by acid beverages over time. Braz Dent J 2010;21:337‑45.
teeth: An in-vitro study. Contemp Clin Dent 2014;5:75-80.
31. Titley KC, Chernecky R, Rossouw PE, Kulkarni GV. The effect
of various storage methods and media on shear‑bond strengths Source of Support: Nil. Conflict of Interest: None declared.

Dispatch and return notification by E-mail

The journal now sends email notification to its members on dispatch of a print issue. The notification is sent to those members who have provided
their email address to the association/journal office. The email alerts you about an outdated address and return of issue due to incomplete/incorrect
address.
If you wish to receive such email notification, please send your email along with the membership number and full mailing address to the editorial
office by email.

Contemporary Clinical Dentistry | Jan-Mar 2014 | Vol 5 | Issue 1 80