COMPARATIVE EVALUATION OF THE REMINERALIZING …

I

COMPARATIVE EVALUATION OF THE REMINERALIZING

POTENTIAL OF THREE COMMERCIALLY AVAILABLE

REMINERALIZING PRODUCTS: A SEM STUDY

By

Dr. NILEENA MARY CHERIAN

Dissertation Submitted to the

Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore

In partial fulfillment of the requirements for the degree of

MASTER OF DENTAL SURGERY

in the speciality of

CONSERVATIVE DENTISTRY AND ENDODONTICS

Under the guidance of

Prof. (Dr). GIRISH T N

Professor

DEPARTMENT OF CONSERVATIVE DENTISTRY AND

ENDODONTICS

COORG INSTITUTE OF DENTAL SCIENCES

VIRAJPET-571 218

2017– 2020

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

VIII

LIST OF ABBREVIATIONS

1. CPP-ACP - Casein Phosphopeptide-Amorphous Calcium Phosphate

2. TCP - Tricalcium Phosphate

3. fTCP – Functionalized Tricalcium Phosphate

4. SEM- Scanning Electron Microscope

5. EDX- Energy Dispersive X-ray

6. Ca - Calcium

7. P - Phosphorous

8. CaCl2- Calcium Chloride

9. KH2PO4 – Potassium dihydrogen phosphate

10. KOH- Potassium hydroxide

IX

LIST OF TABLES

Table. No. Title Page No.

1

Distribution of mean and standard deviation of Ca and

P (wt%) after demineralization 13

2

Comparison of Ca and P (wt%) after remineralization

in between groups analysed by ANOVA

14

3

Comparison of Ca and P (wt%) between individual

groups after remineralization analysed by Post hoc

Tukey

14

4

Comparing the Ca (wt%) after remineralization and

after demineralization in the groups by Paired t test 16

5

Comparing the P (wt%) after remineralization and after

demineralization in the groups by Paired t test

17

6

The mean increase of Ca and P (wt%) in the groups by

ANOVA

18

7 Comparing the mean increase in Ca and P (wt%) in

between groups using Post hoc Tukey

18

X

LIST OF FIGURES

Figure

No.

Title Page No.

1 Samples 45

2 Remineralizing agents used in the study

45

3 SEM Machine (Zeiss EVO LS15) 46

4 EDX Detector (Thermo Ultradry) 46

5 Crown portion with a window of 3x3mm 47

6 SEM Image after demineralization 47

7 EDX graph after demineralization 48

8 EDX data after demineralization 48

9 Samples in demineralising solution 49

10 Samples in remineralizing solution 49

11 SEM image after remineralization in Control (Group I) 50

12 SEM image after remineralization in MI (Group II) 50

13 SEM image after remineralization in Remin Pro (Group III) 51

14 SEM image after remineralization in Clinpro (Group IV) 51

15 EDX analysis in Control (Group I) 52

16 EDX analysis in MI (Group II) 52

17 EDX analysis in Remin Pro (Group III) 53

18 EDX analysis in Clinpro (Group IV) 53

XII

ABSTRACT

COMPARATIVE EVALUATION OF THE REMINERALIZING POTENTIAL

OF THREE COMMERCIALLY AVAILABLE REMINERALIZING

PRODUCTS: A SEM STUDY

Background

Dental caries is an infectious microbiological disease of the teeth that results in local

dissolution of the calcified tissues.1. According to the recent approaches prevention

and control of initial carious lesions are done using non-invasive methods. One such

method is topical application of remineralizing agents. Various remineralizing

products are available in market, but their efficacy are uncertain.

Objectives

The objectives of the study were to evaluate the remineralizing potential of MI paste

(CPP ACP), Reminpro (Hydroxyapatite) and Clinpro (TCP) using SEM and EDX.

Methods

Forty extracted human permanent premolars were collected and stored as per OSHA

norms. The samples were decoronated and an acid resistant nail varnish was applied

leaving behind a 3x3 mm window on the labial surface of the tooth. The samples were

soaked in a demineralising solution for 4 days to create an artificial lesion, after which

SEM and EDX analysis were done. The samples were then randomly divided into 4

groups with 10 samples each. The 4 groups were: Control, MI, Remin Pro and

Clinpro. The remineralizing agents were applied according to the group and subjected

to pH cycling for 10 days. After pH cycling the samples were subjected to SEM and

EDX analysis. The data (Ca and P wt%) were obtained and statistically analysed

using ANOVA, Post hoc Tukey and paired t test.

XIII

Results:

Ca and P (wt%) in the samples were increased after remineralization in all the groups.

In the experimental groups, Ca and P increase was more for Remin Pro, followed by

Clinpro and then MI.

Conclusion:

According to the study, the remineralizing potential of Remin Pro was effectively

more, followed by Clinpro and then MI.

Keywords: Remineralization, CPP ACP, Hydroxyapatite, Tricalcium phosphate

Introduction

1

COMPARATIVE EVALUATION OF THE REMINERALIZING

POTENTIAL OF THREE COMMERCIALLY AVAILABLE

REMINERALIZING PRODUCTS: A SEM STUDY

INTRODUCTION

Dental caries is an infectious microbiological disease of the teeth that results in local

dissolution of the calcified tissues.1 The caries process has been thought to be

irreversible, resulting in permanent loss of tooth substance and subsequent

development of a cavity.2

The traditional approach of treating dental caries was to remove the caries affected

enamel or dentine and to replace it with a restorative material.3 This approach resulted

in a considerable loss of tooth structure. As a result of the recent studies, old concepts

have changed and now there is a paradigm shift in the aetiology, diagnosis, preventive

strategies and treatment of dental caries and many materials have been formulated for

its prevention2.

Recent approaches have focused on application of remineralizing agents to incipient

caries, to control demineralization and promote remineralization.

Dental caries is initiated via the demineralization of tooth hard tissues which is a ‘pH

driven phenomenon’. This occurs by organic acids produced from fermentable

carbohydrates by dental plaque and cariogenic bacteria. Demineralization and

remineralization can be considered as a dynamic process, characterized by the flow of

calcium and phosphate out of and back into the tooth enamel, which should be balanced

to prevent the progression of caries.4

The caries process takes place slowly which requires repeated episodes of prolonged

exposure to acidic conditions consistently below the critical pH for enamel dissolution

(pH 5.5, demineralization) with intervening periods of return to the resting pH of plaque

(pH 7.0, remineralization period). Whenever removal of plaque from retentive tooth

areas is inadequate, followed by ingestion of a diet rich in refined carbohydrates, the

dynamic equilibrium between demineralization and remineralization will be tipped

Introduction

2

towards demineralization, with the development of clinically detectable white spot

lesions. 5

Signs of the caries process cover a continuum from the first molecular changes in the

apatite crystals of tooth, to a visible white spot lesion and eventual cavitation.

White spot lesions are the earliest macroscopic evidence of enamel caries. The majority

of demineralization in white-spot lesions occurs in the subsurface region of enamel.

This subsurface demineralization increases porosity and changes the optical properties

of enamel.

Clinically, early carious lesion in enamel is initially seen as a white opaque spot and is

characterized by being softer than the adjacent sound enamel and is increasingly whiter

when dried with air. Typically, the enamel surface layer stays intact during subsurface

demineralization, but without treatment will eventually collapse into a full cavity.5

Remineralization is the natural repair process for caries lesions. This occurs when

calcium and phosphate in water among enamel or dentin crystals recrystallize on the

surface of existing crystal remnants. Remineralization requires saliva or some other

liquid to facilitate the transport of calcium and phosphate ions into the tooth.6

Fluoride is one of the commonly used agents for remineralization of incipient caries or

prevention of new carious lesion. The cariostatic effect of fluoride is primarily due to

its ability to decrease the rate of demineralization by forming fluorohydroxyapatite and

enhancing the remineralization of incipient carious lesions. It was observed that a high

concentration of fluoride containing pastes inhibited enamel demineralization to a

greater extent. 7

CPP ACP is another remineralizing agent that is commercially available. Casein

phosphopeptide is a bovine milk protein that binds and stabilizes calcium and

phosphate ions in an amorphous state. CPP-ACP maintains saturation levels of

minerals, especially calcium and phosphate, at the tooth surface thereby depressing

demineralization and enhancing remineralization process. CPP ACP are used alone or

often complexed with fluorides. 7

TCP is milled with simple organic materials to create a functionalized TCP ingredient

(fTCP) which are used in remineralizing pastes. This process ensures that prior to use,

Introduction

3

the active calcium sites are protected from premature interactions with fluoride. When

it comes in contact with the tooth surface and is moistened by saliva, the protective

barrier breaks down, making the calcium, phosphate and fluoride ions available to the

teeth. The fluoride and calcium then react with weakened enamel to provide a seed for

enhanced mineral growth relative to fluoride alone.7

It has been claimed that hydroxyapatite fills eroded enamel. Hydroxyapatite

containing pastes have been assumed to be suitable for management of dentinal

hypersensitivity, prevention of enamel demineralization and promoting

remineralization of enamel subsurface lesions.8

The newer approach for the treatment of dental caries which includes preventive

measures based on the concept of remineralization provides the base for the present

study.

The efficacy of the remineralizing pastes commercially available are uncertain. Hence

there is a need to find out the remineralizing potential of these products.

This study was done to compare the remineralizing capacity of three commercially

available remineralizing products using Scanning Electron Microscope and Energy

Dispersive X-ray analysis.

Aims & Objectives

4

AIMS AND OBJECTIVES

AIM

To evaluate and compare the remineralizing potential of three commercially available

remineralizing products using SEM and EDX

OBJECTIVES

1) To evaluate under SEM and EDX, the remineralizing potential of MI paste

(CPP ACP based)

2) To evaluate under SEM and EDX, the remineralizing potential of Remin Pro

(Hydroxyapatite based)

3) To evaluate under SEM and EDX, the remineralizing potential of Clinpro

(TCP based)

4) To compare the remineralization potential of all the three

Review of literature

5

REVIEW OF LITERATURE

A review article on the demineralization-remineralization dynamics in teeth and bone

was done by Neel E A et al. This review provided a thorough overview on the theories,

mechanisms and factors affecting the demin-remin dynamics. The role of calcium and

phosphate ions on the maintenance of teeth and bones and on the therapies that reverse

demineralization and boost remineralization were also compiled. 4

A literature review was done on white spot lesions by Roopa K B et al. The authors

have mentioned on the newer aids to diagnose and detect the white spot lesions and also

on the management of the lesions non-invasively through remineralization. 5

Featherstone published an article on remineralization and the need for newer

approaches. The author mentioned about the remineralization process, the caries

balance, effectiveness of fluorides and ways to enhance its effectiveness 6

A literature review was done by Verma A et al in which the authors concluded that the

prevention of dental caries by remineralization is a whole new concept and philosophy

focused on the intervention at the earliest possible stage with long term protection of

the patient as a whole entity. 9

An in vitro study was done by Nhu N V et al on the Effect of Casein Phosphopeptide-

amorphous Calcium Fluoride Phospate on the remineralization of Artificial carious

lesion. The study confirmed that CPP ACPF was effective in preventing the

demineralization of hard tissues and in remineralizing the surface of the carious lesion

at an early stage in permanent teeth. The results showed the capacity of CPP ACPF in

supplying calcium and phosphate to the enamel, decreasing the dissolution of enamel

surface and increasing the remineralization of the enamel surface.10

A study was conducted by Chokshi K et al., to compare and evaluate the

remineralization potential of fluoride varnish, CPP ACP paste and fTCP using confocal

microscope. They concluded that fluoride varnish showed the greatest remineralization

potential of artificial carious lesions followed by CPP ACP paste and fTCP paste

respectively. 2


6

A study was conducted by Gavrila L et al., investigating the surface topography and

comparing the remineralization potential of fluoride gel (Colgate and Carrefour kids),

fluoride and hydroxyapatite and Colgate Plax mouthwash on primary and permanent

teeth. The products containing fluoride and hydroxyapatite showed a higher

remineralization potential when compared to fluoride products. 11

Manarelli M M et al., conducted an in vitro study on the effects of fluoride varnishes

containing sodium trimetaphosphate on bovine enamel remineralization. They

concluded that TMP and fluoride added to varnishes have a synergistic effect against

enamel demineralization.12

An in vitro study was conducted to evaluate and compare the various remineralizing

agents on surface hardness produced by artificially produced enamel lesions by Singla

M G et al. They concluded that remineralizing pastes used in this study (GC tooth

mousse, Clinpro, Colgate Prevident, SHY NM) could effectively remineralize the

artificially produced early enamel lesions; however, none of them could regain the

surface microhardness to the level found at baseline.8

B Meghna et al compared the remineralization potential of CPP ACP, Tricalcium

phosphate and hydroxyapatite on artificial caries like enamel lesions on primary enamel.

The samples were evaluated by polarized light microscopy before and after treatment

and concluded that hydroxyapatite showed better results compared to CPP ACP and

Tricalcium phosphate. 13

Siddhesh B et al conducted an in vitro study to compare and evaluate the

remineralization potential of fluoride, amorphous calcium phosphate-casein

phosphopeptide (ACP-CPP), and combination of hydroxyapatite (HAP) and fluoride

on enamel lesions. Fluor Protector, GC tooth mousse and Clinpro tooth cream were the

remineralizing agents used in the study. The results were that the fluoride varnish

showed effective remineralization and CPP ACP and Hydroxyapatite in combination

with fluoride showed lesser effects.14


7

Liege Helena Freitas Fernandes et al evaluated the in situ effect of tooth pastes

containing CPP ACP and CPP ACPF on initial erosion prevention. CPP ACP, CPP

ACPF, Fluoridated tooth pastes and placebo pastes were used in the study. The CPP-

ACP pastes were able to reduce initial erosive demineralization in relation to fluoride

and placebo pastes. Nevertheless, the formulation of CPP-ACP with fluoride did not

provide an additional benefit.15

An invitro study was done by Singhal R and Rai B to assess and compare the

remineralization potential of three dentifrices with different compositions on artificially

induced carious lesions in vitro by using scanning electron microscopy and polarised

light microscopy. Within the limitations of the study, being a short-term study, low

sample size and in vitro experiment, Incudent toothpaste has exhibited a higher

remineralizing potential as compared to fluoride-based toothpaste in the study.16

A study by Vidya Manoharan et al aimed to quantitatively compare the

remineralization potential of casein phosphopeptide-amorphous calcium fluoride

phosphate (CPP-ACFP) and Novamin on artificially induced enamel subsurface lesions

using scanning electron microscope-energy dispersive X-ray (SEM-EDX). The authors

concluded that even though both CPP-ACFP and Novamin showed remineralization

potential, remineralization was found to be higher in the samples treated with CPP-

ACFP. 17

Enamel demineralization around orthodontic brackets is an important clinical problem.

The study by Soodeh Tahmasbi et al sought to compare the efficacy of sodium fluoride

(NaF), casein phosphopeptide amorphous calcium phosphate fluoride (CPP-ACP-F; MI

Paste Plus) and a water-based cream (Remin Pro), which contains hydroxyapatite and

fluoride for prevention of enamel demineralization. The results showed that 0.05% NaF

was more efficient than Remin Pro and MI Paste Plus for prevention of white spot

lesions. 18

An original research article was done by Soares et al assessing the enamel

remineralization of four different remineralizing agents using SEM. The purpose of

the study was to evaluate the ability of Casein Phosphopeptide-Amorphous Calcium

Phosphate Fluoride (CPP ACPF), Bioactive Glass (BAG), fluoride enhanced


8

Hydroxyapatite (HA) gel and self-assembling peptide P11-4, to remineralise artificial

carious lesions in enamel in vitro using a 30-day pH cycling model through surface

microhardness analysis and SEM.19

An invitro study was done by Chaudhury T et al to evaluate the comparative analysis

of remineralization potential of three different materials – casein

phosphopeptide-amorphous calcium phosphate with fluoride (CPP-ACPF), calcium

sucrose phosphate (CaSP), and bioactive glass on demineralized enamel using light

fluorescence microscopy and confocal laser scanning fluorescence microscopy. The

authors concluded that CaSP (Toothmin) showed better remineralisation potential

compared to the other groups. The authors also mentioned that light fluorescence

microscopy was an efficient diagnostic aid in detecting remineralization and

demineralization. 20

A polarised light microscopic study was done to evaluate the remineralising potential

of four commercially available products namely SHY-NM, GC Tooth Mousse Plus,

ReminPro and Colgate strong teeth on demineralized human teeth. According to the

authors Polarized light microscope was used to assess the lesion depth because the

histological features of dentin and enamel can be visualized better due to its

birefringence property, which is not well appreciated in a transmitted light

microscope. Image J software, a Javabased image processing program was used to

interpret the lesion depth. 21

Materials and Methods

9

MATERIALS AND METHODS

MATERIALS

40 extracted human permanent teeth without any external structural defects (Fig

1)

Demineralizing solution [2.2 mM CaCl2, 2.2 mM KH2PO4, 0.05 M acetic acid

adjusted to a pH of 4 with 1M KOH]

Diamond disc and straight handpiece

Acid resistant nail varnish

Remineralizing solution [1.5 mM calcium chloride, 0.9 mM sodium phosphate,

and 0.15 M potassium chloride, with a pH of 7.0]

Remineralizing products (Fig 2)

1) MI Paste (CPP ACP) [ Lot: 170530B]

2) Remin Pro (Hydroxyapatite + Fluoride) [Lot:50002002]

3) Clinpro (TCP + Fluoride) [Lot: 70201056572]

Scanning Electron Microscope [Zeiss EVO LS 15] (Fig 3)

Energy Dispersive X-ray [Thermo Ultradry EDX Detector] (Fig 4)

METHODOLOGY

Teeth Specimens

Forty extracted human permanent premolar teeth were collected. The extracted teeth

were cleaned, removed off all soft tissues, and stored in saline. The teeth were stored

as per OSHA norms.

Inclusion criteria:

Teeth with intact enamel

Exclusion criteria:

Caries

Abrasions


10

Cracks

Fracture

Fluorosis

Developmental defects

Preparation of the samples

Forty extracted human permanent teeth were selected for the study. The teeth were

decoronated. An acid resistant nail varnish was used to cover the entire surface of the

enamel crowns of each tooth, leaving a window, sized 3x3 mm parallel to each other

(Fig 5). The acid resistant coating was applied two times, the second application was

done after the first coat had dried. The teeth were stored in distilled water at room

temperature until use.

Preparation of the demineralizing solution

Demineralizing solution that contained (2.2mM CaCl2, 2.2mM KH2PO4, 50mM lactic

acid pH at 4.4 with a 1M KOH solution) was freshly prepared

Artificial lesion formation

Each of the enamel samples were then immersed in 40 ml of demineralizing solution

for 4 days at a temperature of 37°C, to induce artificial caries formation, simulating

an area of demineralization. After four days the teeth were removed from the

demineralizing solution. The samples were washed thoroughly in deionized water and

subjected to SEM imaging for evaluating the surface changes (Fig 6) and EDX

analysis (Fig 7, Fig 8) to evaluate the calcium and phosphorous levels in the

demineralized samples to establish the baseline value and the samples were grouped

randomly.

Grouping of the samples

The samples were randomly divided into 4 groups with 10 teeth in each group,

according to the remineralising agents used.


11

Group I: No remineralising agents used

Group II: MI paste (CPP ACP)

Group III: Remin Pro (Hydroxyapatite)

Group IV: Clinpro (TCP)

pH cycling

pH cycling model was adapted to simulate the dynamic process of demineralization

and remineralization that occurs in the oral cavity. Each of the enamel samples were

treated with the respective remineralizing agents for a period of 2 min, following

which the samples were immersed in 20 ml of demineralizing solution for a period of

3 hours (Fig 9). This was followed-up with treatment of the samples again with

respective remineralizing agents for 2 min. All the enamel samples were immersed in

30 ml of remineralizing solution for a period of 17 h (Fig 10). After each step the

samples were washed thoroughly with deionized water.

The remineralizing solution was replaced every 48 h and the demineralizing solution

replaced every 5 days. The pH cycling was carried out for a period of 10 days.

After the pH cycling the samples were subjected to SEM for evaluating the surface

changes (Fig 11,12,13,14) and EDX analysis to measure the mineral contents in the

samples. (Fig 15,16,17,18)

SEM Analysis

The experimental window was analyzed under SEM. Samples were mounted on the

aluminum holder stubs using a double sticky carbon tape and examined in a scanning

microscope at 15 kV. (Zeiss EVO LS 15)

EDX Analysis

EDX analysis of the samples after remineralization were done. The Ca and P (wt %)

in the samples after remineralizing agents were evaluated.


12

Statistical Analysis

The data was collected, coded and fed in SPSS (IBM version 23). Descriptive statistics

were calculated. Inferential statistics included ANOVA, Post hoc Tukey test and Paired

t test. Level of significance was set at .05 at 95% confidence level.

Sample Size Estimation

13

SAMPLE SIZE ESTIMATION

Sample size calculated based on a study conducted by Shaik Z A et al titled: Quantitative

analysis of remineralization of artificial carious lesions with commercially available newer

remineralizing agents using SEM-EDX – In vitro study.

1. Sample size calculated based on the comparison of mean Ca-P between groups, sample

size was calculated using the following formula:

N = (Zα + Zβ)2× 2σ2 /d2

Zα = 1.96

Zβ = 0.67 (75% power of the study)

σ2 = 0.1132

d = 0.4158

N = 9.057 = 9 per group. (Which was rounded off to 10 per group)

Results

14

RESULTS

The present study evaluated the remineralizing potential of MI paste (CPP-ACP),

Remin Pro (Hydroxyapatite) and Clinpro (Tricalcium phosphate) using SEM and EDX.

The data was collected for the calcium and phosphorous (wt%) after demineralization

and remineralization in the samples using EDX.

The observations and results are divided into two parts:

1) Statistical analysis for Ca and P (wt%) obtained from EDX

2) SEM images for the surface changes

Statistical methods applied for the study were:

Statistical Analysis

The data was collected and fed in SPSS (IBM version 23) for the statistical analysis.

Descriptive statistics was calculated.

Inferential statistics included ANOVA, Post hoc Tukey and paired t test. Level of

significance was set at .05 at 95% confidence level.

RESULT DESCRIPTION

Table 1: Distribution of mean and standard deviation of Ca and P (wt%) after

demineralization

Demineralization Mean Standard deviation

Ca

Control 13.3340 .73129

MI 12.8430 .50279

Reminpro 13.1160 .51928

Clinpro 13.2270 .87430

P

Control 9.5460 1.18161

MI 9.4070 .99335

Reminpro 8.8670 .84211

Clinpro 9.5510 .62965

Results

15

Table 2: Comparison of Ca and P (wt%) after remineralization in between groups

analysed by ANOVA

Remineralization Mean Standard

deviation F Significance

Ca

Control 20.6720 2.50648

12.694 0.000 (H.S) MI 24.5960 .73610

Reminpro 29.3420 5.70853

Clinpro 25.8320 .95514

P

Control 12.9820 .89394

10.919 0.000 (H.S) MI 13.7520 .80134

Reminpro 15.0450 1.00882

Clinpro 15.1050 1.21139

The Ca and P (wt%) in the remineralized samples were analysed using one-way

ANOVA. Highly significant data were obtained in between the groups. All the groups

showed an increase in the Ca and P (wt%). Hence was followed by Post hoc Tukey for

individual group wise comparison.

Table 3: Comparison of Ca and P (wt%) between individual groups after

remineralization analysed by Post hoc Tukey

Mean

difference

Standard

error Significance

95% Confidence

Interval

Lower

bound

Upper

bound

Ca

Control

MI -3.92400 1.41993 .042(S) -7.7482 -.0998

Reminpro -8.67000 1.41993 .000(H.S) -

12.4942 -4.8458

Clinpro -5.16000 1.41993 .005(H.S) -8.9842 -1.3358

Mi Reminpro -4.74600 1.41993 .010(S) -8.5702 -.9218

Clinpro -1.23600 1.41993 .820(N.S) -5.0602 2.5882

Reminpro Clinpro 3.51000 1.41993 .082(N.S) -.3142 7.3342

P

Control

MI -.77000 .44308 .320(N.S) -1.9633 .4233

Reminpro -2.06300 .44308 .000(H.S) -3.2563 -.8697

Clinpro -2.12300 .44308 .000(H.S) -3.3163 -.9297

Mi Reminpro -1.29300 .44308 .029(S) -2.4863 -.0997

Clinpro -1.35300 .44308 .021(S) -2.5463 -.1597

Reminpro Clinpro -.06000 .44308 .999(N.S) -1.2533 1.1333

Results

16

Ca (wt%) in the Control group was compared with other three experimental groups, MI

group showed a significant value of 0.042 and highly significant values of 0.000 and

0.005 was obtained in the Remin Pro group and Clinpro group respectively.

Between the experimental groups a significant value of 0.010 was obtained between MI

and Remin Pro, whereas no significant value was obtained between Clinpro and Remin

Pro.

Comparing the P (wt%) between Control and the experimental groups, a highly

significant values of 0.000 was obtained in Remin Pro and Clinpro. No statistically

significant data was obtained in MI.

Between the experimental groups, a significant value of 0.029 was obtained between

Remin Pro and MI. Even between Clinpro and MI a significant value of 0.021 was

noted. Remin Pro and Clinpro had no statistically significant data.

Graph 1: Comparing the Calcium and Phosphorous (wt%) in groups after

remineralization

0

5

10

15

20

25

30

CALCIUM P

20.672

12.982

24.596

13.752

29.342

15.045

25.832

15.105

REMINERALIZATION

CONTROL

MI

REMINPRO

CLINPRO

Results

17

Table 4: Comparing the Ca (wt%) after remineralization and after


Calcium Mean Standard

deviation T Significance

Control Remineralization 20.6720 2.50648

9.452 0.000 (H.S) Demineralization 13.3340 .73129

MI Remineralization 24.5960 .73610


Reminpro Remineralization 29.3420 5.70853


Clinpro Remineralization 25.8320 .95514


Highly significant data was obtained after demineralization and after remineralization

in all the groups.

The T values were 9.452, 54.304, 8.617 and 30.978 in Control, MI, Remin Pro and

Clinpro respectively.

Graph 2: Comparing the Ca (wt%) after demineralization and after

remineralization

0

5

10

15

20

25

30

CONTROL MI REMINPRO CLINPRO

20.672

24.596

29.342

25.832

13.334 12.843 13.116 13.227

CALCIUM

REMINERALIZATION

DEMINERALIZATION

Results

18

Table 5: Comparing the P (wt%) after remineralization and after


P Mean Standard

deviation T Significance

Control Remineralization 12.9820 .89394

7.445 0.000 (H.S) Demineralization 9.5460 1.18161

MI Remineralization 13.7520 .80134


Reminpro Remineralization 15.0450 1.00882


Clinpro Remineralization 15.1050 1.21139


Highly significant data was obtained after demineralization and after remineralization

in all the groups. The T values were 7.445, 9.912, 11.773 and 12.960 for Control, MI,

Remin Pro and Clinpro respectively.

Graph 3: Comparing the P(wt%) in the groups after remineralization and after

demineralization

0

2

4

6

8

10

12

14

16

CONTROL MI REMINPRO CLINPRO

12.98213.752

15.045 15.105

9.546 9.4078.867

9.551

P

REMINERALIZATION

DEMINERALIZATION

Results

19

Table 6: The mean increase of Ca and P (wt%) in the groups by ANOVA

Mean increase Mean Standard

deviation F Significance

Ca

Control 7.3380 2.45493

12.236 0.000 (H.S) MI 11.7520 .68252

Reminpro 16.2260 5.95487

Clinpro 12.6050 1.28672

P

Control 3.4340 1.45841

6.971 0.001 (H.S) MI 4.3450 1.38622

Reminpro 6.1780 1.65939

Clinpro 5.5540 1.35519

Highly significant values were obtained for Ca and P (wt%) in all the groups, hence

was followed by Post hoc Tukey test for individual comparisons

Table 7: Comparing the mean increase in Ca and P (wt%) in between groups using

Post hoc Tukey

Mean

difference

Standard

error Significance

95% Confidence

Interval

Lower

bound

Upper

bound

Ca

Control

MI -4.41400 1.47663 .025(S) -8.3909 -.4371

Reminpro -8.88800 1.47663 .000(H.S) -12.8649 -4.9111

Clinpro -5.26700 1.47663 .006(H.S) -9.2439 -1.2901

Mi Reminpro -4.47400 1.47663 .022(S) -8.4509 -.4971

Clinpro -.85300 1.47663 .938(N.S) -4.8299 3.1239

Reminpro Clinpro 3.62100 1.47663 .085(N.S) -.3559 7.5979

P

Control

MI -.91100 .65722 .516(N.S) -2.6810 .8590

Reminpro -2.74400 .65722 .001(H.S) -4.5140 -.9740

Clinpro -2.12000 .65722 .014(S) -3.8900 -.3500

Mi Reminpro -1.83300 .65722 .040(S) -3.6030 -.0630

Clinpro -1.20900 .65722 .272(N.S) -2.9790 .5610

Reminpro Clinpro .62400 .65722 .778(N.S) -1.1460 2.3940

Results

20

Comparing the mean increase of Ca (wt%) between the Control group and the three

experimental group. A significance of 0.025 was obtained in MI and highly significant

values of 0.000 and 0.006 were obtained in Remin Pro and Clinpro.

In between the experimental group, a significance of 0.022 was obtained between MI

and Reminpro, whereas no significant data was obtained comparing MI and Clinpro

and Clinpro and Remin Pro

Comparing the mean increase of P (wt%) between the Control and the three

experimental groups highly significant value of 0.001 was obtained in Remin pro and

a significant value of 0.014 was obtained in Clinpro. No significance was noted between

Control and MI.

In between the experimental groups, a significant value of 0.040 was obtained only

between MI and Remin Pro.

Graph 4: Comparing the mean increase in Ca and P (wt%) in the groups

0

2

4

6

8

10

12

14

16

18

CALCIUM P

7.338

3.434

11.752

4.345

16.226

6.178

12.605

5.554

MEAN INCREASE

CONTROL

MI

REMINPRO

CLINPRO

Results

21

SEM Images for surface changes

SEM image after demineralization (Fig 6)

SEM images taken after demineralization revealed loss of surface integrity. Clear

destruction of the enamel surface was noted, resulting in significant depressions and

irregularities.

Results

22

SEM Image after remineralization

Group I (Control) (Fig 11)

Results

23

Group II (MI) (Fig 12)

Group III (Remin Pro) (Fig 13)

Results

24

Group IV (Clinpro) (Fig 14)

Areas of calcified deposits were noted in all the groups after remineralization.

Few deposits were noted in the MI and the control group compared to Clinpro and

Remin Pro.

Discussion

25

DISCUSSION

Dental caries is considered as a dynamic disease process where an equilibrium exists

between the pathological and protective factors causing demineralization and

remineralization respectively. The pathological factors include frequent intake of

sugars, acidic foods and beverages, inhibition of salivary function and acidogenic

bacteria whereas the protective factors are antibacterial agents, composition and rate

of salivary flow, fluoride intake from diet and other extrinsic sources.5

The caries process takes place slowly and requires repeated and prolonged exposure to

acidic conditions consistently below the critical pH for enamel dissolution (pH 5.5,

demineralization) with intervening periods of return to the resting pH of plaque (pH 7.0,

remineralization period). The process is said to be continuous, which begins with

demineralization, then formation of early enamel lesions, followed by dentinal

involvement and later cavitation.5

The critical pH of dental enamel is 5.5, as though this were a fixed value, independent

of the composition of the solution to which enamel is exposed. In fact, the critical pH

varies over a wide range, its value depending on the concentrations of calcium and

phosphate in the solution.

If the pH of the solution is above the critical pH, then the solution is supersaturated

with respect to the mineral, and more mineral will tend to precipitate out.

Conversely, if the pH of the solution is less than the critical pH, the solution is

unsaturated, and the mineral will tend to dissolve until the solution becomes saturated.

The concept of critical pH is applicable only to solutions that are in contact with a

particular mineral, such as enamel. 22

Whenever removal of plaque from retentive tooth areas is inadequate, followed by

ingestion of a diet rich in refined carbohydrates, the dynamic equilibrium between

demineralization and remineralization will be tipped towards demineralization, with the

development of clinically detectable white spot lesions. 5

White spot lesions, implies that there is a subsurface area with most of the mineral

loss beneath a relatively intact enamel surface. Clinically, early caries lesion in

Discussion

26

enamel is initially seen as a white opaque spot and is characterized by being softer

than the adjacent sound enamel and is increasingly whiter when dried with air.5

In a white-spot caries lesion, the decalcification has occurred below the surface, and

the lesion is covered by a virtually intact surface zone of enamel with a thickness of

about 0.03 mm. There is very good clinical evidence that such lesions can be

remineralized if the surface remains intact, provided they are kept free of plaque and

if salivary flow is adequate. 5

Remineralization occurs when calcium and phosphate in the water among the enamel

or dentin crystals recrystallize on the surfaces of existing crystal remnants. The

calcium and phosphate come primarily from saliva. This process has been known for

a long time and in recent decades has been studied extensively.6

The mineral formed during remineralization is more resistant to acid than the original

enamel or dentin mineral, especially if fluoride is present to enhance remineralization

and to be incorporated into the new crystal surfaces.6 Remineralization needs

bioavailable calcium and phosphate, and is greatly enhanced by the presence of

fluoride even at sub-ppm levels.23

In the recent years, the primary focus is on prevention and early detection of initial

carious lesions, various non-invasive/minimal intervention modalities have been

advocated. One such non-invasive method is the topical application of remineralizing

agents. Thus, in the present study three such commercially available remineralizing

agents were evaluated to check their efficacy.

Some of the remineralizing agents commercially available are fluorides, CPP ACP,

nanohydroxyapatite, bioactive glass, tricalcium phosphate, ACP technology, Xylitol,

calcium sodium phosphosilicate.

In the present study the remineralizing potential of CPP ACP based Recaldent

technology MI paste, nanohydroxyapatite based Remin Pro and Tricalcium Phosphate

based Clinpro were evaluated.

Discussion

27

MI Paste which is a CPP-based product and particularly CPP-ACFP are the basis for

the anticariogenicity and dental remineralization (calcium, phosphate, fluoride, and

water) on the tooth surface and in the dental biofilm. The anticaries action of CPP

derivates has a topical effect based on the following: modulation of bioavailable

calcium phosphate levels because they maintain ionic phosphate and calcium

supersaturation; buffering effects on plaque; increased remineralization and reduction

of the hydroxyapatite solution; and difficulty for Streptococcus mutans and

Streptococcus sobrinus to adhere and grow.24

These complexes are found in ACP in the dental biofilm and increase calcium

phosphate levels which serve as a reservoir for free calcium and phosphate ions.

Once present in the enamel subsurface lesion, the CPP-ACP would release the weakly

bound calcium and phosphate ions which would then deposit into crystal voids. The

release of the calcium and phosphate ions would be thermodynamically driven. The

CPPs have a high binding affinity for apatite (Cross et al., 2007); hence, on entering

the lesion, the CPPs would bind to the more thermodynamically favored surface of an

apatite crystal face.25

The final effect is, saliva and plaque are kept in calcium and phosphate

supersaturation with respect to the enamel which allows a reduction in

demineralization and favours remineralization. On the other hand, inhibiting adhesion

of cariogenic bacteria to the hydroxyapatite making it possible to modulate the

activity of plaque bacteria and favour colonization by less cariogenic bacteria. This

can help to reduce acid formation in the biofilm and reduce enamel

demineralization.24

Prolonging contact time with tooth surfaces by including these bio nanocomplexes in

chewing gums and toothpastes has proven effective in experimental studies on the

remineralization of subsurface lesions and reduction of demineralization where

portions of enamel previously in contact with CPP-ACP based substances were

subjected to artificial demineralization.24

Discussion

28

The combination of CPP-ACP nanocomplexes and fluoride in toothpaste provide a

greater concentration of fluoride ions in the dental biolfilm and a greater increase in

remineralization than if only fluoride toothpaste is applied.24

Remin Pro(VOCO, Germany) is another type of remineralizing paste which in

contrast to CPP-ACP products, contains calcium and phosphate in the hydroxyapatite

form. In addition, Fluoride and Xylitol have also been included in this product.

Hydroxyapatite fills eroded enamel, Fluoride gets converted to fluorapatite when it

comes in contact with saliva; thus, strengthens the tooth and renders it more resistant

to acid attacks. Xylitol reduces the harmful effects of bacteria and their metabolic

product lactic acid. It has been recommended to prevent enamel demineralization and

to promote remineralization of enamel subsurface lesions. 8

Tricalcium phosphate (TCP) has been considered as one possible means for

enhancing the levels of calcium in plaque and saliva. Combining calcium phosphate

and fluoride ions in oral care products is problematic and can lead to loss of

bioavailable fluoride ion due to a reaction between the calcium phosphate phase and

the fluoride ion. In an approach to overcome this incompatibility of calcium

phosphates and fluoride ions, new technologies have been developed. This technology

supports functional tricalcium phosphate (fTCP) where tricalcium phosphate particles

have been ball milled with sodium lauryl sulphate, and has been included in a tooth

crème with sodium fluoride marketed as Clinpro tooth crème (3 M ESPE)13

The methodology followed in our study was similar to that of the studies done by

Soares R et al19 and Bajaj M et al13 . The samples were immersed in demineralizing

solution initially to create an artificial lesion and pH cycling model was done to

simulate the dynamic process of demineralization and remineralization that occurs in

the oral cavity. The pH cycling protocol adopted for this study was based on the

model described by Featherstone JDB et al.,.26

This pH cycling model has been utilised successfully to review the anti caries

potential of dentifrice formulations since it simulates the oral cavity. In the protocol

adopted, dynamic cycles of demineralization and remineralization was done by

immersing the samples in demineralizing and remineralizing solution. The

composition of the demineralizing and remineralizing solution in the present study

Discussion

29

was similar to that in the study done by Buzalaf M et al 27. The demineralizing

solution was adjusted to a pH of 4 and the remineralizing solution to 7. The

remineralizing toothpastes were topically applied on the samples.

Various techniques have been used to assess the enamel remineralization. It can be

quantitatively assessed by measuring the mineral content, microhardness or

qualitatively using confocal microscopy, polarised light microscopy, scanning

electron microscopy.

In this study qualitatively the surface changes were assessed using SEM and

quantitively the mineral content calcium and phosphorous was assessed using EDX.

This was similar to that utilized by Manoharan V et al 17 and M N Hegde et al 28.

SEM is one of the most sensitive, time-tested techniques to assess the

demineralization and remineralization of the carious lesions in vitro, as reported in

earlier studies, while in most studies using SEM, samples are coated with metals such

as gold or palladium to improve image quality. Harding et al and Nicolae et al.

studied the samples using SEM without metal sputtering so that they could be

observed again, if necessary, once the study ended. This version was considered in

our study as the samples had to be evaluated after demineralization and re-evaluated

after remineralization.29

EDX has been used for elemental analysis at the ultrastructural level. It is a

microanalytical technique that is used in conjunction with SEM wherein SEM does

the structural analysis and the elemental analysis is done by EDX. The principle is

based on the energy emitted in the form of X-ray photons when electrons from

external sources collide with the atoms in a material, thus generating characteristic X-

rays of that element. The EDX X-ray detector measures the number of emitted X-rays

v/s their energy. The energy of the X-ray is characteristic of the element from which

the X-ray was emitted. A spectrum of the energy v/s relative counts of the detected X-

rays is obtained and evaluated for qualitative and quantitative determinations of the

elements present in the specimen using a computer-based program.28

In the present study, remineralizing pastes MI, Remin Pro and Clinpro were compared

to assess their remineralizing potential. EDX analysis was done to measure the Ca and

P content in the samples.

Discussion

30

While comparing the remineralizing pastes with the control group a statistically

significant increase in Ca and P content was noted in all the three experimental groups

with significance of 0.042 in MI, 0.000 in Remin Pro and 0.005 in Clinpro.

Comparing MI with Remin Pro, a statistically significant increase in Ca and P was

noted with a p -value of 0.010. Hence a better remineralizing potential was seen in the

Remin Pro group. The result was in concurrence with the findings by Sandeep T et

al30, where the Hydroxyapatite based cream was marginally more effective than the

CPP ACPF based paste. Other studies by authors Heravi F et al 8 and Ebrahimi M et al

31 showed an increased remineralizing potential in MI and Remin Pro but were not

statistically significant.

Comparing MI and Clinpro, no statistically significant data was obtained in the Ca

content, whereas the P content was increased significantly with a p value of 0.021 in

the Clinpro group.

Comparing Clinpro and Remin Pro, in our present study no significant difference was

noted in the remineralizing potential. On the contrary, in a study by Rao R et al32, it

was noted that Clinpro showed a statistically significant remineralizing potential

compared to Remin Pro. Surface Microhardness was assessed in their study using

Vicker’s Hardness test.

So, in our study, better remineralizing potential was observed in Remin Pro, the

possible reason for the increase in Ca and P may be due to the presence of

hydroxyapatite (Ca and P) in its composition. Significant increase in Ca content was

not noted in Clinpro, the possible reason being the known instability of fluoride ions

in oral care formulations containing poorly soluble calcium-based abrasives.

The Clinpro product contains sodium fluoride, hence the fluoride ion would be

susceptible to the reduction in bioavailability in the presence of added calcium

phosphate without a stabilizer. fTCP is poorly soluble and together with the large

particle size and low amount added would explain the poor release from the product

and the inability to significantly increase salivary calcium and inorganic phosphate

levels.33

No statistically significant data was obtained in MI paste. These results might be

different due to shorter treatment applications and immediate acid challenge. CPP-

ACP may have been incorporated into the lesion but not activated when it was

Discussion

31

necessary or even washed away in the demineralizing solution. This may be due to a

different time between the release of ACP from CPP during the acid challenge and the

timing of a gradient necessary to deposit calcium and phosphate into the lesion during

remineralization.34

More clinical studies are to be conducted inorder to know their efficacy in the clinical

scenario.

Conclusion

32

CONCLUSION

The recent approaches aim in the prevention and minimal intervention treatment of

dental caries rather than the old concept of ‘extension to prevention’. Thus, newer

studies focus on the preventive treatment strategies of early carious lesion and methods

to regain the lost mineral content.

In the present study remineralizing potential of three commercially available

remineralizing pastes – MI, Remin Pro and Clinpro were evaluated.

In accordance to the results obtained in the study all the three experimental groups

showed a significant increase in Ca and P content. The remineralizing potential of

Remin Pro was effectively more, followed by Clinpro and then MI paste.

More research using an in-situ model simulating the oral environment, using natural

saliva and plaque, or a clinical study need to be performed to support the results

obtained in the study.

Summary

33

SUMMARY

The present study was done to evaluate the remineralizing potential of three

remineralizing pastes MI (CPP-ACP), Remin Pro (Hydroxyapatite) and Clinpro

(TCP) using SEM and EDX. SEM imaging was done to evaluate the surface changes

and EDX analysis was done to measure Ca and P content.

Teeth samples were taken according to the inclusion criteria. The samples were

coated with an acid resistant nail varnish leaving behind a 3x3 mm window on the

labial surface. The samples were soaked in demineralizing solution to create an

artificial lesion and then viewed under SEM and EDX analysis was done. After which

the samples were grouped and pH cycling was done for 10 days.

After pH cycling the samples were viewed under SEM for evaluation of surface

changes and subjected to EDX analysis to measure the calcium and phosphorous

levels. The data was obtained and statistically analysed using ANOVA, Tukey post

hoc test and paired t test.

The results obtained in the study were,

Ca and P (wt%) in the enamel samples were increased after remineralization in

all the groups compared after demineralization. All the groups showed

statistically significant data after demineralization and after remineralization.

Comparing the Control (Group I) with the other three experimental groups,

there was a significant increase in Ca content in all the three groups MI,

Remin Pro and Clinpro. Even though there was an increase in P content in all

the experimental groups, statistically significant data was obtained in Remin

Pro and Clinpro.

In the experimental groups, Remin pro showed an increase in Ca and P which

was statistically significant, followed by Clinpro and then MI.

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34

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38

CONSENT FORM TEMPLATE

I, Dr Nileena Mary Cherian, Postgraduate student, Department of Conservative Dentistry

and Endodontics, Coorg Institute of Dental Sciences, Virajpet, conducted a study titled,

COMPARATIVE EVALUATION OF THE REMINERALIZING POTENTIAL OF

THREE COMMERCIALLY AVAILABLE REMINERALIZING PRODUCTS: A SEM

STUDY on 40 human teeth samples.

This proposal has been reviewed and approved by Institutional Review Board of Coorg

Institute of Dental Sciences, Virajpet, which is a committee whose task is to make sure that

research participants are protected from harm.

CERTIFICATE OF CONSENT

I have read the foregoing information, or it has been read to me. I have/had the opportunity

to ask questions about it and any questions that I have asked have been answered to my

satisfaction.

I consent voluntarily to participate as a participant in this research. I understand that I have

the right to withdraw from the study at any time before the publication of the data. I have

been assured that the data will be kept confidential and anonymous. I also give consent for

my data to be published or presented if the above conditions are met.

Name of Participant

Signature of Participant

Date

39

If illiterate

I have witnessed the accurate reading of the consent form to the potential participant,

and the individual has/had the opportunity to ask questions. I confirm that the individual

has given consent freely.

Name of witness Thumb print of participant

Signature of witness

Date

THE CONSENT FORM WAS NOT USED IN THE PRESENT STUDY AS THIS WAS AN

INVITRO STUDY

Ethical Committee Clearance Certificate

40

ETHICAL COMMITTEE CLEARANCE CERTIFICATE

Proforma Data

41

PROFORMA DATA

1.Spread sheet for Ca and P(wt%) after demineralization-

Group I (Control) Group II (MI) Group III(Reminpro) Group IV ( Clinpro)

S.NO Ca P Ca P Ca P Ca P

1

2

3

4

5

6

7

8

9

10

Proforma Data

42

2) Spread sheet for Ca and P(wt%) after remineralization-

Group I (Control) Group II (MI) Group III(Reminpro) Group IV ( Clinpro)

S.NO Ca P Ca P Ca P Ca P

1

2

3

4

5

6

7

8

9

10

Figures

45

FIGURES

Figure 1: Samples

Figure 2: Remineralizing agents used in the study

Figures

46

Figure 3: SEM Machine (Zeiss EVO LS15)

Figure 4: EDX Detector (Thermo Ultradry)

Figures

47

Figure 5: Crown portion with a window of 3x3mm

Figure 6: SEM Image after demineralization

Figures

48

Figure 7: EDX graph after demineralization

Figure 8: EDX data after demineralization

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Figure 9: Samples in demineralising solution

Figure 10: Samples in remineralizing solution

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Figure 11: SEM image after remineralization in Control (Group I)

Figure 12: SEM image after remineralization in MI (Group II)

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Figure 13: SEM image after remineralization in Remin Pro (Group III)

Figure 14: SEM image after remineralization in Clinpro (Group IV)

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Figure 15: EDX analysis in Control (Group I)

Figure 16: EDX analysis in MI (Group II)

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Figure 17: EDX analysis in Remin Pro (Group III)

Figure 18: EDX analysis in Clinpro (Group IV)

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COMPARATIVE EVALUATION OF THE REMINERALIZING …