Qualitative analysis of re mineralized carious lesions subjected to fluoride supplement through confocal laser scanning microscope

doi:10.4236/ojst.2011.13010

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Open Journal of Stomatology, 2011, 1, 55-60

doi:10.4236/ojst.2011.13010 Published Online September 2011 (http://www.SciRP.org/journal/OJST/ OJST

Published Online September 2011 in SciRes. http://www.scirp.org/journal/OJST

Qualitative analysis of remineralized carious lesions

subjected to fluoride supplement through confocal laser

scanning microscope

K. Shashikala*, N. V. Sheela

Department of Conservative Dentistry and Endodontics, R. V. Dental College& Hospital, Bangalore, India.

Email: *[email protected]

Received 10 June 2011; revised 25 July 2011; accepted 3 August 2011.

ABSTRACT

Aim: 1] Comparative evaluation of the linear depth

of induced remineralized lesions after subjecting to

fluoride supplements and 2] To assess the average

fluorescence at both the demineralized and the re-

mineralized zones in all the three study groups under

confocal laser scanning microscope. Method: Forty

five sound human premolars extracted for orthodon-

tic reasons were decoronated 1 mm below the ce-

mento-enamel junction and coated with nail varnish

except for a 3 × 3 mm window on the buccal surface.

The samples were placed in 50 ml of de mineralizing

solution at pH 4.6 for 96 hours. Following deminer-

alization, the lower half of the 3 × 3 mm window in

all the samples were covered with nail varnish to

serve as control. The samples were randomly divided

into three groups of fifteen teeth each (n = 15) and

specimens in group A[Nfd] were remineralized using

non-fluoridated dentifrice [control], those in groups

B [Fd5] a nd group C [Fd10] using 500 ppm and 1000

ppm of fluoride containing dentifrice, respectively.

The specimens were subjected to a 20 day reminer-

alization treatment regimen and were sectioned into

100 µm thick sections and two images were captured

on the buccal surface from either side of the midpoint

of occluso-cervical length using confocal laser scan-

ning microscope [CLSM]. Results: were tabulated

and statistically analyzed by Anova. Study concluded

that 1000 ppm fluoridated dentifrice showed a

greater degree of remineralization than other groups

and confocal laser scanning microscopes gives prom-

ising results in the diagnosis of early enamel lesions

over the conventional methods.

Keywords: Dental Caries; Fluoridated Dentifrice;

Confocal Laser Scanning Microscope

1. INTRODUCTION

Dental caries is a complex disease process that afflicts a

large population of the world, regardless of gender, age

and ethnicity, although it does tend to affect more indi-

viduals with a low socio-economic status to a greater

extent. Tooth minerals are lost and regained constantly in

the human oral environment. The tooth health is hence

dependent upon eq uilibrium of this mineral exchange. A

break in the equilibrium causes the tooth to either de-

mineralize or remineralize depending upon the concen-

tration of the mineral saturation in the oral cavity [1 ].

Over the last few decades, fluoride in various forms

has been proven to reduce caries in both the primary and

permanent dentitions when used in a variety of ways.

This is because it acts as a catalyst and influences reac-

tion rates with dissolution and transformation of hy-

droxyapatite to fluorapa tite that resists the demineraliza-

tion process in the tooth. Various topical agents like

fluoridated solutions, gels, mouth rinses and dentifrices

have been used to hasten the remineralization of these

carious lesions [2]. Fluoridated dentifrice in various

concentrations has been used to bring about reminerali-

zation of the carious lesion since it is a most common

and easily available vehicle that is used to cleanse the

teeth worldwide and can deliver fluoride topically to the

oral cavity. Remineralization of the carious lesions

caused by the action of fluoride supplements have been

analyzed by various qualitative and quantative techni-

ques of measurement of tooth mineral changes that in-

clude Polarized Light Microscopy (PLM), light scatter-

ing, Polarization-Sensitive Optical Coherence Tomo-

graphy, Transverse Microradiography, Energy Disper-

sive X-ray analysis, cross-sectional microhardness de-

termination and Confocal Laser Scanning Microscopy.

Confocal laser scanning microscopy is a powerful imag-

ing techniq ue based upon opti cal behavi o r o f li g ht within

specimens. Generally, confocal analysis features excita-

K. Shashikala et al. / Open Journal of Stomatology 1 (2011) 55-60

tion (such as fluorescence) although emission detection

is also possible [3]. The optical property of this tech-

nique helps in the detection of carious and non-carious

lesions of the tooth [4]. To date, not much information is

available on the usage of confocal microscope for the

analysis of early enamel lesions of the tooth.

Therefore, the aim of the present study is 1) Compara-

tive evaluation of the linear depth of induced remineral-

ized lesions [measured in microns] after subjecting to

fluoride supplements and 2) To assess the average fluo-

rescence at both the demineralized and the remineralized

zones in all the three study groups under confocal laser

scanning microscope.

2. MATERIALS AND METHODS

Forty five healthy human premolars which were ex-

tracted for orthodontic purposes and with no patient

identifiers were collected from the Department of Oral

and maxillofacial Surgery. The teeth were cleaned thor-

oughly to remove any blood, soft tissue or extrinsic de-

posits with hand scaler. They were stored in 100 ml of

10% buffered formalin (pH 6.8 to 7.0) for a period of

three months until they were used for the study. Teeth

were sectioned 1mm below the cemento-enamel junction

with a slow speed diamond disc. The roots were dis-

carded and the crowns were used for the study.

2.1. Artificial Smooth-Surface Enamel Lesion

Formation (Demineralization Procedure)

All the samples were covered with nail varnish leaving a

window of 3 × 3 mm on the buccal surface and were

kept in a 50 ml of de mineralizing solution in a sterile

glass beaker, which was a mixture of calcium chloride

[2.0 mmol/L], tri sodium phosphate [2.0 mmol/L] in

acetate buffer [75 mmol/L] solution at pH 4.6 for 96

hours. Following demineralization, the lower half of the

3 × 3 mm window in all the samples were covered with

nail varnish to serve as control. The samples were ran-

domly divided into three groups of fifteen teeth each [n

= 15] before they were being subjected to the pH cycle.

Group Nfd: Remineralization of the lesion using non-

fluoridated dentifrice tooth paste [Meswak Dabur, India]

was used as a control.

Group Fd5: Remineralization of the lesion using 500

ppm of fluoride containing Dentifrice Bubble Fruit tooth

paste [Colgate, India].

Group Fd10: Remineral iz at ion of t he le si on u si ng 100 0

ppm of fluoride containing Dentifrice-Colgate Total too-

th paste [Colgate, India].

2.2. Stimulated Whole Saliva Collection

Five healthy female adult volunteers in the age group of

18 yrs - 25 yrs who had full complement of teeth with no

past caries experience were chosen for the collection of

saliva between 9 am to 11 am, after obtaining clearance

from institutional ethical committee and written in-

formed consents from participating individuals. The se-

lected subjects were not on any medication that influ-

enced the salivary flow rate or had any salivary pathol-

ogy, with no faulty dental restorations, no current caries

activity, and no active periodontal disease, and were

neither with dental prosthesis nor with orthodontic ther-

apy. They were made to brush the teeth with a non-

fluoridated dentifrice and later made to rinse thoroughly

prior to the collection of saliva. They were then made to

chew on paraffin wax and empty the stimulated saliva

into sterile containers in about 5 mins. The 15 ml of the

sample was collected and stored under refrigeration until

use.

2.3. The Ph Cycling Regimen (Remineralization

Procedure)

Group Nfd: Sample of 15 teeth were subjected to a 20

day remineralization treatment regimen by immersion in

fresh non-fluoridated dentifrice slurry prepared by add-

ing 5 g of dentifrice in 10 ml of water in a glass beaker

for 2 mins, followed by rin sing with distilled water. The

remineralizing period wherein the teeth were immersed

in 15 ml of collected saliva sample was for 2 hrs.

The teeth were then subjected to 4 hrs of acid chal-

lenge in order to simulate the oral environment like the

in-vivo conditions, where samples were subjected to 15

ml of demineralizing solution, followed by 2 hrs of im-

mersion in 15 ml of saliva and then subjecting it to the

dentifrice for 2 mins again and then immersion in saliva

for the rest 16 hrs. The saliva samples were changed

each day during the acid challenge period. The proce-

dure was repeated for Groups Fd5 and Fd10 specimens,

wherein the dentifrices used contained 500 and 1000

ppm of fluoride respectively, unlike the non-fluoridated

dentifrice used in Gro up NFd.

2.4. Post-Treatment Analysis

Following the treatment period the specimens were mo-

unted in self-cure acrylic resin. The samples were sec-

tioned longitudinally perpendicular to the varnished area

so that each section included the varnish-covered base-

line lesion area and the uncovered, post-treatment lesion

area with a hard tissue microtome (Leica SP 1600) to

obtain specimens that were 100 microns thick.

The sections were polished with an abrasive stone and

were then stained with freshly prepared 0.1 mM Rhoda-

mine B solution for 1 hr. The stained samples were

washed thoroughly with phosphate buffer solution until

there was no dye leaching out of the sample. All samples

were mounted on frosted glass slides with 80% glycerol

mountant and the edges of the cover slip were coated

K. Shashikala et al. / Open Journal of Stomatology 1 (2011) 55-60

OJST

with transparent nail enamel for further analysis th rough

the Confocal Laser Scanning Microscope, Leica TCS SL

inverted microscope.

The images with CLSM were captured from the buc-

cal surface that is one each from either side of the mid-

point measured from the occluso-cervical length of the

tooth at [5×] magnification and Argon laser was used at

488 nm wavelength for excitation and emission range of

498 - 514 nm wavelength. Two images were captured on

the buccal surface from either side of the midpoint of

occluso-cervical length. The buccal surface areas were

scanned between 10 and 50 microns below the cut sur-

face and the superficial areas were not scanned because

of the concern regarding the smear layer. The captured

images were calibrated for linear depth of fluorescence

and also the average/total fluorescence of the lesion us-

ing the Leica TCS SL in-built software, Germany.

3. RESULTS

A Comparative evaluation of all the samples was under-

taken to study, 1) The linear depth of lesions in microns

[µm], as seen through con focal laser scanning micro-

scope and 2) To assess the average fluorescence at both

the demineralized and the remineralized zones in all the

three study groups under confocal laser scanning micro-

scope.

The induced carious lesion depths were assessed un-

der CLSM by measuring from the enamel surface to the

deeper zone of the lesion and an average of ten meas-

urements were taken from every image that was captured

both in the demineralized and remineralized zone of the

3 × 3 mm window of every specimen of all the three

groups. The measurements were tabulated and statistically

analyzed by Anova and student t test (Tables 1 and 2).

The extent of 0.01 mM Rhodamine B dye penetration

seen as fluorescence in the specimens under Argon Laser

were also calibrated and recorded with the in-built soft-

ware [Leica TCS SL, Germany]. The average of the

mean amplitude obtained from the images of the speci-

mens of the three groups was also tabulated and ana-

lyzed (Figures 2 and 3).

Confocal microscopic evaluation in the present study

shows that the highest value for demineralization after

96 hrs was seen in the control group, which was 183.41

± 7.81 µm and the lowest was for group NF5 [500 ppm

dentifrice] which was 180.91 ± 7.78 µm. Similarly the

highest value of remineralization was 156.74 ± 7.95 µm

seen in the group NFd specimens showing that the val-

ues were significant [p < 0.001] and the remineralization

values were 124.07 ± 3.46 µm lowest for group Fd10

[1000 ppm dentifrice]. A p-value of 0.616 was obtained

for the demineralization under CLSM (Figure 1).

Descriptive statistical analysis was carried out in the

present study. Results on continuous measurements were

presented on Mean  SD (Min-Max) and results on

categorical measurements are presented in Number (%).

Significance was assessed at 5% level of significance.

Analysis of variance (ANOVA) was used to find the

significance of study parameters between three or more

groups of specimens, Student t test (two tailed, inde-

pendent) was used to find the significance of study pa-

rameters on continuous scale between two groups (Inter

group analysis) on metric parameters, Student t test (two

tailed, dependent) was used to find the significance of

study parameters on continuous scale within each group.

4. DISCUSSION

The use of artificial caries systems has greatly increased

Table 1. Evaluation of linear depth of lesion in µm as seen through confocal laser scanning microscope for both

demineralized and remineralized areas.

Zone Group NFd [Control]Group Fd5 Group Fd10 P value

Demineralised 183.41 ± 7.81 180.91 ± 7.78 181.54 ± 5.84 0.616

Reminerali ze d 156.74 ± 7.95 144.65 ± 5.71 124.07 ± 3.46 < 0.001**

Difference 26.67 ± 13.01 36.26 ± 7.57 57.47 ± 6.67 -

P value < 0.001** < 0.001** < 0.001** -

Table 2. Evaluation of average fluorescence in µm under Con focal laser scanning microscope.

Zone Group Nfd [Control ] Group Fd5 Group Fd10 P va lue

Demineralized 83.41 ± 7.81 80.31 ± 8.95 80.07 ± 8 .41 0.363

Reminerali ze d 54.07 ± 6.44 40.29 ± 4.77 25.54 ± 4.63 < 0.0014**

Difference 29.34 ± 9.13 40.03 ± 7.87 54.53 ± 9.7 3 -

P value < 0.001** < 0.001** < 0.001** -

+ Suggestive significance (P value: 0.05 < P < 0.10); * Moderately significant (P value: 0.01 < P  0.05) ** strongly significant (P

value: P  0.0 1) .

K. Shashikala et al. / Open Journal of Stomatology 1 (2011) 55-60

100

150

200

250

Group AGroup BGroup C

con focal l aser scannin g m icro scop e

Demineralised

Rem ineralize d

100

Group AGroup BGroup C

average flu orescence in confocal laser

Dem ineralis ed

Rem ineralize d

(a) (b)

Figure 1. (a) Average linear depth of lesion in µm as seen through confocal laser scanning microscope for both de mineralized and re

mineralized areas. (b) Average fluorescence in µm as seen through the confocal laser scanning microscope for both de mineralized

and re mineralized areas.

(a) (b) (c) (d) (e)

Figure 2. CLSM images [5×] of occlusal aspect of the 3 × 3 mm window of specimens. (a) Nfd group.(b) Fd5 group (c) Fd10 group

showing 2D and (d) 3D reconstruction of the images at every 10 µ stack, arrows indicate de mineralized and arrow heads indicate re

mineralized zones. (e) Image of group Fd10 specimen [20 × zoom] showing demineralized and re mineralized zones and the enamel

rods.

(a) (b)

Figure 3. (a) Images showing the method of calibration with the Leica TCS SL in-built software. (b) Calibrated Image showing the

graph of the intensity of fluorescence along the ten measurements (ROI 1-10) and the table showing the length measurements and

means amplitude along with minimum and maximum amplitude of fluorescence.

K. Shashikala et al. / Open Journal of Stomatology 1 (2011) 55-60 59

our knowledge of the demineralization and remineraliza-

tion process [13]. The administration of fluoride has

been proposed, and used, as a method of reducing

enamel susceptibility to decalcification since the time it

was discovered in the early 20th century by eminent

investigators like McKay and Black [5]. Fluoride affects

the caries process by enabling the formation of high

quality fluorapatite that aids remineralization and inhib-

its glycolysis of plaque microorganisms [6]. The early

attempts to incorporate fluoride into toothpastes which

dates back to the 1960s and it is one of the most com-

mon and effective modes of topical fluoride delivery [7].

Hence, in the present study a non-fluoridated denti-

frice has been used as a control along with two higher

concentrations of fluoride, that is 500 ppm and 1000

ppm for comparison. The study was designed in such a

way that it simulated an oral environment subjected to

acid and remineralization twice a day.

Monitoring demineralization and remineralization of

dental hard tissues is essential for the prevention and

minimally invasive treatment of dental caries and hy-

persensitivity [8]. Light based methods used for the de-

tection of dental caries induced enamel demineralization

are Conventional radiography, micro radiography, Digi-

tal Imaging Fiber Optic Trans illumination Imaging

[DIFOTI], laser induced fluorescence [DIAGNO dent],

and quantitative light-induced fluorescence [QLF]. Con-

ventional microscopy suffers from the problem as light

scattering, namely multiple scattering from objects that

are out of focus within the illuminated region prevents

imaging deep within a sample. Further, if care is not

taken, optical microscopy can lead to the observation of

certain artifacts which in turn leads to incorrect physical

interpretation of the system in question [3 ,9].

Confocal microscopy and its application in dentistry

have been advocated by Watson [9]. CLSM overcomes

the limitations of conventional wide-field microscopy

through: high resolution fluorescence, high resolution

3-D imaging, non-destructive imaging technique and

in-situ characterization of tissue microstructure images.

Fontana performed a study to correlate area of lesion,

average fluorescence and total fluorescence obtained by

con focal microscopy to lesion depth and mineral loss

obtained from micro radiography and polarized light

microscopy. The findings show that when a 0.1 mM so-

lution of rhodamine B dye was used, the lesion area cor-

related well with the mineral loss obtained from micro

radiography [10].

However, the average lesion fluorescence best repre-

sented mineral loss, based on their hypothesis that rho-

damine B penetrates the voids and pores created during

enamel demineralization which is a better method of

evaluation over the birefringence through CLSM. Ca-

bezas also conducted a correlation study between micro

radiography and con focal microscopy for measurement

of enamel remineralization. As described by Fontana, 0.1

mM rhodamine B dye was used and lesion area, total

fluorescence and average fluorescence were evaluated.

Lesion area was the confocal parameter that showed the

strongest correlation with microradiography, followed by

total lesion fluorescence [8,10].

The present study showed a lesser degree of reminer-

alization with the non-fluoridated dentifrices which was

similar to the study by Gonzalez [10], who had used 250

ppm and 1100 ppm dentifrices along with a placebo con-

trol. It was also seen in the study that the linear depth

measurements of the lesion both in the de mineralized

zone and the re mineralized zone varied significantly

under the con focal microscopic. This is on par with the

analysis by the earlier studies [11,12] showing that con

focal microscopy is an advanced tool to diagnose and

measure early enamel lesions both in-vivo and in-vitro

without requiring the tooth to be sectioned.

Fluorides are effective means of remineralization of

carious lesions in higher concentrations (1000 ppm) as

compared to the non-fluoridated control or 500 ppm

concentration [13]. Diagnosis of early enamel carious

lesions still remains a challenge to dentists worldwide.

Various in-vitro studies show promising results with con

focal laser scanning microscope as a diagnostic aid for

early carious lesion detection. This appro ach will enable

the operator to circumvent errors due to variation in the

thickness of enamel at different locations of the crown.

Recent evolution in techniques and technologies have

facilitated a relatively wide spread adoption of this im-

aging diagnostic modality with increased “user friendli-

ness and flexibility”. Study concluded that 1000 ppm

fluoridated dentifrice showed a greater degree of remin-

eralization than other groups and con focal laser scan-

ning microscopes gives promising results in the diagno-

sis of early enamel lesions over the conventional meth-

ods.

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