Comparative Analysis Of Remineralized Early Enamel Carious Lesions Subjected To Variable Concentrations Of Fluoride Supplement Through Polarized Light Microscope

INTRODUCTION

           Dental caries is an infectious microbiologic disease of the teeth that results in localized dissolution and destruction of the calcified tissues.¹It is a complex disease process known to afflict a large population of the world.²

           Tooth minerals are lost and regained constantly in the human oral environment. The tooth health is hence dependent upon equilibrium of this mineral exchange. A break in the equilibrium causes the tooth to either demineralize or remineralize depending upon the concentration of the mineral saturation in the oral cavity.^2,3,4

           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 reaction rates with dissolution and transformation of hydroxyapatite to fluorapatite that resists the demineralization 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. Fluoridated dentifrice in various concentrations has been used to bring about remineralization 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 quantitative techniques of measurement of tooth mineral changes that include Polarized Light Microscopy (PLM), light scattering, Polarization- Sensitive Optical Coherence Tomography, Transverse Microradiography, Energy Dispersive X-ray analysis, cross-sectional microhardness determination and Confocal Laser Scanning Microscopy.⁵

           Under polarized light microscopy, the qualitative evaluation of the zones of demineralization (body of the lesion and translucent zone) and remineralization (surface zone and dark zone) are assessed with respect to the area occupied, width and depth. With this, the pore volume changes associated with demineralization and remineralization within various zones of carious lesion can be determined through birefringence. The optical property of this technique helps in the detection of carious and noncarious lesions of the tooth.

           Therefore, the aim of the present study is to evaluate the remineralization of early enamel carious lesions through polarized light microscopy after subjecting it to various concentrations of fluoridated dentifrice.

Materials and methods:

Method of collection of teeth and sample preparation:

           Forty five healthy human premolars, extracted for orthodontic purposes were collected, cleaned thoroughly and 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 1 mm below the cementoenamel junction with a slow speed diamond disc and the crowns were used for the study.

Artificial smooth-surface enamel lesion formation (Demineralization procedure):

           All the samples were covered with nail varnish leaving a window of 3 x 3mm on the buccal surface and were kept in a 50 ml of demineralizing solution in a sterile glass beaker, which was a mixture of calciumchloride (2.0 mmol/L) , trisodiumphosphate (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 x 3 mm window were covered with nail varnish to serve as control (Fig 1). The samples were randomly divided into three groups of fifteen teeth each (n=15).

Group A: Remineralization using non-fluoridated dentifrice -                                  Meswak tooth paste from Dabur was used as a control.

Group B: Remineralization using 500 ppm of fluoride containing Dentifrice -         Colgate Bubble Fruit tooth paste

Group C: Remineralization using 1000 ppm of fluoride containing Dentifrice-        Colgate Total tooth paste

Stimulated whole saliva collection:

           Five healthy female adult volunteers in the age group of 18 - 25yrs who have full complement of teeth with no past caries experience or prosthesis were chosen for the collection of saliva between 9 am to 11 am, after obtaining an informed consent. They were made to brush the teeth with a nonfluoridated dentifrice and 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 5mins and 15 ml of the collected sample was stored under refrigeration until use.

The pH cycling regimen (Remineralization procedure):

           Specimens of Group-A were subjected to a 20 day remineralization treatment regimen by immersion in saliva samples and fresh nonfluoridated dentifrice slurry prepared by adding 5 gm of dentifrice in 10 ml of water in a beaker as shown in Table 1. The procedure was repeated for Group B and Group C specimens, with 500 ppm and 1000 ppm fluoridated dentifrices, respectively.

Post-treatment analysis:

           The specimens were mounted in self-cure acrylic resin, sectioned longitudinally perpend icular to the varnished area with a hard tissue microtome(Leica SP 1600) (Fig 1) to obtain specimens of 100 microns thickness, polished with an abrasive stone, stained with freshly prepared 0.1mM Rhodamine B solution for 1hr and washed thoroughly with phosphate buffer solution and were mounted on frosted glass slides with 80% glycerol mountant for further analysis through the Polarized Light Microscope (PLM), Olympus BX51 model with a 1 CCD c-mount adapter.

           The imaging with PLM was done under 4X magnification and two images were captured from the buccal surface, (one each from either side of the midpoint measured from the occluso-cervical length of the tooth) and calibrated for linear depth of the lesion from the enamel surface using Image Pro Express software (Fig 2).

Results:

A Comparative evaluation study with 45 samples was undertaken to study the linear depth of lesion in μm as seen through polarized light microscope.

The averages of ten measurements in each image were tabulated and statistically analyzed (Table 2, Graph 1).

           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, Student t test (two tailed, independent) was used to find the significance of study parameters 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.

DISCUSSION

           The use of artificial caries systems has greatly increased our knowledge of the demineralization and remineralization process. Factors that affect the phase of mineralization include the oral pH, the contents and concentration of saliva, the oral bacteria present, frequency of sucrose ingestion, presence of fluoride or other chemicals and the duration of time all of these factors are present.^6,7 Both of the de- and remineralization phases can be occurring at the same time in different parts of the mouth.

           The bacterial plaque produces organic acids which can cause dissolution of the mineral content of the enamel. During periods of high bacterial metabolic activity and low pH conditions, demineralization and remineralization can occur at various depths in the early carious lesion.

           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 20^th century by eminent investigators like McKay and Black. Fluoride affects the caries process by enabling the formation of high quality fluorapatite that aids remineralization and inhibits glycolysis of plaque microorganisms. Several methods of fluoride administration have been investigated including professionally applied gels and varnishes, home rinses and fluoride containing etchants, bonding agents, cementing media and elastic modules.

           The early attempts to incorporate fluoride into toothpastes which dates back to the 1960s, and it is known to react with calcium to form calcium fluoride.^16,19,21 Hence, in the present study a nonfluoridated dentifrice has been used as a control along with two higher concentrations of fluoride, i .e 500 ppm and 1000 ppm for comparison.

           Simulation of the natural mouth environment has forced the researchers to use pH-cycling techniques and pH-cycle creating models have been accepted as a good evaluating method of the caries process and also provide standard study conditions. In this study, the experimental set-up was arranged in such a way that it simulated an oral environment subjected to acid and remineralization twice a day. In order to accomplish this, cariogenic acid, flouride remineralization solution and saliva were applied on the initially demineralized sample surfaces. In enamel “calcification” produces a mineral increase from 45 wt% to 96 – 98 wt% and a significant rise in Ca:P molar ratio. This ultimately results in the most highly mineralized and hardest skeletal tissue in the body.^8,9

           Clinical visual methods of detecting caries of macroscopically intact occlusal surfaces have been shown to have relatively poor sensitivity. Light based methods used for the detection of dental caries induced enamel demineralization are conventional radiography, micro radiography, Digital Imaging Fiber Optic Transillumination Imaging (DIFOTI),laser induced fluorescence (DIAGNOdent),and quantitative light-induced fluorescence (QLF).^10,11

           Micro-radiography has been suggested as the 'gold standard' in caries detection. It allows the determination of the mineral loss, lesion depth and body of the lesion. However, because of its destructive nature and hazards of ionizing radiation, it cannot be used in clinical situations.^10,11

           Conventional 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. Most of the above disadvantages can be avoided by using fibre optic visible light spectroscopy, polarized light microscopy and confocal laser scanning microscopy.

           The images viewed in the polarized light microscope will be a reduction, cancellation or pseudo-isotropy, or a reversal of the intrinsic birefringence of the enamel. The percentage of volume of spaces can be calculated from the observed birefringence using the known intrinsic birefringence of the enamel. Changes in carious enamel can be determined using various staining media with differing molecular sizes and refractive indices. 12 For this purpose 0.1mM of Rhodamine B dye has been used in this study.

           Fontana¹³ performed a study to correlate area of lesion, average fluorescence and total fluorescence obtained by confocal microscopy to lesion depth and mineral loss obtained from microradiography and polarized light microscopy. The findings show that when a 0.1 mMsolution of rhodamine B dye was used, the lesion area correlated well with the mineral loss obtained from microradiography. However, the average lesion fluorescence best represented mineral loss, based on their hypothesis that rhodamine B penetrates the voids and pores created during enamel deminer alization.¹³

           In the present study the highest values of linear depth for demineralization under PLM was seen in the group A [non-fluoridated dentifrice (control)] which was found to be 184.68±6.43 μm(Table 5) and the highest value for the remineralization was also seen in group A and was found to be 156.07±4.76 μm which was significantly higher than that for the group C. The differences in demineralized and remineralized zones were 59.47±8.83 μm which was also significantly higher for the group C. These findings suggest that the remineralization was more promising with the 1000 ppm fluoridated dentifrice. These results were similar to the values obtained by Celso Silva Queiroz¹⁴ who used 500ppm and 1100 ppm dentifrices on bovine teeth and VLN Kumar¹⁵ who used 1100 ppm dentrifrice and casein phosphopeptide-amorphous calcium phosphate along with a placebo as control. All these findings were on par with the analysis by the earlier studies showing that PLM is an advanced tool to diagnose and measure early enamel lesions.^{13,14,15,16,17,18,19,20,21,22}

CONCLUSION

           Fluorides supplements show promising remine ralization of the early enamel carious lesions and polarized light microscopy is one of the valuable tools for its diagnosis.

Clinical significance

Diagnosis of early enamel carious lesions still remains a challenge to dentists worldwide. Various in-vitro studies show promising results with polarized light microscope for the diagnosis of early carious lesion. This approach 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 imaging modality with increased “user friendliness and flexibility”. Further studies are necessary for evaluation of teeth both in-vitro and in-vivo with PLM.

Acknowledgements

We thank Dr. Madhura, Reader, Department of Oral & Maxillofacial Pathology, D A P M R.V. Dental College & Hospital, for helping me with the study.