Comparative evaluation of remineralizing potential of commercially available agents MI paste, Remin pro, and Clinpro using Scanning Electron Microscope and Energy Dispersive X-ray: An in vitro study.

BACKGROUND: According to recent approaches, the prevention of initial dental caries is achieved using certain noninvasive methods. One such method is the topical application of remineralizing agents. Various remineralizing products are commercially available, but their efficacy is uncertain. AIM: The aim of the study was to evaluate the remineralizing potential of MI, Reminpro, and Clinpro using scanning electron microscope (SEM) and energy dispersive X-ray (EDX).
METHODOLOGY: Forty extracted human premolars were collected. The samples were decoronated and the crown portion was coated with nail varnish leaving behind a 3 mm × 3 mm window on the labial surface. The samples were soaked in demineralizing solution for 4 days to create an artificial lesion, followed by SEM-EDX analysis. The samples were then randomly divided into 4 groups Control, MI, Remin Pro, and Clinpro with ten samples each. The remineralizing agents were applied according to the group and subjected to pH cycling for 21 days which was followed by SEM-EDX analysis. Data obtained were statistically analyzed using SPSS IBM version 23 (ANOVA, post hoc-Tukey, and Paired-t-test).
RESULTS: Ca and P were increased after remineralization in all groups. Between groups, the increase was more for ReminPro, followed by Clinpro and MI.
CONCLUSION: In this study, the remineralizing potential of Remin Pro was effectively more, followed by Clinpro and MI. Copyright:

INTRODUCTION

Dental caries is an infectious microbial disease of the teeth that results in localized dissolution of the calcified tissues.[1] The progression of caries is known to be an irreversible process, leading to permanent loss of tooth substance and subsequent development of a cavity.[2] Dental caries commences by demineralization of hard tissues which is a “pH driven phenomenon.” This mainly occurs due to the action of organic acids, produced by dental plaque and cariogenic bacteria from fermentable carbohydrates. Inward and outward movement of calcium and phosphate from the tooth enamel leads to the dynamic process of demineralization and remineralization. Thus, any imbalance in this process will lead to the formation of caries.[3]

The traditional approach involves the treatment of dental caries by removing the affected enamel or dentine followed by its replacement with a restorative material.[4]

Preventive measures based on the concept of remineralization have emerged as one of the most recent approaches to treat incipient dental caries without cavitation and this provides the base for the present study. There is a need to understand the efficacy of commercially available remineralizing pastes as the remineralization potential of these products remains uncertain. This study aims to compare the remineralization capacity of three commercially available products using the scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis. The null hypothesis for the present study is that there is no difference in the remineralizing potential of MI, Clinpro, and Reminpro.

METHODOLOGY

The study was approved by the institutional review board with the reference number IRB/CIDS/113/2017.

Sample size calculation

The sample size was calculated with the formula:

N = (Zα + Zß)2 × 2 σ2/d2.

Teeth specimens

Forty extracted human permanent premolar teeth were collected. The extracted teeth were cleaned and disinfected in autoclave according to Occupational Safety and Health Administration regulations OSHA. The teeth were then stored in distilled water. Only teeth with intact enamel were included in the study. Any teeth with caries, abrasions, cracks, fracture, fluorosis, or developmental defects were excluded.

Preparation of the samples

The teeth specimens selected for the study were decoronated at the level of the cementoenamel junction. An acid-resistant nail varnish was used to cover the entire surface of the crowns of each tooth, leaving a window measuring 3 mm × 3 mm. A second application was applied 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 2.2 mM CaCl2, 2.2 mM KH2PO4, 50 mM lactic acid pH at 4.4 with a 1M KOH solution.

Preparation of the remineralizing solution

Remineralizing solutions that contained 1.5 mM calcium chloride, 0.9 mM sodium phosphate, and 0.15 M potassium chloride, with a pH of 7.0 were freshly prepared.

Remineralizing agents used

MI paste

Remin pro

Clinpro.

Artificial lesion formation

The teeth samples were kept at 37°C in 40 ml of demineralizing solution for 4 days. This protocol was followed to artificially form incipient caries. After 4 days the teeth were removed from the demineralizing solution and rinsed thoroughly in deionized water. The samples were then subjected to SEM imaging for evaluating the surface changes and EDX analysis to evaluate the calcium and phosphorous levels in the demineralized samples to establish the baseline value.

Grouping of the samples

The samples were divided randomly into four groups with ten teeth in each group, according to the remineralizing agents used. The first group was the control (no remineralizing agents used), the second group was treated with MI paste (casein phosphopeptide-amorphous calcium phosphate [CPP-ACP]), the third group with Remin Pro (hydroxyapatite), and the fourth group with Clinpro (tricalcium phosphate [TCP]).

pH cycling

pH cycling was done to mimic the process of demineralization and remineralization that occurs in the oral cavity.

The remineralizing agent was applied on the teeth samples and rubbed with an applicator tip for a period of 2 min, following which the samples were immersed in 20 ml of demineralizing solution for a period of 3 h. After the 3 h immersion in demineralizing solution, the samples were again subjected to treatment with respective remineralizing agent for 2 min. All the samples were then immersed in 30 ml of remineralizing solution for a period of 17 h. This cycle of demineralizing-remineralizing was continued for a period of 21 days.

For the control group, the samples were periodically immersed in demineralizing and remineralizing solutions without the application of the remineralizing agent.

After each step, the samples were washed thoroughly with deionized water. The remineralizing solution was replaced every 48 h and the demineralizing solution every 5 days.

After the pH cycling was carried out for 21 days the samples were subjected to SEM for evaluating the surface changes and EDX analysis to measure the mineral contents in the samples. All the sample groups were coded to prevent bias during analysis.

Scanning electron microscope analysis

The experimental samples were analyzed under SEM (Zeiss EVO LS 15). Samples were mounted on aluminum stubs using a double sticky carbon tape and surface morphology was analyzed under a scanning microscope at 15 kV.

Energy dispersive X-ray analysis

EDX analysis (Thermo Ultradry EDX Detector) of samples after remineralization was carried out to evaluate the Ca and P (wt %) in the samples after treatment with remineralizing agents.

Statistical analysis

Statistical Analysis was done using SPSS (IBM SPSS Statistics 23, IBM Corp., Armonk, NY,USA). Inferential statistics included ANOVA, Post hoc Tukey test, and Paired t-test. The level of significance was set at. 05 at 95% confidence level.

RESULTS

The present study evaluated the remineralizing potential of MI paste, ReminPro, and Clinpro using SEM and EDX.

The data were obtained for the calcium and phosphorous (wt %) after demineralization and remineralization in the samples using EDX.

The observations and results are divided into two parts:

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

SEM images for the surface changes.

Result description

Statistical analysis for Ca and P (wt %) obtained from energy dispersive X-ray

The Ca and P (wt %) in the remineralized samples were analyzed using one-way ANOVA. The significant difference in data were obtained in between the groups. All the groups showed an increase in the Ca and P (wt %). Hence, it was followed by Post hoc Tukey for individual group-wise comparison [Table 1].

Table 1

Comparison of Ca and P (wt %) between individual groups after remineralization analysed by post hoc Tukey

	Mean difference	SE	Significance	95% CI
				Lower bound	Upper bound
Ca
Control
MI	−3.92400	1.41993	0.042 (S)	−7.7482	−0.0998
Reminpro	−8.67000	1.41993	0.000 (HS)	−12.4942	−4.8458
Clinpro	−5.16000	1.41993	0.005 (HS)	−8.9842	−1.3358
MI
Reminpro	−4.74600	1.41993	0.010 (S)	−8.5702	−0.9218
Clinpro	−1.23600	1.41993	0.820 (NS)	−5.0602	2.5882
Reminpro
Clinpro	3.51000	1.41993	0.082 (NS)	−0.3142	7.3342
P
Control
MI	−0.77000	0.44308	0.320 (NS)	−1.9633	0.4233
Reminpro	−2.06300	0.44308	0.000 (HS)	−3.2563	−0.8697
Clinpro	−2.12300	0.44308	0.000 (HS)	−3.3163	−0.9297
MI
Reminpro	−1.29300	0.44308	0.029 (S)	−2.4863	−0.0997
Clinpro	−1.35300	0.44308	0.021 (S)	−2.5463	−0.1597
Reminpro
Clinpro	−0.06000	0.44308	0.999 (NS)	−1.2533	1.1333

NS: Not significant, S: Significant, HS: Highly significant, SE: Standard error, CI: Confidence interval

Ca (wt %) in the control group was compared with other three experimental groups, all the three groups MI group (0.042), Reminpro (0.000), and Clinpro (0.005) showed significant values.

On comparing the Ca wt % between the inter experimental group, a significant value of 0.010 was obtained for Reminpro on comparison with MI, whereas there was no significant data observed between MI Clinpro and Clinpro Reminpro.

Comparing the P (wt %) between control and the experimental groups, statistically significant values of 0.000 were obtained in Remin Pro and Clinpro, respectively. No significant value was observed in MI.

On comparing the inter-experimental groups, between MI and Reminpro a significant value of 0.029 was observed for Remin Pro. In between Clinpro and MI, a significant value of 0.021 was noted for Clinpro. Remin Pro and Clinpro had no statistically significant data.

The Ca and P (wt %) after demineralization and after remineralization was statistically analyzed using paired t-test. Highly significant values for obtained in all the experimental groups [Table 2].

Table 2

Comparing the Ca and P (wt %) after demineralization and after remineralization using paired t-test

	Mean	SD	t	Significance
Calcium
Control
Remineralization	20.6720	2.50648	9.452	0.000 (HS)
Demineralization	13.3340	0.73129
MI
Remineralization	24.5960	0.73610	54.304	0.000 (HS)
Demineralization	12.8430	0.50279
Reminpro
Remineralization	29.3420	5.70853	8.617	0.000 (HS)
Demineralization	13.1160	0.51928
Clinpro
Remineralization	25.8320	0.95514	30.978	0.000 (HS)
Demineralization	13.2270	0.87430
Phosphorus
Control
Remineralization	12.9820	0.89394	7.445	0.000 (HS)
Demineralization	9.5460	1.18161
MI
Remineralization	13.7520	0.80134	9.912	0.000 (HS)
Demineralization	9.4070	0.99335
Reminpro
Remineralization	15.0450	1.00882	11.773	0.000 (HS)
Demineralization	8.8670	0.84211
Clinpro
Remineralization	15.1050	1.21139	12.960	0.000 (HS)
Demineralization	9.5510	0.62965

HS: Highly significant, SD: Standard deviation

Scanning electron microscope images for surface changes

Scanning electron microscope image after demineralization SEM images taken after demineralization revealed clear destruction of the enamel surface, resulting in significant depressions and irregularities.

Scanning electron microscope image after remineralization Areas of calcified deposits were noted in all the groups after remineralization. Amorphous calcific deposits were scattered on the surface of remineralization along the prismatic borders. Few crystals were noted in the MI and the control group compared to Clinpro and Remin Pro [Figure 1].

Figure 1

Scanning electron microscope images after demineralization and remineralization

DISCUSSION

Dental caries are regarded as a dynamic disease process where an equilibrium exists between the pathological and protective factors instigating demineralization and remineralization respectively. The pathological factors include frequent intake of sugars, acidic food 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 dissolution of enamel takes place at a critical pH of 5.5, irrespective of the solution it is exposed to. Depending on the calcium and phosphate concentrations in the solution, the critical pH might vary. The solution becomes supersaturated with the mineral when its pH is above the critical pH, resulting in precipitation of minerals out of the solution,

Remineralization results in the production of minerals that are more resistant to acid than the original enamel or dentin mineral, mainly due to the incorporation of fluoride into new crystal surfaces.[6] The availability of calcium and phosphate in the oral environment is essential for remineralization and is greatly augmented by the presence of fluoride even in minimal concentrations.[7]

MI paste is a CPP-based product and CPP-ACP is the basis for anti-cariogenicity and remineralization (calcium, phosphate, fluoride, and water) on the tooth surface and dental biofilm. CPP derivatives demonstrate the anticaries activity with topical effect by modulation of bioavailable calcium phosphate levels to maintain ionic phosphate and calcium supersaturation, buffering effects on plaque, increased remineralization, and reduction of the hydroxyapatite solution. Moreover, they have the ability to prevent the adherence and growth of Streptococcus mutans and Streptococcus sobrinus.[8]

When CPP-ACP is present in enamel subsurface lesion, it releases loosely bound calcium and phosphate ions to fill these subsurface voids. This release of calcium and phosphate ions would be a thermodynamically driven process. CPPs on entering a lesion would bind to more thermodynamically preferred surface of an apatite crystal face as they have extreme binding affinity for apatite.[9]

Combining CPP-ACP nanocomplexes and fluoride in toothpaste results in greater concentration of fluoride ions in the dental biofilm providing increased remineralization which is better than fluoride toothpaste applied alone.[8]

The methodology adopted in this study was comparable with various other studies[1011] in which the samples were initially immersed in demineralizing solution to develop an artificial lesion followed by pH cycling model to simulate the dynamic process of demineralization and remineralization that takes place within the oral cavity. The pH cycling protocol adopted for this study was based on the model described by Featherstone and Zero.[12]

SEM was used in various in vitro studies to analyze the demineralization and remineralization of the carious lesions. Most of it reported the use of SEM with metal sputtering (Gold and Palladium) for image enhancement. A study by Kamath et al. reports the use of SEM without metal sputtering for re-observation of samples after the study ended. This was considered in the present study as the samples required to be re-examined after demineralization and remineralization.[13] Good quality SEM images were obtained even without gold sputtering according to Shehadat et al.[14] A carbon tape was used to aid in the conduction in the present study.

Comparing MI with Remin Pro, a statistically significant increase in Ca and P was noted with a P = 0.010. Results showed better remineralizing potential in the Remin Pro group which was in concurrence with the findings by Thakur et al.,[15] where the hydroxyapatite based cream was marginally more effective than the CPP ACPF based paste. Various other studies[1617] showed an increased remineralizing potential in MI and Remin Pro though not statistically significant.

The Ca content was not statistically significant while comparing MI and Clinpro, whereas the P content was increased significantly with a P = 0.021 in the Clinpro group.

Comparing Clinpro and Remin Pro, in our present study no significant difference was noted in the remineralizing potential. Conversely, in a study[18] it was demonstrated that Clinpro showed improved remineralizing potential that was statistically significant in comparison to Remin Pro.

In this study, Remin Pro showed better remineralizing potential and the possible reason for this increase in Ca and P may be due to the existence of hydroxyapatite in its composition. A significantly higher amount of Ca content was not noted in Clinpro, and could be due to the known instability of fluoride ions in oral formulations that contains poorly soluble calcium-based abrasives. Clinpro contains sodium fluoride, making the fluoride ions susceptible to reduction in bioavailability in the presence of added calcium phosphate without a stabilizer. Various studies have reported that fTCP is poorly soluble and a large particle, this would explain the poor release of Ca and P from the product.[19]

MI paste did not show any statistically significant data in the present study. The variability of the results may be due to the washing away off loosely bound calcium and phosphate, due to the immediate demineralization process.[20]

It's a challenge to replicate the exact oral microflora and the interaction with the tooth. Some factors such as plaque, oral salivary content have an effect on the demin-remin cycle. All these factors interaction is difficult to replicate as these factors vary from individuals to individuals race to race. More clinical studies are to be conducted in order to know their efficacy in the clinical scenario.

CONCLUSION

The present approach in restorative dentistry aims at “prevention and minimal intervention” in contrast to the earlier concept of “extension for prevention.” Hence, most research is focussed on the strategy of preventive treatment of early carious lesion and also on methods to regain lost minerals.

The results of this study showed significant increase in Ca and P content in all the three experimental groups. The highest remineralization potential was observed with Reminpro, followed by Clinpro and MI paste.

Further research, simulating in vivo conditions need to be undertaken to extrapolate the results obtained in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.