Comparative evaluation of remineralizing potential of three agents on artificially demineralized human enamel: An in vitro study.

INTRODUCTION: Remineralization as a treatment procedure has received much attention from clinicians. The objective of this in vitro study was to find out the efficacy of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF), and tricalcium phosphate fluoride (TCP-F) in remineralizing enamel surface on which artificial caries lesion had been created. The changes were analyzed using DIAGNOdent(®) (KaVo) and scanning electron microscope (SEM).
MATERIALS AND METHODS: A total of 52 premolars and 24 molars were selected and classified into four groups of 13 premolars and 6 molars in each: I (CPP-ACP), II (CPP-ACPF), III (TCP-F), and IV (artificial saliva). All the samples were assessed using DIAGNOdent at the baseline and after demineralization and remineralization. Ten samples were randomly selected from each group baseline after demineralization and after remineralization for surface evaluation using SEM.
RESULTS: Statistical analysis showed that all the experimental groups had a significantly higher amount of remineralization except for group IV.
CONCLUSION: All the three experimental groups showed a statistically significant amount of remineralization. However, because of the added benefit of fluoride (NaF 0.2%), CPP-ACPF (Tooth Mousse-Plus(®)) and TCP-F showed marginally more amount of remineralization than did CPP-ACP (Tooth Mousse(®)). Remineralization efficacy was TCP-F > CPP-ACPF > CPP-ACP.

INTRODUCTION

Dental caries is a most common disease that affects a large number of people. It is a worldwide public health problem, affecting numerous urban and rural communities.[1-3]

White-spot lesions are the earliest macroscopic evidence of enamel caries.[4] Typically, the enamel surface layer stays intact during subsurface demineralization, but, without treatment, will eventually collapse into a full cavity.[5] Near-neutral pH of saliva is endowed with a natural buffering capacity. Natural demineralization of tooth at an early stage is reversed by saliva, which contains calcium ions, phosphate ions, buffering agents, fluoride, and other substances.[6]

The strategy for aided remineralization is to have ions directly delivered to where and when they are needed most. Several mechanisms are available for aided remineralization. The most well known is the delivery of topical fluoride, which has been proven to be a highly effective measure for prevention of caries.[78] More recently, this has led to introduction of new materials containing calcium and phosphate ions.[9]

Different technologies are used for remineralization of early enamel caries lesions. One technology involves,first, casein phosphopeptide stabilized amorphous calcium phosphate (CPP-ACP), the second is an unstabilized amorphous calcium phosphate (ACP), and the third is a bioactive glass containing calcium sodium phosphosilicate. A new concept involves tricalcium phosphate. Since all systems rely on calcium and phosphate compounds, their effect is mainly based on an enhancement of the natural capacity of saliva to remineralize mineral loss.[10]

Considering these factors, an in vitro clinical trial was planned to compare the remineralizing potential of three agents (CPP-ACP, CPP-ACP + fluoride, and tricalcium phosphate + fluoride) on artificial enamel carious lesion by using KaVo DIAGNOdent® pen and environmental scanning electron microscopy (E-SEM).

MATERIALS AND METHODS

The study was done using sound permanent extracted teeth. Caries-free premolars extracted for orthodontic reasons and caries-free molars requiring extractions for impaction reasons were included in the study. Teeth with any visible or detectable caries, teeth with any hypoplastic lesions, teeth with any white spot lesions, and teeth showing a DIAGNOdent score more than 7 (as a DIAGNOdent score between 3 and 7 indicates normal enamel) were excluded from the study. The extracted teeth were stored in 10% formalin immediately after extraction. The teeth were thoroughly cleaned of its debris, calculus, and soft tissues. The buccal surfaces of all the teeth were polished using micromotor, contra-angled handpiece, polishing brush, polishing cup, and polishing paste. The polished extracted teeth were randomly grouped into four using simple randomized sampling.

GROUP I: CPP-ACP (GC Tooth Mousse, Tokyo, Japan)

GROUP II: CPP-ACP + Fluoride (GC Tooth Mousse plus, Tokyo, Japan)

GROUP III: Tricalcium Phosphate (Clinpro Tooth crème, 3M ESPE, Australia)

GROUP IV: Control (No agent used).

Each extracted tooth was coated with nail varnish, leaving an enamel window of 3 mm × 3 mm on the buccal surface in the middle one-third of the crown. For ease of identification, four different colors of nail varnishes were used. One window was made on each premolar while two windows were made on the buccal surfaces of molars and these were counted as two samples. Each window in all the groups was numbered 1 to 25 using the area on root portion. All the samples were examined using DIAGNOdent (KaVo, Biberach, Germany) to assess for any surface changes present on the labial window. In this study, type B probe was used. As recommended by the manufacturer, prior to every measurement session, the instrument was calibrated against its own ceramic standards. The labial window area was carefully scanned using the type B probe by holding the tip in close contact with the tooth surface and tilting the tip around the measuring area in order to collect the fluorescence from all directions.

Samples showing a moment value between 3 and 7 on the digital display were selected. Samples showing a value greater than 7 were discarded and replaced by teeth having a moment value 3 to 7. The baseline values of the four groups were then recorded.

Ten selected samples from each group (total 40) were also assessed by environmental scanning electron microscope (E-SEM) (Quanta 200™ FEI ICON Analytical Company, India).

A demineralizing solution and artificial saliva were then prepared in Department of Biochemistry, Rural Medical College, Loni. A digital pH meter was used to check pH during and after preparation of solution]. Each time before checking pH, the instrument was calibrated using phosphate buffer solution of pH 7.0. The composition of demineralizing solution and artificial saliva used was as follows:

Demineralizing solution:

The final pH was adjusted to 4.5 with 50% sodium hydroxide (NaOH).

2.2 mM calcium chloride (CaCl2.2H2O)

2.2 mM monosodium phosphate (NaH2PO4.7H2O)

0.05 M lactic acid

Artificial saliva:

The final pH was adjusted to 7.00 at 37 C° with 85% lactic acid.

2.200 g/L gastric mucin,

0.381g/L sodium chloride (NaCl)

0.213 g/L calcium chloride (CaCl2.2H2O)

0.738 g/L potassium hydrogen phosphate (K2HPO4.3H2O)

1.114 g/L potassium chloride (KCl).

All the samples were then immersed into a glass container containing 50 ml of demineralizing solution for a period of 48 h at 37 C° using an incubator (OSWORLD™, model no: JRIC-9, by M/S Commander Diagnostics, India). This demineralizing procedure was intended to produce a consistent subsurface lesion. After 48 h of incubation in the demineralizing solution, the teeth were washed with deionized water, dried with the help of an air syringe, and placed in four different clean glass containers until further evaluation.

The teeth were evaluated with DIAGNOdent and the samples showing a moment value of 9 and above on the digital display were taken for further evaluation. This value indicated the presence of a subsurface lesion on the tooth surface. The samples were also assessed using E-SEM.

The samples in each group were treated with the respective remineralizing agent (except for the control group) at every 24 h for 7 days, with the help of cotton applicator tip. Samples in experimental groups were rubbed with respective remineralizing agent for 4 min, washed with deionized water, and placed in artificial saliva. In the control group, samples were washed with only deionized water and placed in artificial saliva. Artificial saliva was changed every 24 h just before immersion of freshly treated samples.

After 7 cycles of remineralization, the surface was assessed using DIAGNOdent to record the values. The samples were also assessed using E-SEM.

The DIAGNOdent values obtained were tabulated and statistically analyzed using Student's paired ‘t’ test for intragroup comparison and one-way ANOVA test was used for intergroup comparison. P values less than 0.05 were considered to be statistically significant.

OBSERVATIONS AND RESULTS

Observations and results are divided into two parts: (1) Statistical analysis for DIAGNOdent scores and (2) E-SEM microphotographs.

Statistical analysis was done for intragroup, intergroup between two groups, and multiple group comparison of DIAGNOdent score.

By applying Students paired ‘t’ test, a highly significant increase in the mean values of DIAGNOdent score of enamel from baseline to after demineralization was seen. There was a highly significant decrease in the mean values of DIAGNOdent score of enamel from after demineralization to 7 days after remineralization in all the three study groups [Tables 1–4]. This proves that all the three remineralizing agents used in this study are effective in remineralizing artificial enamel caries. Intergroup comparison between groups I-III, groups I-IV, groups II-IV, and groups III-IV showed statistically significant improvement in DIAGNOdent scores at each stage in the study [Tables 5 and 6].

Table 1

Change in DIAGNOdent score from before demineralization to after demineralization and 7 days after remineralization in group I

Table 4

Change in DIAGNOdent score from before demineralization to after demineralization and 7 days after remineralization in group IV

Table 5

Between-groups statistical comparison of relative improvement in DIAGNOdent score at each stage of study

Table 6

Between groups statistical comparison of relative percentage improvement in DIAGNOdent score at each stage of study

Tables 1–3 show the comparison of relative percentage improvement in DIAGNOdent score at each stage of the study by applying one-way ANOVA with Tukey's correction for multiple comparisons; P values less than 0.05 are considered to be statistically significant.

Table 3

Change in DIAGNOdent score from before demineralization to after demineralization and 7 days after remineralization in group III

Environmental scanning electron microscopy analysis

All E-SEM microphotographs taken before demineralization showed normal enamel surface (consistently smooth) in all the groups. Microphotographs taken after 48 h of demineralization showed irregular surface with loss of some surface enamel in all the study groups. Few specimens also showed an etching pattern after demineralization.

Remineralization of demineralized enamel surface is seen after treatment with all the three agents for 7 days. Instead of large remineralization globules, the remineralized enamel showed regular surface. Treatment of demineralized enamel by using CPP-ACP + Fluoride also showed improvement and remineralization was more than in group I. Remineralization in TCP + fluoride group was better than in the other two experimental groups. Enamel surface in this group had maximum remineralization when observed in SEM microphotographs. Enamel samples in group IV did not show any improvement in the demineralized lesions [Table 4].

DISCUSSION

The present study was carried out to evaluate the efficacy of CPP-ACP, CPP-ACP + F, and TCP + F on early enamel caries in vitro with the help of DIAGNOdent and E-SEM. Evaluation of demineralization and remineralization was done at baseline, after 48 h of demineralization, and 7 days of remineralization.

Assessment of in vitro demineralization and remineralization can be done using different methods. The methods used are SEM/ESEM,[11-13] DIAGNOdent,[13] surface microhardness,[1415] etc., Researchers have used one or more of the above-mentioned parameters. In the present study, E-SEM and DIAGNOdent are used to assess demineralization and remineralization.

DIAGNOdent laser fluorescence is a noninvasive method used to measure early demineralization of tooth. The organic and inorganic materials present on the tooth surface absorb the laser light and emit fluorescence in the infrared region of the spectrum. In the presence of caries, light with a higher wavelength is reemitted, and the changes are registered in a digital number scale, with an audible sound indicating the fluorescence increase.[13] Promising results have been published with this Laser Flourescence device.[1617]

Many in vitro studies have proved that CPP-ACP have a remarkable ability to remineralize caries. While in one study authors have concluded that although CPP-ACP can remineralize surface lesion, it is not effective in remineralizing the early enamel caries at the subsurface level.[18]

Fluoride, when added to CPP-ACP, gives a synergistic effect on remineralization of early carious lesion. Elsayad reported that addition of fluoride to CPP-ACP could give a synergistic effect on enamel remineralization.[19] Karlinsey found CPP-ACP + fluoride to be effective in remineralizing bovine enamel specimens.[20] In the present research also CPP-ACP + fluoride is found to be more effective than CPP-ACP alone with no significant difference. The DIAGNOdent score was improved by 7.16 ± 5.32 in group II, that is, 34.42% improvement after remineralization for 7 days. The results are similar to the study done by Jayrajan et al.[13]

In the present research, group III has shown 54.24% of remineralization in 7 days with an improved DIAGNOdent score by 9.48 ± 4.46. TCP + fluoride is found to be effective than both CPP-ACP (significant) and CPP-ACP + fluoride (not significant). Higher concentration of calcium ion in TCP and addition of fluoride in group III might have led to better remineralization capacity than CPP-ACP and CPP-ACP + fluoride. Also both fluoride containing groups showed better performance than CPP-ACP alone.

Fluoride present in the oral fluids alters the continuously occurring dissolution and reprecipitation processes at the tooth–oral fluid interface. Remineralization of incipient caries lesions is accelerated by trace amounts of fluoride.[21] High concentration fluoride therapies lead to deposition of aggregates of calcium fluoride on the surface, which then acts as a reservoir of fluoride. The rate of fluoride release is enhanced at lower pH levels.[22] A pH less than 5 causes loss of adsorbed phosphate and triggers a slow dissolution of the calcium fluoride.[23] To increase its surface area, nano-sized particles of calcium fluoride have been prepared, with a diameter of about 41 nm.[24] Such particles are many times larger than those in Recaldent™ (CPP-ACP or CPP-ACPF), where the nanoclusters are only 2 nm in diameter. In laboratory studies where there is no saliva or plaque present and prolonged contact with remineralizing agents is assured, artificial solutions containing calcium and phosphate and fluoride (at levels of 1 ppm) can result in mineral gain in natural and laboratory-created white spot carious lesions over a 4-week period.[25]

Some studies on nonfluoride caries-preventive agents showed poor evidence, and where the panelists could not reliably estimate the benefit versus the basis of the findings of published studies, the panelists concluded that there was insufficient evidence of nonfluoride caries-protective agents. According to them, in such cases, clinicians and patients alike should understand fully the uncertainty in the evidence as well as any potential risks of using or not using a particular intervention.[25]

Drawbacks of the present study include period of remineralization used in the study was 7 days, which could not remineralize artificial caries completely, thus the period of application for complete remineralization cannot be described for remineralizing agents used in the study. Although surface remineralization was confirmed, enamel subsurface remineralization was not evaluated in the study. One must bear in mind that remineralization in vitro may be quite different when compared with dynamic complex biological system, which usually occurs in oral cavity in vivo. Thus, direct extrapolations to clinical conditions must be exercised with caution because of obvious limitations of in vitro studies.

There is a great need for improved and novel remineralization methods to alter the caries balance for the better, especially in individuals with a high cariogenic bacterial challenge.

CONCLUSION

All the three remineralizing agents in the study could effectively remineralize artificial enamel caries

TCP + fluoride-based products performed better than CPP-ACP-based products in remineralizing artificial enamel caries

Incorporation of fluoride into CPP-ACP compounds enhanced the remineralizing capacity

Complete remineralization did not occur by all the three remineralizing agents in 7 days.

Table 2

Change in DIAGNOdent score from before demineralization to after demineralization and 7 days after remineralization in group II