Comparative evaluation of the remineralizing potential of different calcium and fluoride-based delivery systems on artificially demineralized enamel surface; an in vitro study.

Background: Enamel is the hardest and vastly mineralized structure of the human body. Inorganic salts make up the majority of it. Water and ions pass right through the enamel. The enamel undergoes demineralization and remineralization cycle throughout life. Recently, the interest in the development of calcium, phosphate, and fluoride remineralization technology has been increased leading to the development and reintroduction of various remineralizing agents such as fluoride, casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF), nano-hydroxyapatite (nano-HA), xylitol, bioglass, Ozone, etc. Aim: The aim of this study is to evaluate and compare the potential remineralization of CPP-ACPF, calcium sucrose phosphate (CaSP), amine fluoride (AmF), and nano-HA. Settings and Design: The design of this research is experimental. This is an in vitro study. The research was carried out at the Dental Teaching Institute. This study involved human teeth. No demographic data were obtained. One hundred and twenty premolars were selected from the tooth bank of the Department of Oral and Maxillofacial Surgery. The teeth included in the study were noncarious, nonrestored, and nonfractured extracted teeth. Teeth excluded from the study were extracted teeth with any visible or detectable caries, white spot lesions, microcracks, attrition, abrasion, erosion, or abfraction. Materials and
Methods: One hundred and twenty enamel samples were taken; they were divided into six groups (n = 20). The demineralization process was carried out on Groups II, III, IV, V, and VI. The remineralization process was carried out on Groups III, IV, V, and VI for 14 days using CASP, AmF, and nano-HA, respectively. No surface treatment was performed in Group I causing it a positive control group, whereas Group II was considered a negative control with only enamel surface demineralization and no remineralization. The microhardness of enamel was measured using Vickers microhardness testing machine after a 14-day remineralization regimen. Statistical Analysis: In the statistical analysis, one-way analysis of variance and post hoc Tukey's tests were performed.
Results: The mean microhardness values in declining order: positive control > nano-HA > AmF > CaSP > CPP-ACPF > negative control.
Conclusion: All remineralizing agents exhibited enhanced surface remineralization. Nano-HA showed the highest remineralization potential followed by AmF, CaSP, and CPPACPF. Copyright:

INTRODUCTION

Dental caries is a frequent oral problem affecting a vast population. Numerous communities are affected by this worldwide public health problem. The onset of early enamel caries is caused by a drop in oral pH below 5. This pH value is low enough to initiate enamel demineralization. Enamel subsurface layer gets demineralized during the early stages of demineralization.[1]

A carious lesion occurs when there is an organization of a distinctive bacterial population, which is capable of demineralizing enamel under a specific modified environment within the mouth. This demineralization manifests as a white opaque spot clinically, particularly when air-dried.[2]

However, demineralization is opposed by neutralizing the oral pH. This can be achieved by remineralization. The environment around the lesion becomes richer in calcium and phosphate ions due to remineralization. Remineralization involves the formation of partially dissolved apatite crystals and the prevention of future caries development.[1] At an early-stage natural demineralization of the tooth is reversed by saliva, which contains calcium, phosphate, fluoride ions, buffering agents, and other substances.[3]

There are abundant ways for rapid remineralization. It consists of a mechanism to deliver ions to the distressed area. Remineralizing agents such as fluoride are extensively used on the enamel surface.[4] When acid attacks the enamel surface, the pH of the surrounding starts to increase and the fluoride in the microenvironment stops dissolving enamel, stopping the demineralization.[2]

Milk and milk products can cause the cessation of initiation of dental caries. The anticariogenic property of milk is provided by casein, calcium, and phosphate, imparting its action. As a potential remineralizing agent calcium sucrose phosphate (CaSP) was recently reinstated. It decomposes into calcium, phosphate, and sucrose ions, which boosts remineralization.[4]

Nano-hydroxyapatite (nano-HA) is a calcium-phosphate compound that is bioactive and biocompatible. It shows a strong affinity to the tooth and can strongly adsorb on the enamel surface, thereby remineralizing the initial carious lesion.[5]

This study emphasizes the evaluation and comparison of the enamel remineralizing potential of casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF), CaSP, amine fluoride (AmF), and nano-HA using surface microhardness (SMH) analysis.

MATERIALS AND METHODS

A total of 120 intact maxillary premolars, freshly extracted for orthodontic purposes were selected. Teeth with any white spot lesions, any visible or detectable caries were excluded from the study. To decorate all the teeth at the cementoenamel junction, a diamond disc fixed on a straight handpiece was utilized.[6] Enamel samples were embedded in self-cure acrylic resin and the enamel surface was kept exposed. These samples were placed in deionized water until their next use. One hundred and twenty enamel samples were prepared and randomized into six groups each with 20 samples.

The demineralizing solution was prepared within the Biochemistry Department.

During and after solution preparation, a digital pH meter (BSR Technologies Pvt. Ltd., Nashik, Maharashtra, India) was used to assess the pH.

The demineralizing solution was composed of:

2.2 mM monosodium phosphate, NaH2PO4·7H2O (Research-Lab Chem Industries Pvt. Ltd., Mumbai, Maharashtra, India)

2.2 mM calcium chloride, CaCl2·2H2O (Cero Chemicals Pvt. Ltd., Navi Mumbai, Maharashtra, India)

0.05 M lactic acid, C3H6O3(Modern Industries Pvt. Ltd., Nashik, Maharashtra, India).

After pH was adjusted to 4.5 with sodium hydroxide (Loba Chemie Pvt. Ltd., Mumbai, Maharashtra, India), all samples of Groups II, III, IV, V, and VI were immersed in 50 mL of demineralizing solution for 72 h at 37°C in the universal incubator. The goal of this demineralization treatment was to create a homogeneous subsurface lesion. The teeth samples were then cleaned with deionized water, dried, and placed in respective glass containers until further evaluation.[3] In Groups III, IV, V, and VI, the samples were remineralized every 24 h for 14 days. Samples were applied with appropriate remineralizing agents for 3 min using a tip applicator, cleansed with water, and then put in artificial saliva. After each remineralizing cycle, all samples were stored in an incubator at 37°C. Control samples were merely placed into artificial saliva after being washed with deionized water. Every 24 h, artificial saliva was replenished before the submersion of recently processed samples.[3] The SMH of the specimens was determined using a Vickers microhardness testing machine (Prima, Germany) following 14 days of remineralization. For 10 s, 100 g steady load was applied continuously to the surface of the specimens using an elongated diamond pyramid indenter under a ×40 objective lens. The accuracy of indentation length values was determined under ×400 high magnification. A built-in microscope was used to measure the depth of the indentations, and the values were converted to Vickers microhardness values. Each specimen was indented five times and the mean was calculated. Statistical evaluation was carried out with the use of one-way analysis of variance (ANOVA), post hoc Tukey's test using SPSS 19.0 software program (SPSS Inc., Chicago, IL, USA).

Statistical methods

Across study groups, the SMH of samples was compared. A one-way ANOVA test was used to evaluate the mean and standard deviation (SD) of microhardness of samples for each group. Tukey's post hoc test was used to compare mean microhardness between groups on a pair-wise basis. The statistical significance was set at 5%, and the analysis was carried out with the SPSS 19.0 program (SPSS Inc., Chicago, IL, USA).

RESULTS

Table 1 displays the statistical parameters for each group, such as the mean and SD of sample microhardness. Positive control had the greatest mean of 279.42 VHN, while negative control had the lowest mean of 198.76 VHN. In the case of materials used, nano-HA indicated the highest mean of 266.43 VHN, followed by AmF with a mean value of 236.61 VHN, followed by CaSP with a mean value of 224.19 VHN, and CPP-ACPF with the least mean value of 212.62 VHN [Table 1]. The mean microhardness value difference between the groups was significant statistically, according to the test. Accordingly, Table 2 displays a pair-wise comparison of microhardness among groups to see which ones differed considerably from the others. The test indicated that the positive control group's mean microhardness was considerably higher than all other groups, with P 0.05 for all comparisons. Nano-HA had a higher mean microhardness than the other three experimental groups, although the difference was statistically insignificant, as P > 0.05 revealed. However, it was significantly different from Group II in terms of mean (P < 0.05). The implied microhardness of CPP-ACPF, CaSP, and AmF becomes insignificantly different (P > 0.05), however, differed appreciably from Group II. In conclusion, this study shows that the overall difference in mean microhardness between groups is primarily due to Group I and Group II, with the averages of the four experimental groups slightly different from each other.

Table 1

One-way analysis of variance test

Microhardness (VHN)	n	Mean±SD	F	df	P
Group I (control)	20	279.42±22.391	38.79	5	<0.001 (highly significant)
Group II (demineralization)	20	198.76±15.660
Group III (CPP-ACPF)	20	212.62±24.384
Group IV (CaSP)	20	224.19±21.703
Group V (AmF)	20	236.61±23.339
Group VI (Nano-HA)	20	266.43±25.793
Total	120	236.34±36.103

P>0.05 - not significant; P<0.05 - significant; P<0.001 - highly significant. SD: Standard deviation, CPP: Casein phosphopeptide, ACPF: Amorphous calcium phosphate fluoride, CaSP: Calcium sucrose phosphate, AmF: Amine fluoride, HA: Hydroxyapatite, VHN: Vickers Hardness Number

Table 2

Tukey’s post hoc tests-multiple comparisons (Bonferroni test)

I Groups	J Groups	Mean difference (I-J)	P
Group I (control)	Group II (demineralization)	80.66*	<0.001
	Group III (CPP-ACPF)	66.80*	<0.001
	Group IV (CaSP)	55.23*	<0.001
	Group V (AmF)	42.81*	<0.001
	Group VI (Nano-HA)	12.99	1.000
Group II (demineralization)	Group III (CPP-ACPF)	−13.86	0.799
	Group IV (CaSP)	−25.43*	0.008
	Group V (AmF)	−37.85*	<0.001
	Group VI (Nano-HA)	−67.68*	<0.001
Group III (CPP-ACPF)	Group IV (CaSP)	−11.57	1.000
	Group V (AmF)	−23.99*	0.015
	Group VI (Nano-HA)	−53.82*	<0.001
Group IV (CaSP)	Group V (AmF)	−12.42	1.000
	Group VI (Nano-HA)	−42.24*	<0.001
Group V (AmF)	Group VI (Nano-HA)	−29.83*	0.001

*The mean difference is significant at the 0.05 level. CPP: Casein phosphopeptide, ACPF: Amorphous calcium phosphate fluoride, CaSP: Calcium sucrose phosphate, AmF: Amine fluoride, HA: Hydroxyapatite

DISCUSSION

Dental caries is one of the most prevalent causes of tooth loss in people of all ages and genders. Myriad studies have been performed that have improved our understanding of dental caries and reduced its incidence. Dental caries still perpetuates to be a prominent dental illness, confirming to the World Oral Health Report.[7]

Central to the present vision is the skill to detect caries lesions at an early stage and properly quantify the degree of mineral loss, ensuring that the right intervention is instituted.[8] Enamel subsurface lesions must be diagnosed as early as possible for the simplest possible remineralization of enamel and restoration of function of the tooth.[9]

To imitate oral circumstances, the specimens were submerged in the demineralizing solution for 48 h at 37°C in a universal incubator. This caused a deep demineralization with a healthy superficial surface, simulating an early enamel lesion.[2]

In the present study, four different calcium and fluoride-based ion delivery systems were used for the remineralization of subsurface carious lesions, i.e., (CPP-ACPF), (CaSP), (AmF), and (nano-HA). All the four remineralizing agents used were in the form of standard dentifrices.

Due to the importance of surface intactness, SMH measurement was a viable and quick approach for this experimental design. As a result, the SMH values for each specimen were assessed in the current investigation.[4]

The mean microhardness of the positive control group (Group I) in the current study was substantially higher than all other groups, indicating the validity of the study.

CPP-ACPF is a calcium phosphate supersaturated solution that contains both amorphous and crystalline phases. It is rich in fluoride content. It has a stabilized formula that prevents calcium phosphate from precipitating on its own. The ability to remineralize is corresponding to the amount of free calcium and phosphate ions equilibrated by CPP.[10] According to Cochrane et al., CPP-ACP and CPP-ACFP penetrate the porosity of the enamel subsurface lesion and diffuse into the body of the lesion along a concentration gradient.[11] CPP-ACPF has the lowest mean microhardness of 212.62 MPa in this investigation. It has been claimed that for CPP-ACP molecules to become activated, they must be exposed to an acidic environment, which should dissociate ACP from casein. Only once were samples subjected to the demineralization cycle. They were rinsed with fabricated saliva when it was essential to activate them. This might explain why CPP-ACPF has the lowest microhardness value.[1213]

CaSP creates an aqueous solution that consists of a high concentration of calcium and phosphate without incidence of precipitation. It acts as a perfect carrier for calcium and phosphate in water. CaSP showed enhanced SMH in this investigation, with a mean value of 224.19 MPa. CaSP works by quickly adsorbing sucrose phosphate ions on the enamel surface, slowing the acid dissolution of HA, and allowing for fast remineralization with calcium and phosphate ions through the common ion effect.[6]

AmF used in this study is an organic fluoride. AmF showed better SMH in this investigation, with a mean value of 236.61MPa. According to Shani S et al., amine-fluorides reduce caries by inhibiting the acid produced by plaque bacteria, reducing enamel solubility, preventing bacterial adhesion to teeth, and affecting the vitality of bacteria.[14]

Because of its distinctive potential for remineralization, nano-HA has been intensively explored as a biomimetic material for the repair of dental enamel suffering from mineral loss in recent years. It has also been demonstrated that it could be exercised as an efficacious anticaries agent.[15]

In this study, nano-HA showed the highest SMH with a mean value of 266.43 MPa. Nanosized particles can penetrate the enamel pores and are the natural tooth mineral phase as far as crystal size is considered. Moreover, the high surface area of the nanosized particles imparts a good mechanical strength to dental enamel. Therefore, nano-HA is expected to assist in the remineralization of an early caries lesion.[16] This has been supported by a similar study conducted by Amaechi et al.[17] who compared nano-HA with CPP-ACP and fluoride varnish and exhibited that the SMH of nano-HA was higher than the remaining groups. Various studies conducted by Kim et al.[16] and others by Jeong et al.[18] also evaluated the effect of nano-HA on remineralization using Vickers hardness number and SEM to evaluate enamel surface and found similar results.[16]

Artificial enamel caries was not entirely remineralized after 14 days of remineralization. It was recognized as one of the flaws of this study. As a result, for all the remineralizing agents employed the length of treatment for full remineralization could not be established.

Even though surface remineralization was validated, subsurface remineralization of enamel was not assessed. Within the constraints of this in vitro investigation, it may be concluded that the application of CPP-ACPF, CaSP, AmF, and nano-HA results in remineralization. However, throughout the 14 days, full remineralization did not occur.

CONCLUSION

Surface remineralization was enhanced with all remineralizing agents. Within 14 days, however, there was no absolute remineralization

Nano-HA, followed by AmF, CaSP, and CPP-ACPF, had the greatest potential for remineralization

CPP-ACPF has the least remineralizing potential as compared to other groups, used in the study

SMH analysis has confirmed the formation of HA crystals in all the groups.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.