Comparative evaluation of remineralization potential of nanohydroxyapatite crystals, bioactive glass, casein phosphopeptide-amorphous calcium phosphate, and fluoride on initial enamel lesion (scanning electron microscope analysis) - An in vitro study.

AIM: The aim of this in vitro study was to evaluate and compare the remineralization potential of four different remineralizing agents, i.e., nanohydroxyapatite crystals, bioactive glass, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and fluoride on initial enamel lesion.
MATERIALS AND METHODS: Sixty human maxillary central incisors were used in the present study. Samples were randomly divided into four groups (n = 15). Group 1: nanohydroxyapatite-containing dentifrice (Acclaim); Group 2: bioactive glass containing-dentifrice (SHY-NM); Group 3: CPP-ACP-containing dentifrice; and Group 4: fluoride-containing dentifrice. Baseline microhardness was checked, followed by immersion of teeth samples in demineralizing and remineralizing solution. This was followed by a pH cycle of 10 days. Data were analyzed using ANOVA and Bonferroni method. After this, scanning electron microscopic analysis was done to evaluate remineralization.
RESULTS: Statistical analysis of data was conducted using ANOVA, and multiple comparisons within groups were done using the Bonferroni method (post hoc tests). The decision criterion was to reject the null hypothesis if P < 0.05. If there was a significant difference between the groups, multiple comparisons (post hoc test) using the Bonferroni test were carried out.
CONCLUSION: There is a significant difference in mean microhardness between the groups after remineralization. The mean value was found to be highest for nanohydroxyapatite, bioactive glass, CPP-ACP, and fluoride in descending order. Copyright:

INTRODUCTION

Dental caries in enamel is unique among diseases as enamel is both acellular and avascular.[1]

The current consensus in caries management is that they should be detected at the earliest to prevent surgical intervention. Over the past few decades, fluoride was used in its various forms, which resulted in a widespread reduction in caries due to its extensive use in the form of a toothpaste. In recent years, other remineralizing agents have come up, which helps in remodeling the hydroxyapatite crystals and promote remineralization along with reducing the apatite crystal dissolution.[2] Some of these remineralizing agents are nanohydroxyapatite, bioactive glass, and casein phosphopeptide-amorphous calcium phosphate (CPP-ACP).

MATERIALS AND METHODS

A total of sixty human maxillary central incisors were collected. Teeth were cleaned thoroughly to remove any deposits. Samples were decoronated 1 mm below the cementoenamel junction with a diamond disc [Figure 1]. The crowns were subjected to removal of 200 mm of surface enamel on the labial aspect to eliminate variability in samples. A 4 × 4 mm working window was marked on the labial surface of all samples, which was considered for remineralization, and the rest of the area of the crown was covered with nail varnish. Samples were randomly divided into four groups (n = 15). Group 1: nanohydroxyapatite-containing dentifrice; Group 2: bioactive glass-containing dentifrice; Group 3: CPP-ACP-containing dentifrice; and Group 4: fluoride-containing dentifrice.

Figure 1

Decoronated samples

Baseline surface microhardness measurement

Baseline surface microhardness (B-SMH) was checked with Vickers microhardness testing machine for all tooth samples in the area of the working window. The indentations were made with Vickers microhardness testing machine at the rate of 25 g load for 5 s.[3]

Preparation of demineralizing solution

The buffered demineralizing solution was prepared using analytical grade chemicals and deionized water. The demineralizing solution contained 2.2 mM calcium chloride, 2.2 mM sodium phosphate, and 0.05 M acetic acid; the pH was adjusted with 1 M potassium hydroxide to 4.4.

Preparation of artificial carious lesion

Samples were kept in demineralizing solution for 96 h to produce an artificial carious lesion in enamel. Demineralizing surface microhardness was checked with Vickers testing machine similar to B-SMH.

Preparation of remineralizing solution

The remineralizing solution was prepared with 1.5 mM calcium chloride, 0.9 mM sodium, and 0.15 M potassium chloride, which had a pH of 7.0.

Toothpaste preparation

Dentifrice supernatant was prepared by suspending 12 g of the respective dentifrice in 36 ml deionized water to create a 1:3 dilution. The suspensions were thoroughly mixed and then centrifuged at 3500 rpm for 20 min at room temperature, once daily before starting pH cycling.

The pH cycling model

The specimens were placed in a pH cycling system on a cylindrical beaker for 10 days.

Scanning electron microscopic analysis

After the pH cycling for 10 days, scanning electron microscopic (SEM) analysis was done to evaluate remineralization [Figure 2].

Figure 2

Scanning electron microscopic images (a) nanohydroxyapatite, (b) bioactive glass, (c) casein phosphopeptide-amorphous calcium phosphate, (d) fluoride

RESULTS

The present study compared the remineralizing potential of four agents, i.e., nanohydroxyapatite-containing dentifrice, bioactive glass-containing dentifrice, CPP-ACP-containing dentifrice, and fluoride-containing dentifrice on initial enamel caries [Figure 3].

Figure 3

Comparison of four groups with respect to baseline, after demineralization, and after remineralization

There is a significant difference in mean microhardness between the groups after remineralization, which was found to be statistically significant at P < 0.05, indicating changes in the mineralization of teeth samples as shown in Figure 1and Table 1.

Table 1

Comparison of baseline, after demineralization and after re-mineralization scores in four groups by paired t test

Groups	Points	Mean	Std.Dv.	Mean Diff.	SD Diff.	% of change	Paired t	P
Fluoride	Baseline	236.54	75.60
	After demineralization	196.81	63.54	39.73	96.29	16.80	1.5981	0.1323
	Baseline	236.54	75.60
	After re-mineralization	201.86	38.77	34.68	80.29	14.66	1.6729	0.1165
	After demineralization	196.81	63.54
	After re-mineralization	201.86	38.77	-5.05	71.31	-2.57	-0.2745	0.7877
Cpp-acp	Baseline	219.70	81.07
	After demineralization	233.84	77.01	-14.14	94.44	-6.44	-0.5799	0.5712
	Baseline	219.70	81.07
	After re-mineralization	240.71	58.26	-21.01	102.64	-9.57	-0.7930	0.4410
	After demineralization	233.84	77.01
	After re-mineralization	240.71	58.26	-6.87	55.35	-2.94	-0.4811	0.6379
Bioactive	Baseline	235.46	74.46
	After demineralization	226.77	53.50	8.69	115.28	3.69	0.2921	0.7745
	Baseline	235.46	74.46
	After re-mineralization	278.79	49.51	-43.32	78.11	-18.40	-2.1482	0.0497*
	After demineralization	226.77	53.50
	After re-mineralization	278.79	49.51	-52.02	91.71	-22.94	-2.1967	0.0454*
Nano	Baseline	230.39	74.75
	After demineralization	232.57	83.59	-2.18	130.71	-0.94	-0.0645	0.9495
	Baseline	230.39	74.75
	After re-mineralization	329.82	69.75	-99.42	99.16	-43.15	-3.8832	0.0017*
	After demineralization	232.57	83.59
	After re-mineralization	329.82	69.75	-97.25	81.75	-41.81	-4.6070	0.0004*

DISCUSSION

Dental caries is one of the causes of tooth loss for all human beings across age and gender.[4] It is a highly prevalent multifactorial disease and although in most developed countries. The signs of caries process cover a continuum from the first molecular changes in apatite crystals of the tooth, to a visible white spot lesion, to dentin involvement, and eventual cavitation.

Progression through these stages requires a continual imbalance between pathological and protective factors that result in the dissolution of apatite crystals.[1]

Remineralization of white spot lesions and carious lesions may be possible with a variety of currently available agents containing nanohydroxyapatite, fluoride, bioavailable calcium phosphate, CPP-ACP, and self-assembling peptide.[25] Instead of using the traditional pH cycling method, a modified version was utilized in our study, which included a 3 h demineralization cycle twice a day, with one 2 h and one intervening overnight remineralization cycle, respectively. To replicate early morning, midday, and before bedtime toothbrushing, toothpaste was applied thrice daily.[4] Brushing time of 1 min was utilized in the present study to replicate real-life situations similar to a study done by Kumar et al.[6] who treated the samples for 60 s, which showed similar results, wherein CPP-ACP was better than fluoride.[6] SEM utilized in the study is the most recent technology that will reveal distinct coatings of hydroxyapatite crystals deposited on the treated enamel surface.[7] This technology has been used by many investigators such as Narayana et al.,[8] Swarup and Rao,[9] and Huang et al.[10] For SEM, samples were thoroughly vacuum dried and gold sputtered, and then images of the surface enamel were recorded at various magnification.[11]

Nanohydroxyapatite also acts as a filler because it repairs small holes and depressions on enamel surface, a function enhanced by the small size of the particles that compose it.[110]

By penetrating the enamel pores, nanohydroxyapatite crystals act as a template in the precipitation process and promote crystal integrity and growth.

Moreover, nanohydroxyapatite is hydrophilic and has a greater surface area than conventional hydroxyapatite crystals, so they have better wettability and form a thin layer on enamel surface that bonds to tooth structure.[12]

The ACLAIM toothpaste used in this study contains the synthetic biomimetic hydroxyapatite. The function of this hydroxyapatite is to protect the teeth with the creation of a new layer of synthetic enamel around the tooth.[1314]

In our study, nanohydroxyapatite has proved to be a better remineralizing agent, shown by the highest mean hardness value when measured with Vickers microhardness testing machine.

This has been supported by a similar study conducted by alaudin SS et al.[15] who compared nanohydroxyapatite with CPP-ACP and fluoride varnish and showed that the surface microhardness value for nanohydroxyapatite was higher than the other groups.

Various studies conducted by Kim et al.[16] and others by Jeong et al.[17] also evaluated the effect of nanohydroxyapatite on remineralization using Vickers hardness number and SEM to evaluate enamel surface and found similar results.[16]

Bioactive glass used in the study was invented by Dr. Larry Hench in the 1960s. It acts as a biomimetic mineralizer matching the body’s own mineralizing traits and also affecting cell signals, thereby benefitting the restoration of tissue structure and function.

Bioglass in an aqueous environment immediately begins surface reactions in three phases – leaching, exchange of cations and network dissolution of SiO2, and precipitation of calcium-phosphate forming an apatite layer.[517]

SHY-NM used in the study has been demonstrated to show fine particulate bioactive glasses incorporated into an aqueous dentifrice, which has the ability to clinically reduce the tooth hypersensitivity through the occlusion of dentinal tubules by the formation of carbonated hydroxyapatite layer.[818]

A study conducted by[19] investigated the remineralization potential of bioactive glass and CPP-ACP using SEM and found that bioactive glass seals the fissures formed after demineralization more intimately, and the deposits are more angular, whereas those of CPP-ACP are smaller and amorphous which is in accordance with our study.

The result of the current study revealed a considerable amount of increase in surface microhardness of the enamel achieved after treatment with SHY-NM, which contains bioactive glass as shown by the mean hardness value of 278.79. Furthermore, P = 0.0021 showed that the result is significant when compared to CPP-ACP and fluoride.

CPP-ACP was patented in the United Nations in 1991. Casein is present in various dairy products such as milk, milk concentrates, and cheese.[20]

CPP-ACP is a new remineralization technology based on phosphopeptide from milk protein casein.[21] The casein phosphopeptide contains multiple phosphoryl sequences with the ability to stabilize calcium phosphate in nanocomplex in solutions such as ACP. Through their multiple phosphoryl sequences, CPP binds to ACP in a metastable solution preventing the dissolution of calcium and phosphate ions.[522]

The CPP-ACP also acts as a reservoir of bioavailable calcium and phosphate and maintains the solution supersaturated, thus facilitating remineralization.

In a study sodium fluoride showed higher remineralization potential of CPP-ACP crème. Variation in results may be due to the variable duration of pH cycling.

In the present study, statistical analysis by ANOVA and post hoc showed that the remineralizing potential of CPP-ACP is higher than fluoride but not significant at P < 0.05.

Fluoride becomes incorporated into the tooth structure, and hydroxyl group of the hydroxyapatite is replaced by fluoride.[23] The mechanism by which fluoride increases caries resistance may be from both systemic and topical applications.[1024]

Brown et al. believed that the working mode of fluoride was due to change in solubility after incorporation in enamel, but only 10% of fluoride substitutes hydroxyl group in the apatite of surface enamel which cannot be considered the only factor causing cariostatic effect.[25]

Results by statistical analysis revealed lowest mean microhardness for fluoride-containing dentifrices showing that the other three dentifrices have better remineralizing capacity than fluoride. This may be due to the fact that fluoride acts predominately during the growth period of dentition rather than after development.

CONCLUSION

All experimental groups have remineralizing potential as shown by increase in microhardness of the respective groups when compared to their baseline values

Nanohydroxyapatite-containing dentifrice has highest remineralizing potential followed by bioactive glass, CPP-ACP, and fluoride

Fluoride has the least remineralizing potential as compared to other groups, used in the study

SEM analysis has confirmed the formation of hydroxyapatite crystals in all the groups.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.