Comparative evaluation of long-term fluoride release and antibacterial activity of an alkasite, nanoionomer, and glass ionomer restorative material - An in vitro study.

Background: The antibacterial activity of restorative material and the amount of fluoride released are interlinked. Hence, these are the two foremost properties to be studied. Aim: This study aimed to evaluate and compare the amount and pattern of fluoride release from Type IX GIC (GC HS posterior), nanoionomer (Ketac N100), and alkasite (Cention N), and the antibacterial activity against Streptococcus mutans at 24 and 48 h. Settings and Design: This in vitro study was carried out in laboratory settings with six samples of each group for fluoride release using an ion-chromatography (IC) machine and five samples of each group for antibacterial activity using agar plates. Materials and Methodology: Samples of each group, Group I - Type IX GIC, Group II - nanoionomer, and Group III -alkasite, were prepared, immersed in 2 ml of artificial saliva, and fluoride release recorded using IC after 1, 7, 14, and 28 days intervals. The antibacterial activity against S. mutans was evaluated by placing samples of each group in the agar plates and measuring the diameter of zones of inhibition after 24 and 48 h. Statistical Analysis: One-way ANOVA test to check to mean differences between the groups and Tukey's honestly significant difference post hoc test for multiple intergroup comparisons (P = 0.05).
Results: The Type IX GIC showed the highest fluoride release after day1. However, nanoionomer showed the maximum fluoride release for the remaining days. The least amount of fluoride released was from the alkasite throughout the study. The antibacterial activity of nanoionomer was the highest, followed by Type IX GIC and alkasite at both 24 and 48 h. Conclusions: Nanoionomer showed the highest fluoride release and antibacterial activity. Copyright:

INTRODUCTION

Dental caries, the most common disease occurring in the oral cavity, is the foremost cause of oral pain and tooth loss.[1] Conventionally, the surgical model of treatment is followed in caries management that involves complete caries removal and restoring the cavity prepared to standard dimensions with restorative materials.[2] However, the most frequent reason for replacing restoration even after effective restorative procedures is secondary caries.[3]

With the discovery of fluoride and years of continuing research, we now have strong validation on fluoride's remineralizing capacity and its ability to increase the tooth's resistance to demineralization. Besides, with the recent trends of minimally invasive approaches and remineralization strategies, we are focusing on the medical model for preventive and conservative management of caries. Hence, an ideal restorative material should have both fluoride-releasing and antibacterial properties along with others.

Glass ionomer cement (GIC) is considered to be the first choice of material in patients with high caries risk. However, the quest to find newer biomimetic restorative materials with superior properties developed due to the disadvantages of GIC such as sensitivity to moisture in the oral cavity, low initial mechanical properties, inadequate surface properties and esthetics, poor wear resistance, short working time, and brittleness.[4]

To overcome the drawbacks of GIC and with the advancements in nanotechnology, a nano-filled RMGIC–nanoionomer (Ketac N100) got introduced with a structural morphology hybrid of resin-modified GIC and that of nano-filled resin composite. It has claimed to have improved esthetics along with the benefits of GIC, such as fluoride release.[5]

Recently, Cention N, an ”Alkasite” restorative material, similar to compomer or Ormocer, was introduced. It belongs to a new category, a subgroup of the composite material class that claims to release substantial levels of fluoride ions comparable to traditional GICs and acid-neutralizing hydroxyl and calcium ions with its patented alkaline filler.[6]

The cariostatic effect of dental materials is directly related to the amount of fluoride released.[7] According to Delbem et al., due to the release of fluoride, the initiation and propagation of secondary caries are significantly reduced with the use of GICs.[8] However, there is a lack of evidence regarding the effective fluoride release and antibacterial activity of these hybrid materials. Hence, this in vitro study compared the fluoride release of Type IX GIC, nanoionomer, and alkasite restorative material using ion chromatography (IC) and antibacterial activity against S. mutans using agar plate diffusion assay.

The null hypothesis formulated was that the fluoride-release pattern and antibacterial activity of the three materials studied over time were not significantly different.

MATERIALS and Methodology

The materials used were Group I-Type IX GIC (GC gold label HS posterior extra), Group II – nanoionomer (Ketac N100), and Group III – alkasite (Cention N).

Evaluation of fluoride release

The artificial saliva was prepared according to Sato et al.[9]

The composition of artificial saliva is as follows:

Na3PO4– 3.90 mM, NaCl2– 4.29 mM, KCl – 17.98 mM, CaCl2– 1.10 Mm,

MgCl2– 0.08 mM, H2SO4– 0.50 mM, NaHCO3– 3.27 mM, distilled water, and pH was set at a level of 7.2.

Sample preparation for fluoride release

The materials are proportioned, according to the manufacturer's instruction, Hand Mixed using a plastic spatula and mixing pad, and loaded into molds (10-mm diameter and 2-mm depth). The specimen's top surface was covered by a mylar strip and glass slide to remove the excess cement. Waxed dental floss was incorporated into the cement during the setting. The GIC was allowed to set at room temperature for 15 min. The specimens of nanoionomer and alkasite were light cured using dental curing light of intensity 1200 mW/cm2(Woodpecker light cure Unit Mini S) for 20 s. The disks were then removed from the molds, suspended inside a plastic vial containing 2 ml of artificial saliva, and incubated at 37°C. The artificial saliva was changed after 1, 7, 14, and 28 days intervals. The quantity of fluoride released was recorded using an IC machine in parts per million. The final results of fluoride release were reported in μg/cm2 taking into account the surface area and solution volume of each specimen, using the formula:

Fluoride release = F μg/ml × volume of solution/area of sample in cm2

Preparations for agar plate diffusion assay

The antibacterial activity of each material was evaluated using the agar plate diffusion assay. Streptococcus mutans (MTCC) strain was used in the study. The strains were cultured on Mueller–Hinton agar (MHA) at 37°C for 24 h in 5% CO2. Single colonies from plates were transferred into BHI broth and incubated at 37°C for 24 h. The density of the microbial suspension was adjusted to 0.5 McFarland turbidity standards, which correspond to 106(colony-forming unit [CFU]/ml), and inoculated onto the surface of MHA. Five wells were created on each plate.

Specimen preparation

Specimens were prepared as mentioned for fluoride release. After 30 min, they were ultraviolet (UV) sterilized for 10 min, transferred to the bacterial plates with microorganisms, and incubated at 37°C. The diameter of zones of inhibition produced around the specimens was measured at 24 h and 48 h at three different points, and tests were repeated thrice to verify the homogeneity of the results. The zone of inhibition was calculated by subtracting the diameter of specimens using Vernier calipers. All the procedures were conducted using aseptic techniques and in an UV light sterilized biohazard hood.

Statistical analysis

The mean values of fluoride and zone of inhibition recorded for all the materials were analyzed using IBM SPSS Statistics for Windows Version 19 (IBM Corp., Armonk, NY, USA). The values between groups were analyzed using one-way analysis of variance, and multiple intergroup comparisons were analyzed using the Tukey honestly significant difference (HSD) (post hoc) test. The confidence interval was set to 95%, and the P value was set for 0.05.

RESULTS

The results have shown that all materials released maximum fluoride after day 1 which decreased and got stabilized over the 7th, 14th, and 28th days [Graph 1]. Among the groups on day 1, the Type IX GIC released significantly higher fluoride when compared to other groups (P < 0.001) Table 1A and 1B. However, from days 7, 14, and 28, a significantly higher amount of fluoride was released by nanoionomer (P < 0.001). The alkasite released lower amounts of fluoride when compared to other materials at all-time intervals (P < 0.001). The cumulative fluoride release from the nanoionomer (57.8 μg/cm2) was the highest among all the groups studied, followed by Type IX GIC (51.15 μg/cm2) and the alkasite (37.25 μg/cm2), respectively [Graph 3].

Graph 1

Showing mean and standard deviation of fluoride release by groups at different intervals

Table 1A

Mean and standard deviation of fluoride release by groups at different intervals (n=6)

Days	Groups, mean±SD			P
	Group I (type 9 GIC)	Group II (ketac nanoionomer)	Group III (Cention N)
Day 1	24.59±0.92	23.92±1.34	17.94±0.60	<0.001*
Day 7	12.55±0.96	15.76±1.09	9.56±0.90
Day 14	7.31±0.92	9.55±0.78	5.63±0.67
Day 28	6.70±0.92	8.57±0.79	4.12±0.41

*One-way ANOVA test P<0.05: statistically significant. ANOVA: Analysis of variance, SD: Standard deviation, GIC: Glass ionomer cement

Table 1B

Multiple intergroup comparison fluoride release by groups using post hoc tests - Tukey honestly significant difference

Repeated contrast test result for fluoride release
	F	P
Fluoride release
Day 1 versus day 7	875.966	<0.001*
Day 7 versus day 14	751.208	<0.001*
Day 14 versus day 28	103.303	<0.001*
Fluoride release versus groups
Day 1 versus day 7	15.250	<0.001*
Day 7 versus day 14	12.458	0.001*
Day 14 versus day 28	6.747	0.008*

*Tukey post hoc test P<0.05: statistically significant

Graph 3

Showing cumulative mean and standard deviation of fluoride release by groups at different intervals

The analysis of variance comparison of the mean zone of inhibition obtained at 24 and 48 h indicated a statistically significant difference (P = 0.016 and P = 0.001) [Table 2]. However, the Tukey HSD showed no statistically significant difference (P = 0.083). Hence, least significant difference comparison was applied, and nanoionomer exhibited a larger zone of inhibition at all periods Figure 1a and b, indicating a higher antibacterial activity (18.2 mm ± 3.11 and 16.4 mm ± 2.30) at 24 and 48 h, respectively, followed by GIC (14 mm ± 1.58 and 10.8 mm ± 0.83) Figure 2a and b and alkasite (14.4 mm ± 1.14 and 11 mm ± 1.22) Figure 3a and b. Nevertheless, there was a general tendency of decrease in the zone of inhibition between the intervals, thus indicating the lowering of antibacterial activity as a function of time with all the materials [Graph 2].

Table 2

Mean and standard deviation for zone of inhibition by groups

Days (h)	Groups, mean±SD			P
	Type 9 GIC (Group I)	Ketac nanoionomer (Group II)	Cention N (Group III)
24	14.00±1.581	18.20±3.114	14.40±1.140	0.016
48	10.80±0.837	16.40±2.302	11.00±1.225	0.001

*One-way ANOVA test P<0.05: Statistically significant. ANOVA: Analysis of variance, SD: Standard deviation, GIC: Glass ionomer cement

Figure 1

(a) Nanoionomer (Ketac N100), specimen in Streptococcus mutans showing zone of inhibition at 24 h. (b) Nanoionomer (Ketac N100), specimen in Streptococcus mutans showing zone of inhibition at 48 h

Figure 2

(a) Type IX GIC (GC gold label HS posterior extra), specimen in Streptococcus mutans showing zone of inhibition at 24 h. (b) Type IX GIC (GC gold label HS posterior extra), specimen in Streptococcus mutans showing zone of inhibition at 48 h

Figure 3

(a) Alkasite (Cention N)-specimen in Streptococcus mutans showing zone of inhibition at 24 h. (b) Alkasite (Cention N)-specimen in Streptococcus mutans showing zone of inhibition at 48 h

Graph 2

Bar diagram showing mean and standard deviation for zone of inhibition by groups at different intervals

DISCUSSION

Methods such as colorimetric analysis, mass spectrometry, spectrophotometry, chromatography, electroanalysis, capillary electrophoresis, and radioanalytical are available for measuring fluoride ions released from materials.[10] The IC method was chosen over gas chromatography or ion-selective electrode, as this can measure free uncomplexed fluoride ions. It can detect small quantities of fluoride not detected by other methods.[10] The artificial saliva was used as the medium as it is similar to natural saliva and the chemical conditions of the mouth. Further, natural saliva cannot be used due to its unstable nature.[11]

The agar-plate diffusion assay is the most commonly used method to evaluate antibacterial activity due to its convenience and efficiency. However, it is highly dependent on molecular size and the diffusion constant of the antibacterial component, inoculum size, and degree of material/agar contact. Hence, to overcome certain limitations, specimens of equal dimensions, the same degree of material/agar contact, the same amount of inoculum, incubation period, and a smaller number of samples and variables have been used.[12] Streptococcus species were used as they are commonly associated with dental caries, directly correlated to the formation of new caries lesions, and accepted that reducing their number also reduces the caries activity.[13] The specimen geometry, temperature, surface treatment and finishing, storage medium, and experimental designs were standardized for all materials. On the other hand, the composition, powder/liquid ratio, and mixing time vary according to the materials used.

The results have shown that, after day 1, nanoionomer released lesser fluoride than Type IX GIC. However, from days 7, 14, and 28, nanoionomer released higher mean fluoride than Type IX GIC and alkasite. Furthermore, nanoionomer showed higher cumulative and long-term fluoride release. The results of this study are similar to the findings of Paschoal et al.,[4] and Neelakantan et al.,[14] who reported that conventional GIC released the highest fluoride on the first 3 days and thereafter nanoionomer showed the highest release for the remaining days. However, according to a study by Fúcio et al.,[15] Type IX GIC released significantly higher amounts of fluoride than nanoionomer throughout the experimental period.

Nanoionomer sets by both acid–base and light polymerization reaction. The higher release after day 1 may be due to HEMA, which slowly absorbs water, and hence, the higher long-term release and the results obtained.[816] Furthermore, the size of nano-sized glass particles “nanomers” are 5–25 nm and Fluoroaluminosilicate (FAS) glass are 1 μm in comparison to 3.34–9.6 μm size of conventional GIC. Hence, the increased acid–base reactivity from a larger surface area and higher fluoride release from nanoionomer.

However, the higher amount of fluoride released at day1 by GIC may be due to the burst effect. Hence, the high initial fluoride is through superficial rinsing due to the initial acid dissolution of powder particles from the surface. However, over time, fluorides become trapped in the hardening polysalt gel matrix, diffuse through cement pores and fractures, and hence, lower fluoride release.[15] Further, as explained by Tay et al., resin-modified GIC contains a thin hydrogel layer compared to the thicker 300-nm silica gel layer in conventional GIC. This layer on water sorption gets thicker and, henceforth, the reason for lower fluoride release by GIC for the remaining days.[16]

The alkasite showed the lowest fluoride release at all intervals of time. The results of our study correlate with the study by Gupta et al.,[17] who reported that at neutral pH, GIC released significantly higher amounts of fluoride ion when compared to Cention N (self-cure and light cure) at all-time intervals.

It can be attributed to the higher powder liquid (P/L) ratio of 4.6:1 in alkasite compared to 3.6:1 in GIC and 1.6:1 in nanoionomer, and hence, decreased solubility and lower fluoride discharge. Furthermore, of the 78.4% filler content in alkasite, only 24.6% is responsible for fluoride ion release when compared to 99.9% filler content in GIC. Besides, fillers in alkasite are resilient to dissolution due to surface modification. Furthermore, calcium fluoride and calcium phosphate form a 0.5-μm thick surface layer resistant to rinsing with saliva.[6]

According to the results of the agar-plate diffusion assay, all restorative materials evaluated showed antibacterial activity. Nanoionomer produced a greater zone of inhibition at all intervals than the other two materials. The results are similar to a study by Fúcio et al.,[15] in which nanoionomer produced a greater zone of inhibition than Type IX GIC. The larger zone of inhibition exhibited by nanoionomer can be related to the high solubility of the components, which can be associated with the polycarboxylate matrix that is incompletely formed as the polymerization and acid–base reactions try to inhibit each other when they compete simultaneously. Furthermore, a lower P/L ratio of 1.6:1, lower pH, and acid neutralization rate for extended times compared to other materials all attribute to high solubility and hence high antibacterial activity.[15]

The antibacterial activity of GIC was lower than nanoionomer. The antibacterial activity of Type IX GIC is related to the low initial pH of setting reaction, the release of metallic ions (Ca++, Al+++, and OH-), fluoride release, or other chemical components present in the powder of these materials.[18] The results of the present study are similar to the study by da Silva et al.,[18] and Naik et al.,[19] who compared different restorative GIC and reported that all materials showed antibacterial activity. However, the results of this study are contrary to Yap et al., who claimed that despite the presence of fluoride, GIC did not have an antibacterial effect when the specimens were allowed to set.[20]

The alkasite showed a lower mean zone of inhibition, thus indicating lower antibacterial activity. The lower antibacterial activity of alkasite despite the release of acid neutralizing hydroxyl and calcium ions by its patented alkaline filler that aids in neutralizing the excess acidity during acid attacks by cariogenic flora could be attributed to the direct correlation between the amount of fluoride released and the antibacterial effect. The results are similar to a study by Mohamed et al., who reported a weak antibacterial effect of alkasite compared to chlorhexidine.[21]

Hence, there is a significant difference in fluoride release and antibacterial activity of nanoionomer and the other materials tested, and hence, the null hypothesis was rejected.

A constant release of fluoride from restorative materials can have clinical implications. The GIC can act as fluoride reservoirs that absorb fluoride ions from saliva and release them back to saliva, which, in turn, can inhibit secondary caries around restorations. However, to date, whether the amounts of fluoride released from GIC are sufficient to impede dental caries is still a question. However, Cate et al.[22] reported that dentin demineralization could be inhibited clinically with fluoride levels above 1 ppm. Thus, the frequent external application of fluoride may be beneficial to maintain the high fluoride release and provide caries protection.

The limitations in the present study could be the differences in the composition, powder/liquid ratio, and storage medium. Moreover, components of saliva, acquired pellicle, pH, ion concentration, and temperature might decrease the fluoride diffusion from the restorative materials in the oral cavity.

So far, no long-term clinical studies have assessed secondary caries in teeth restored by nanoionomer or alkasite. To infer from these results, clinical studies with saliva, cariogenic bacteria, constant acid challenges, and more research on recharge ability, physical and mechanical properties of these hybrid materials need to be investigated.

CONCLUSIONS

Within the limitations, it can be concluded that among the three restorative materials tested, nanoionomer released the highest amount of daily and cumulative mean fluoride. Furthermore, it showed higher antibacterial activity followed by conventional GIC at all intervals. The alkasite showed the least amount of fluoride release and antibacterial activity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.