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ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 2  |  Page : 158-166
 

Surface remineralization potential of nano-hydroxyapatite, sodium monofluorophosphate, and amine fluoride containing dentifrices on primary and permanent enamel surfaces: An in vitro study


Department of Pedodontics and Preventive Dentistry, Punjab Government Dental College and Hospital, Amritsar, Punjab, India

Date of Web Publication2-Jul-2018

Correspondence Address:
Navneet Grewal
Department of Pedodontics and Preventive Dentistry, Punjab Government Dental College and Hospital, SSSS Chowk, Majitha Road, Amritsar - 143 001, Punjab
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_142_17

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   Abstract 


Background: Organic amine fluorides and nano-hydroxyapatite dentifrices have shown remineralization potential in various studies. However, there is a lack of direct comparison between amine fluoride and nano-hydroxyapatite with conventional inorganic fluorides as sodium monofluorophosphate. Aim: The aim of the study is to evaluate remineralizing efficacy of the three dentifrices on both primary and permanent enamel surfaces. Methods: Enamel sections were obtained from 40 sound molars – 20 primary and 20 permanent molars. Five enamel slabs were prepared from these extracted molars stored in artificial saliva, wherein one slab acted as control, second as demineralized (negative control), and other three slabs were brushed with sodium monofluorophosphate, amine fluoride, and nano-hydroxyapatite dentifrices, respectively, twice daily for 21 days. Scanning electron microscopy- Energy dispersive spectroscopy (SEM-EDS) analysis for surface morphology and calcium and phosphorus content and Vickers surface microhardness (SMH) values were evaluated at baseline, after demineralization, and postapplication of the experimental agents. Results: Highly significant changes in calcium phosphorus ratio and SMH values were seen in enamel slabs treated with nano-hydroxyapatite and amine fluoride dentifrice as compared to sodium monofluorophosphate in both primary and permanent teeth. Surface morphology of enamel slabs treated with amine fluoride most closely resembled natural enamel morphology, while sodium monofluorophosphate-treated surfaces showed globular pattern of remineralization. Deposition of a separate highly mineralized layer over existing surface was seen in nano-hydroxyapatite-treated surfaces. Conclusions: Nano-hydroxyapatite exhibited highest remineralization potential in terms of mineral gain followed by amine fluoride and sodium monofluorophosphate dentifrice.


Keywords: Amine fluoride, nano-hydroxyapatite, remineralization


How to cite this article:
Grewal N, Sharma N, Kaur N. Surface remineralization potential of nano-hydroxyapatite, sodium monofluorophosphate, and amine fluoride containing dentifrices on primary and permanent enamel surfaces: An in vitro study. J Indian Soc Pedod Prev Dent 2018;36:158-66

How to cite this URL:
Grewal N, Sharma N, Kaur N. Surface remineralization potential of nano-hydroxyapatite, sodium monofluorophosphate, and amine fluoride containing dentifrices on primary and permanent enamel surfaces: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2018 [cited 2019 Dec 8];36:158-66. Available from: http://www.jisppd.com/text.asp?2018/36/2/158/235678





   Introduction Top


“A clean tooth never decays” has been a forceful proverb in the early years of oral hygiene promotion. Today, toothbrushing and other mechanical cleaning procedures are considered to be the most reliable means of controlling plaque.[1] For effective caries control, these methods should be combined with the use of anticariogenic agents.[2]

The preventive effect of fluoride dentifrices containing inorganic fluorides such as sodium monofluorophosphate and sodium fluoride has already been established by various authors.[3],[4] Organic form of fluoride such as amine fluoride also gained popularity in caries and plaque inhibition when used through dentifrices. The unique properties of amine fluorides include rapid distribution and greater wettability on enamel surface owing to decrease in its surface-free energy [5] and highly bacteriostatic and bactericidal effects.[6]

In the last decade, nano-hydroxyapatite-containing dentifrices have been introduced claiming superior anticaries activity, protection against hypersensitivity, and maintenance of natural translucent whiteness and gloss of the tooth. Nano-hydroxyapatite is considered one of the most biocompatible and bioactive materials.[7] Its nanoparticles have similarity to apatite crystals of natural enamel in morphology and crystal structure. Various in vitro studies have revealed superior remineralizing efficacy of nano-hydroxyapatite over fluorides in permanent teeth.[8],[9],[10] However, few studies exist regarding the efficacy of these agents in primary teeth.

Therefore, the aim of the present in vitro, prospective comparative study was to compare remineralizing potential of three commercially available dentifrices vis-á-vis nano-hydroxyapatite, amine fluoride, and sodium monofluorophosphate dentifrices on primary and permanent enamel surface. The null hypothesis for the present study was that there was no significant difference in the remineralizing efficacy of the three dentifrices in both primary and permanent teeth.


   Methods Top


The research protocol for the present study was reviewed and approved by the Ethical Committee of Baba Farid University of Health Sciences, Faridkot, Punjab, India, vide letter no: BFUHS/2K15p-TH/7555 dated 6/8/15. A total of 40 sound teeth – 20 primary molars and 20 permanent molars – indicated for extraction were procured from the Departments of Pedodontics and Preventive Dentistry and Oral and Maxillofacial Surgery, Government Dental College and Hospital, Amritsar, and stored in artificial saliva. The teeth selected fulfilled the following inclusion criteria:

  • Sound enamel and dentin surface available for preparation of five enamel sections
  • Teeth free from cracks, stains, and hypomineralized areas or white spot lesions or caries
  • Teeth stored for not >1 month in artificial saliva
  • Teeth without any prior restoration
  • Teeth belonging to healthy donors.


Exclusion criteria for teeth were as follows:

  • Teeth with cracks or discoloration in the area to be utilized for enamel sections
  • Teeth with enamel hypoplasia or any other developmental anomalies
  • Teeth procured from a donor suffering from transmissible or systemic conditions affecting hard tissues.


Preparation of enamel sections and demineralization

The roots of each of the selected molar (both primary and permanent molars) were cut at the cementoenamel junction using a low-speed (15,000–20,000 rpm) diamond disc (Dentoreum) mounted on a straight handpiece (NSK, Japan). The crowns of the permanent and primary molars were then stored in artificial saliva at 4°C in the tooth bank following the proper protocol of sterilization and storage.[11] Enamel sections were made within a week of their storage. Artificial saliva was prepared as per Se rra and Cury, 1992 recommendation,[12] and it comprised 0.4% carboxymethylcellulose, 6% sorbitol, 0.062% KCl, 0.085% NaCl, 0.005% MgCl2, 0.016% CaCl2, 0.08% K2 HPO4, 0.2% nipagin, and distilled deionized water, maintained at pH 7.23 which was measured using digital pH meter. The crown of each molar was sectioned mesiodistally into buccal and lingual halves. These halves were further sectioned to obtain five tooth slabs using a low-speed (15,000–20,000 rpm) diamond disc. Each permanent tooth slab was carefully shaped into a standardized rectangular form (3 mm × 3 mm × 2 mm), whereas the primary tooth slab was prepared to measure 2 mm × 2 mm × 2 mm. The dimensions of the slabs were checked by double-blind calibrated examiners with the help of a digital Vernier Caliper (INSIZE 1112-150).

The five enamel slabs obtained from each tooth were further divided as follows [Table 1]:
Table 1: Showing schematic illustration of sample distribution

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  • Subgroup 1 (Control group): Neither subjected to demineralization nor remineralization procedure
  • Subgroup 2 (Negative control): Demineralized enamel slab
  • Subgroup 3: Demineralized enamel slab to be brushed with sodium monofluorophosphate-containing dentifrice
  • Subgroup 4: Demineralized enamel slab to be brushed with amine fluoride-containing dentifrice
  • Subgroup 5: Demineralized enamel slab to be brushed with nano-hydroxyapatite-containing dentifrice.


Of five enamel slabs, the control group tooth slab was kept in a sterile specimen container in a humidified environment containing artificial saliva. The remaining four slabs were embedded in self-cure resin such that only the surface enamel was exposed. These were submerged in 0.1 mol/l lactic acid solution at pH 5.0 for 4.5 h to create surface softened lesions as described by White et al.[13]

Treatment of the enamel sections with dentifrices

The remaining three enamel sections of each permanent and primary tooth were randomly divided into three subgroups and brushed twice daily with individual dentifrices for 21 days. A standardized brushing procedure using electronic toothbrush (Philips Sonicare with standard ProResults brush head) was carried out for 2-min duration used in side-to-side (sonic) mode. After 2 min, the enamel slab was transferred to a calibrated glass beaker containing 10 ml of tap water and gently agitated for 1 min to simulate the in vivo postbrushing rinsing protocol.[14] It was then rinsed under tap water and transferred into a sterile specimen container containing artificial saliva until next brushing period. The fluoride content of tap water as measured by fluoride ion analyzer was 0.13 ppm. The artificial saliva solutions were changed every 2 days. The above-mentioned brushing regimen was repeated twice daily for 21 days for all three subgroups each using sodium monofluorophosphate (Colgate dental cream), amine fluoride (Elgydium™ sensitive), and nano-hydroxyapatite dentifrice (Apagard M-plus).

Scanning electron microscope–energy dispersive X-ray spectroscopy and Vickers surface microhardness analysis

After 21 days, both primary and permanent enamel slabs from individual groups were examined under scanning electron microscope (SEM) (JEOL JSM 6510 LV) at ×1000 and ×2000 to assess morphological changes. Energy-dispersive X-ray spectroscopy (EDS) analysis was used to measure mineral content vis-á-vis calcium and phosphorus in various groups of primary and permanent teeth. The findings of calcium and phosphorus content in sound, demineralized, and remineralized tooth slabs after the use of respective agents were tabulated for further statistical evaluation. Surface microhardness (SMH) values were measured using Vickers SMH tester (Laizhou Huayan Model HV-1000). Vickers indenter with a linear loading segment of 500 gf was applied for 10 s on the enamel surface. Three indentations were made on the surface, and mean of the three readings was taken as the final value. The measurement of SMH in sound, demineralized, and remineralized tooth slabs after the use of respective agents was tabulated and put to statistical analysis.

The pH of artificial saliva used for storing enamel slabs was measured before the start of experiment using digital pH meter (ELICO, INDIA L1 127). The solutions were changed after every 2 days, and analysis of pH was done so as to maintain a constant pH value for the next experimental period. pH and fluoride content of artificial saliva used for storing enamel sections were measured in the beginning, during and after the experimental period using digital pH meter and fluoride ion analyzer (Thermo Orion 9409BN).

Statistical analysis

Evaluation of normality distribution of the data in various subgroups of both primary and permanent teeth revealed that the data were normally distributed. Comparison of calcium-phosphorus ratio, microhardness values, pH, and fluoride content of artificial saliva solutions before and after treatment with the three test dentifrices was done by paired t-test using SPSS software version 17.5 (IBM, Armonk, NY, United states of America). Level of significance was set at ά = 0.05 (two-sided).


   Results Top


Energy dispersive spectroscopy analysis

In primary teeth, there was a highly significant increase in the mean calcium-phosphorus ratio in all the three subgroups vis-a-vis sodium monofluorophosphate (Subgroup 3), amine fluoride (Subgroup 4), and nano-hydroxyapatite (Subgroup 5) as compared to baseline (P < 0.001). Highly significant difference was observed between Subgroup 3 and Subgroup 5 (P < 0.001) and significant difference between Subgroup 4 and Subgroup 5 (P < 0.05) [Table 2].
Table 2: Intergroup comparison of mean calcium-phosphorus ratio in various enamel slabs of primary teeth

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In permanent teeth, highly significant difference between Subgroup 5 compared to Subgroup 3 and Subgroup 4 was observed (P < 0.001) [Table 3]. The mean Ca:Pin Subgroup 3 was comparable to baseline in permanent teeth (P > 0.05), whereas it was higher for Subgroup 3 compared to baseline in primary teeth (P < 0.001). This difference in comparison between primary and permanent teeth could be explained on the basis of lower Ca:Pin primary teeth which would allow more imbibition of Ca and P ions on enamel as opposed to permanent teeth with high Ca:P.[15]
Table 3: Intergroup comparison of mean calcium and phosphorus ratio in various enamel slabs of permanent teeth

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Scanning electron microscopy evaluation

SEM images evaluated at ×1000 and ×2000 magnification revealed that the surface topography of sound enamel had smooth and intact surface with honeycomb appearance [Figure 1] and [Figure 2]. Loss of mineral content giving a porous appearance and depressions in defined honeycomb pattern was seen in demineralized enamel surfaces [Figure 3] and [Figure 4]. All the three dentifrices had remineralized the demineralized surfaces. However, enamel slabs treated with sodium monofluorophosphate showed a homogeneous pattern of remineralization with formation of globular structures [Figure 5] and [Figure 6]. Treatment with amine fluoride restored the normal smooth morphology of enamel comparable to sound enamel slab [Figure 7] and [Figure 8]. The surface appeared to be homogeneous with honeycomb pattern of natural enamel. In nano-hydroxyapatite group, deposition of thick homogeneous apatite layer could be observed on enamel surface which did not mimic the natural enamel morphology. The porous demineralized surface appeared to be completely covered by this layer [Figure 9] and [Figure 10].
Figure 1: Baseline (×1000)

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Figure 2: Baseline (×2000)

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Figure 3: Demineralized (×1000)

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}
Figure 4: Demineralized (×2000)

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Figure 5: Sodium monofluorophosphate (×1000)

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Figure 6: Sodium monofluorophosphate (×2000)

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Figure 7: Amine fluoride (×1000)

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Figure 8: Amine fluoride (×2000)

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Figure 9: Nano-hydroxyapatite (×1000)

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Figure 10: Nano-hydroxyapatite (×2000)

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Surface microhardness analysis

The results of SMH values also closely followed the trends of calcium-phosphorus ratio. In the primary teeth, the decreasing order of mean SMH was as follows: nano-hydroxyapatite > amine fluoride > sodium monofluorophosphate > baseline > demineralized [Table 4]. In the permanent teeth, this order was as follows: nano-hydroxyapatite > amine fluoride > sodium monofluorophosphate = baseline > demineralized [Table 5].
Table 4: Intergroup comparison of mean surface microhardness values in various enamel slabs of primary teeth

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Table 5: Intergroup comparison of mean surface microhardness values in various enamel slabs of permanent teeth

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Fluoride and pH analysis

The enamel slabs belonging to various groups were kept in artificial saliva maintained at neutral pH (7.23) at baseline with 0.02-ppm fluoride content throughout the study period so as not to influence the final results of the study. After treatment with respective dentifrices, in primary teeth, the mean pH of artificial saliva solution was 6.62 ± 0.41 in sodium monofluorophosphate and 7.06 ± 0.53 in nano-hydroxyapatite group. The pH in amine fluoride group decreased to 4.96 ± 0.52 (acidic range). The pH values in permanent teeth also followed the same order as described for primary teeth with mean pH of artificial saliva in sodium monofluorophosphate and nano-hydroxyapatite ranging 6.88 ± 0.41 and 7.26 ± 0.46, respectively, whereas it decreased to 4.95 ± 0.42 in amine fluoride group.

At the end of experimental period, highest mean fluoride content was seen in amine fluoride group (12.63 ± 1.50) followed by sodium monofluorophosphate (10.29 ± 0.92) and nano-hydroxyapatite (3.95 ± 0.93) in primary teeth. In the permanent teeth, the decreasing order of mean fluoride content was also similar, i.e., amine fluoride (13.10 ± 1.11) > sodium monofluorophosphate (11.14 ± 0.74) > nano-hydroxyapatite (3.98 ± 0.53).


   Discussion Top


Remineralization was evident with all the three dentifrices, but the highest mineral gain was observed with nano-hydroxyapatite followed by amine fluoride and sodium monofluorophosphate as indicated by calcium-phosphorus ratio and SMH values. Thus, the null hypothesis (stating that there was no difference in remineralizing efficacy among the three dentifrices) was rejected.

The structure of an in vitro study closely resembles that of a clinical trial. The merits of in vitro studies compared to that of a clinical trial include control over independent variables, unforeseen bias, and ease of operation, thus improving internal validity of the results.[16] Therefore, an in vitro model was selected for the present study to minimize several individual factors such as diet, composition of saliva, salivary flow and buffering capacity, plaque removal strategies, and brushing frequency, which could have a potential impact on remineralization.

Both primary and permanent teeth were chosen to assess the three agents as the microstructure and mineral composition of primary and permanent teeth are different. Calcium and phosphorus contents are higher in permanent as compared to primary teeth besides other histological differences.[15] In the present study, the baseline Ca:Pin primary enamel slabs (1.41 ± 0.04) was lower than that of permanent enamel slabs (1.64 ± 0.02). Furthermore, there are few studies on comparative evaluation of remineralizing agents on primary teeth.

Geddes [17] stated that lactic acid is the acid produced in maximum proportions in human oral cavity besides other organic acids such as acetic acid, propionic acid, and isobutyric acid. Hence, lactic acid was the demineralizing agent of choice as it demonstrated an in vivo-like environment under in vitro conditions.

The present study employed one of the most widely used techniques, i.e., SEM with EDS to quantify the amount of mineral gain in a given tooth sample along with the qualitative analysis of its surface topography. Vickers SMH test was employed in the present study to evaluate SMH of enamel slabs before and after application of respective dentifrices. It has been proposed that in tooth hardness studies, the Vickers indenter is more useful than the Knoop indenter because a square shape produced by the indentation on a nonflat surface is easily detected.[18]

pH of saliva is a major factor in dynamics of demineralization and remineralization equilibrium. After treatment with the respective dentifrices, the mean pH in sodium monofluorophosphate and nano-hydroxyapatite was observed in neutral pH range and that of amine fluoride decreased to acidic range. Lower pH value accelerates dissolution of enamel apatite and provides more free Ca ions, consequently favoring precipitation of calcium fluoride-like material in a fluoride-containing solution.[19] Below the critical pH, hydroxyapatite is dissolved, but the released mineral ions could be reprecipitated as fluorapatite or a mixed fluorhydroxyapatite. This mechanism prevents loss of mineral ions, while providing additional protection to mineral crystallites by laying fluoride-rich outer layers onto the apatite crystallites.[20] This difference in pH would partially explain higher remineralizing efficacy of amine fluoride over sodium monofluorophosphate. Furthermore, the mean fluoride concentrations in artificial saliva solutions of amine fluoride group were higher as compared to sodium monofluorophosphate. This could be explained on the basis of dissociation of fluoride compounds and free fluoride concentrations in the solution, which is more for amine fluoride than sodium monofluorophosphate in the aqueous solution.[21]

Treatment of enamel sections with nano-hydroxyapatite produced the highest mineral gain. It is known from previous research that nano-hydroxyapatite particles have similarity to apatite crystals of natural enamel in morphology, physical properties, crystal structure, and similar Ca:P as natural enamel, thus explaining the higher Ca:P and SMH in nano-hydroxyapatite group.[8]

The surface morphology observed through SEM revealed that the surface topography after the use of amine fluoride most closely resembled the natural enamel [Figure 7] and [Figure 8]. This would provide a favorable clinical scenario in everyday situation for fighting against dental decay in healthy individuals. Deposition of a new layer over existing enamel in nano-hydroxyapatite suggests the bulk precipitation of hydroxyapatite crystals [Figure 9] and [Figure 10]. The addition of reinforced mineral-rich layer over the existing surface would provide real enamel repair in conditions of severe enamel loss or damage as seen in erosion (due to various intrinsic or extrinsic factors), abrasions, white spot lesions, postfixed orthodontic treatment, and dentinal hypersensitivity. The role of nano-hydroxyapatite can be preserved as a therapeutic dentifrice for these conditions. Li et al.[22] demonstrated resistance of nano-hydroxyapatite coated enamel layer to acid dissolution and its negligible average mass loss rate, thus proving its supremacy in providing enamel remineralization. The results of in vitro studies have to be further evaluated through randomized controlled clinical trials to assess the efficacy of nano-hydroxyapatite in clinical conditions and compare it with the conventional inorganic fluoride and organic amine fluoride dentifrices.


   Conclusions Top


It is time to shift our focus from the traditional dentifrice formulations and adopt more biocompatible and bioactive dentifrices with superior remineralizing efficacy over fluoride-containing dentifrices. Considering the cost-effectiveness of both nano-hydroxyapatite and amine fluoride dentifrice, amine fluoride would provide a cost-effective alternative. However, on the basis of results of the current study, nano-hydroxyapatite dentifrices have shown superior remineralizing efficacy over amine fluoride and sodium monofluorophosphate dentifrices.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3, [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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