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| Year : 2019 | Volume
: 30
| Issue : 4 | Page : 590-594 |
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| In Vitro evaluation of remineralization potential of novamin on artificially induced carious lesions in primary teeth using scanning electron microscope and vickers hardness |
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Anshul Gangwar, Kaushal Kishor Jha, Jyoti Thakur, Madhuri Nath
Department of Pedodontics and Preventive Dentistry, Institute of Dental Sciences, Bareilly, Uttar Pradesh, India
Click here for correspondence address and email
| Date of Submission | 06-May-2016 |
| Date of Decision | 03-Nov-2018 |
| Date of Acceptance | 10-Jul-2019 |
| Date of Web Publication | 18-Nov-2019 |
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 Abstract | | |
Aims: The objective of this in vitro study was to find out the efficacy of Novamin in remineralizing enamel surface on which artificial caries lesion had been created. The changes were analyzed using Vickers Hardness Testing Machine and Scanning electron microscope (SEM). Settings and Design: A total of 40 primary teeth were selected and divided into two groups: Control group and SHY NM (Novamin). All the samples were subjected to ph cycle protocol and assessed using Vickers Hardness Testing Machine and Scanning electron microscope. Each demineralized sample was randomly divided into two groups: Gp I –Control group, Gp II – Novamin (SHY NM). After 10-day period of ph cycle, the obtained data was analyzed statistically. Statistical Analysis Used: Pre and post groups were compared by paired t test. The significance of mean difference between the groups was done by Tukey's post hoc test after ascertaining normality by Shapiro-Wilk (W) test and homogeneity of variance by Levene's test. A two-tailed P value less than 0.05 (P < 0.05) was considered statistically significant. All analyses were performed on SPSS software (Windows version 17.0). Results: Statistical analysis showed that bioactive glass (novamin) remineralizes artificially induced carious lesion in primary teeth. Conclusions: SHY NM (Novamin) exhibited superior remineralization potential.
Keywords: Bioactive glass, enamel, remineralization, scanning electron microscope, SHY NM (Novamin), vickers hardness testing machine
How to cite this article:
Gangwar A, Jha KK, Thakur J, Nath M. In Vitro evaluation of remineralization potential of novamin on artificially induced carious lesions in primary teeth using scanning electron microscope and vickers hardness. Indian J Dent Res 2019;30:590-4 |
How to cite this URL:
Gangwar A, Jha KK, Thakur J, Nath M. In Vitro evaluation of remineralization potential of novamin on artificially induced carious lesions in primary teeth using scanning electron microscope and vickers hardness. Indian J Dent Res [serial online] 2019 [cited 2023 Mar 30];30:590-4. Available from: https://www.ijdr.in/text.asp?2019/30/4/590/271056 |
| Introduction | |  |
Worldwide contribution of dental caries to the burden of oral diseases is about 10 times higher than that of periodontal disease and other common oral conditions.[1] Owing to its globally high prevalence, dental caries is a 'pandemic' disease characterized by a high percentage of untreated carious cavities causing pain, discomfort, and functional limitations.[2] Untreated carious cavities, furthermore, has significant impact on the general health of children and on social and economic wellbeing of communities.[3]
A surgical approach for the elimination of carious lesion was developed only a century ago; this approach was necessary during that time, because there was no valid alternative. The focus on caries has recently shifted to the development of methodologies for the detection of the early stages of caries lesions and the use of non-invasive treatment for these lesions.
The non-invasive treatment of early carious lesions by remineralization has the potential to be a major advance in the clinical management of the disease. Remineralization of white-spot lesions and early carious lesions may be possible with a variety of currently available agents containing novamin, fluoride, bioavailable calcium and phosphate, and casein phosphopeptide in-amorphous calcium phosphate, self-assembling peptide.[4] The current concept further bridges the traditional gap between prevention, non-invasive, and surgical procedures which is just what dentistry needs for the current age.
Bioactive glass (Bioglass ®) was invented by Dr. Larry Hench in 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.[4] Novamin ®, a trade name for bioactive glass, is manufactured by Novamin Technologies Inc. (Alachua, FL, USA). It has been demonstrated that, fine particulate bioactive glasses (<90 μm) incorporated into an aqueous dentifrice has the ability to clinically reduce the tooth hypersensitivity through the occlusion of dentinal tubules by the formation of the CAP layer.[5]
To assess the remineralization potential of bioactive glass, an in vitro clinical trial was planned to evaluate the remineralizing ability of SHY NM (Novamin) on artificial enamel carious lesion by using Vickers hardness number and scanning electron microscopy (SEM).
| Materials and Method | | |
Forty freshly extracted sound human primary anterior teeth extracted due to physiological mobility or retained in {THE} the permanent dentition were used in this study after clinical and radiographic examinations. Teeth with any visible/discoloration/detectable caries, teeth with hypoplastic/white spot lesion, enamel cracks/fracture, developmental defects, and teeth with any restoration/pulp therapy were not used in the study. The teeth were collected from healthy individuals after parent's consent. The samples were washed and ultrasonicated to remove any debris and stored in normal saline until their use.
The root of the teeth was separated from the crown portion at the cemento-enamel junction (CEJ) using a diamond-coated disc. The labial surface of all the samples were progressively ground flat and hand polished with the aqueous slurry of progressively finer grades of silicon carbide, up to 4000 grit in order to obtain flat surface and about 150 micrometer thickness of the enamel tissue was removed from the original tooth surface. In order to prevent dehydration the specimens were stored in normal saline. Each sample was embedded in the self-cured acrylic resin. In order to create the artificial carious lesions, an acid resistant nail varnish was applied around the exposed tooth surfaces, leaving a 4 × 4 mm window of the enamel exposed in the centre.[6]
Then, the baseline enamel SMH was measured.
Demineralizing solution was prepared using 2.2 mM CaCl2.2H2O (calcium chloride), 2.2 mM NaH2 PO4.7H2O (monosodium phosphate) and 0.05 M Lactic Acid. Each ingredient was added separately to deionized water under continuous stirring and was allowed to dissolve completely before the next ingredient was added. The solution was maintained at 37°C and the pH was adjusted to 4.5 using 50% NaOH solution.[7]
Artificial saliva was prepared by mixing 2.200 g/L Gastric Mucin, 0.381 g/L NaCl (sodium chloride), 0.213 g/L CaCl2.2H2O (calcium chloride), 0.738 g/L K2 HPO4.3H2O (potassium hydrogen phosphate), and 1.114 g/L KCl (potassium chloride). Each ingredient was added separately to Deionized water under continuous stirring and was allowed to dissolve completely before the next ingredient was added. The solution was maintained at 37°C and the pH was adjusted to 7.00 using 85% lactic acid.[7]
Remineralization/demineralization study protocol
The samples were randomly divided into two groups (20 each): Group I -Control (brushed with DI water) and Group II -SHY NM (Novamin)
The specimens of the enamel blocks were immersed in the demineralized solution. The solution was stirred and the demineralization was performed at 37°C for 48 hours and the enamel SMH was measured after demineralization.
The specimens underwent were subjected to the remineralization process twice a day (09:00 am, 4:00 pm) for 10 days.[8]
- 09:00 am: All the teeth were removed from artificial saliva, brushed using a soft-bristled powered toothbrush with respective remineralizing agents for 2 minutes, and gently rinsed with deionized (DI) water.
- 09:30 am–16:00 pm: All teeth soaked in artificial saliva at 37°C.
- 4:00 pm: All teeth were removed from artificial saliva, brushed using a soft-bristled powered toothbrush with respective remineralizing agents for 2 minutes, and gently rinsed with deionized (DI) water.
- 04:30 pm–09:00 am: All teeth were again soaked in artificial saliva at 37°C.
Group I (control) received no intervention throughout the cycling regime. The demineralization solution and artificial saliva were changed daily. After the 10-day period of pH cycling regime the groups were evaluated on the basis of surface microhardness and SEM appearance of enamel surface.
SEM observation
At the end of the 10-day of pH cycle, the remaining 20 teeth (n = 10) were mounted for scanning electron microscopy (SEM) analysis (80000 × magnification).
All the teeth from each group were mounted on carbon mounts and coated with a gold/palladium alloy coating by a process called sputtering.
Hardness testing
Vicker's hardness test to check the microhardness of enamel surface was done. The testing was done with a FIE microhardness tester India. Twenty samples out of forty (n = 10) were placed on the tester after leveling the dental stone block so that a plane is achieved.
The diamond tip was used to create a nanoindent. Under a 100x microscope, the sample positioning was done so that the indent falls on the enamel portion of the section. A load of 100 gf for 15 s was applied, and the rhomboid indent is measured for length, and depth and the hardness values were calculated.
| Statistical Analysis | | |
Data was summarized as Mean ± SD (standard deviation). Pre and post groups were compared by paired t test. The significance of mean difference between the groups was done by Tukey's post hoc test after ascertaining normality by Shapiro-Wilk (W) test and homogeneity of variance by Levene's test. A two-tailed P value less than 0.05 (P < 0.05) was considered statistically significant. All analyses were performed on SPSS software (Windows version 17.0).
| Results | | |
Micro-morphological surface observations of the enamel surfaces under SEM
- Under 80000 × magnification in the sound enamel the crystals were homogeneously arranged with a clear outline and rods were orderly placed in the sound enamel and crystals were homogeneously arranged with a clear outline [Figure 1].
- The demineralized enamel showed a rough surface with a honeycomb appearance, which is a peculiar characteristics of carious enamel [Figure 2].
- Shallow depressions and fine porosities within these depressions were observed in group I [Figure 3].
- Samples treated with Novamin (Group II) showed a nearly smooth surface, with complete obtrusion of inter-rod spaces in some fields. The rods appeared as they were fused together with some globules deposited on the surface, relatively no evidence of porosities or irregularities [Figure 4].
Surface microhardness evaluation
The surface hardness of baseline enamel (i.e. before demineralization) and after demineralization are summarized in [Table 1] and also shown graphically in [Graph 1]. The hardness of all teeth samples before demineralization ranged from 315-349 kg/mm 2 with mean (±SD) 331.43 ± 9.09 kg/mm 2 while after demineralization it ranged from 234 to 261 kg/mm 2 with mean (±SD) 244.65 ± 6.93 kg/mm 2. | Table 1: Vicker's Hardness (Mean±SD) of extracted normal teeth before and after demineralization
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The initial hardness decreased comparatively after demineralization. Comparing the mean hardness before and after demineralization, paired t test showed significant decrease (26.2%) in hardness after demineralization as compared to before demineralization (331.43 ± 9.09 vs. 244.65 ± 6.93, t = 52.35, P < 0.001).
The enamel samples were further randomized equally to treat with one of two treatment protocols [Group I: Control (brushed with DI water), Group II: SHY NM (Novamin). The hardness of groups after treatment is summarized in [Table 2] and also depicted in [Graph 2]. After treatment, the hardness of Group I and Group II, ranged from 283 to 299 kg/mm 2, 307 to 319 kg/mm 2, respectively with mean (±SD) 291.00 ± 5.50 kg/mm 2, 312.20 ± 3.46 kg/mm 2. After treatment, the mean hardness Group II was the highest (i.e. Group I < Group II).
The comparison (P value) of mean difference in hardness of teeth between the groups by Tukey post hoc test is summarized in [Table 3]. | Table 3: Comparison (P value) of mean difference in hardness of teeth between the groups by Tukey post hoc test
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| Discussion | | |
Tooth caries is known as the most prevalent chronic disease with its etiology being quite complex involving interaction between the agent, host, time, and environmental factors. Prevention of dental caries is very essential as it affects self-esteem, quality of life and also indirectly contributes to the decrease in nation's productivity.[9]
The primary anterior teeth were chosen in this study for induction of artificial caries like lesion because the organic content of the primary tooth enamel is higher than that of the permanent tooth making the primary tooth enamel softer and more porous and consequently more susceptible to caries compared to the permanent enamel.
Vicker's hardness method was used to check microhardness because it was non-destructive, very reliable, rapid and economical as compared to other hardness tests. The square shaped indent obtained was more easy and accurate to measure and detect visually and digitally.
The present in vitro study was planned to assess the efficacy of Novamin as a remineralizing agent using Vickers Hardness Testing Machine and Scanning electron microscope (SEM). Assessment of in vitro demineralization and remineralization can be done using different methods. Many studies have been conducted using one or a combination of different methods like the SEM/ESEM,[10],[11],[12] Diagnodent,[12] surface microhardness,[13],[14] etc., The present study utilized both SEM and surface microhardness to assess remineralization. Under the limitations of the present study it was observed that novamin exhibits significant remineralization.
The surface topographic changes, analyzed by SEM, showed that the enamel surface treated by Novamin has a much smoother and uniform surface.
In our study the mean values for enamel micro hardness at baseline were in the range from 315 VHN to 349 VHN which is within the standard range of 250 VHN to 360 VHN and there was decrease in hardness after demineralization (P < 0.001). In the present study, after remineralization by Group I (control), Group II (SHY NM Novamin) the micro hardness of teeth increased significantly. The enamel micro hardness increased by 47 VHN, 68 VHN. The results of the present study were in agreement with the work of MohantyP et al.[8] who also inferred that novamin enhanced remineralization.
This study used SHY NM which is derived from bioactive glass. Bioglass in an aqueous environment immediately begins surface reaction in three phases-leaching and exchange of cations, network dissolution of SiO2 and precipitation of calcium and phosphate forming an apatite layer. The critical stages for glass surface reactions are the initial Na + and H +/H3O + ion exchange and de-alkalinization of the glass surface layer which is quite rapid, within minutes of implantation and exposure to body fluids.[15] The net negative charge on the surface and loss of sodium causes localized breakdown of the silica network with the resultant formation of (silanol) Si (OH) groups, which then repolymerizes into a silica-rich surface layer.[16] Within 3–6 h in vitro, the calcium phosphate layer will crystallize into the carbonated hydroxyl apatite (CAP) layer, which essentially is the bonding layer. Chemically and structurally, this apatite is nearly identical to bone and tooth mineral.
Novamin is a multi-component, highly biocompatible, inorganic compound made of elements (silicon, calcium, sodium, and phosphorous). The advantage of novamin is not only acceptance within the body, but also its ability to chemically bond structure. It is considered as a breakthrough in remineralization because current systems are dependent on adequate saliva as a source of calcium and phosphate, and bioactive glasses are enriched with these ions. To the best of our knowledge, the harmful effect of Novamin is still unknown.
| Conclusion | | |
This invitro study inferred that SHY NM (Novamin) could effectively remineralize artificial enamel. Thus, the use of bioactive glass (Novamin Technology) in remineralization of enamel is quite promising, especially in patients with systemic problems, but further research needs to be undertaken to prove its efficacy.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Dr. Anshul Gangwar
Institute of Dental Sciences, 113 North City, Pilibhit Road, Bareilly, Uttar Pradesh - 243 122
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ijdr.IJDR_326_16
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3] |
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