ORIGINAL RESEARCH |
https://doi.org/10.5005/jp-journals-10015-2245 |
Comparative Evaluation of Remineralizing Efficacy of Nanohydroxyapatite Dentifrices on Artificial Carious Lesion in Primary Teeth: An In Vitro Study
1,3-6Department of Pediatric and Preventive Dentistry, K M Shah Dental College and Hospital, Sumandeep Vidyapeeth, An Institution Deemed to be University, Vadodara, Gujarat, India
2Department of Public Health Dentistry, K M Shah Dental College and Hospital, Sumandeep Vidyapeeth, An Institution Deemed to be University, Vadodara, Gujarat, India
Corresponding Author: Pratik B Kariya, Department of Pediatric and Preventive Dentistry, K M Shah Dental College and Hospital, Sumandeep Vidyapeeth, An Institution Deemed to be University, Vadodara, Gujarat, India, Phone:+91 9558330199, e-mail: prateek.kariya@gmail.com
Received on: 03 May 2023; Accepted on: 01 June 2023; Published on: 22 August 2023
ABSTRACT
Aim: The study was conducted with the aim to compare the efficacy of sodium monofluorophosphate and nanohydroxyapatite-containing toothpaste in remineralizing artificial caries on extracted primary teeth using the Vickers surface microhardness test.
Materials and methods: This experimental in vitro study was conducted on 30 freshly extracted primary teeth from individuals requiring extractions as part of their dental treatment. The lottery method was used to randomly divide the samples into two groups remineralization using nanohydroxyapatite (nano-HA) group (coded group I) and remineralization using the sodium monofluorophosphate group (group II). All the samples were subjected to a demineralization regimen using a demineralizing agent for 48 hours. Then all the samples were subjected to remineralizing regime using sodium fluoride (NaF) toothpaste (226 ppm) and nano-HA toothpaste for 28 days. Surface microhardness for remineralized samples was recorded using the Vickers microhardness test by utilizing a 25 gm load for a duration of 5 seconds. The results were statistically analyzed using the analysis of variance test and post hoc test at a 5% level of significance.
Results: The baseline microhardness expressed as Vickers hardness number (VHN) for the nano-HA group (group I) was 341.73 ± 5.07; 256.54 ± 5.10 VHN after demineralization and 321.27 ± 6.48 VHN after remineralization, respectively. For the sodium monofluorophosphate group (group II), the microhardness value was found to be 365.56 ± 5.69 VHN at baseline, 275.65 ± 5.22 VHN after demineralization, and 313.10 ± 8.02 VHN postremineralization.
Conclusion: Nanohydroxyapatite (nano-HA) causes remineralization and increases the microhardness of the tooth at the microstructural level. Therefore, it is a novel method to remineralize initial carious lesions.
Clinical significance: Remineralization therapy is a contemporary approach for carious lesions in early detection and a noninvasive or minimally invasive treatment approach, which is crucial for the dental profession in shifting their therapeutic approach to a new paradigm. The present study showed that nano-HA toothpaste has the potential to remineralize the artificial carious lesion.
How to cite this article: Kariya PB, Singh S, Bargale S, et al. Comparative Evaluation of Remineralizing Efficacy of Nanohydroxyapatite Dentifrices on Artificial Carious Lesion in Primary Teeth: An In Vitro Study. World J Dent 2023;14(6):498-502.
Source of support: Nil
Conflict of interest: None
Keywords: Artificial carious lesion, Dentifrice, Nanohydroxyapatite, Primary teeth.
INTRODUCTION
Enamel is the most rigid structure of the tooth, which is functionally organized to provide an integrated mechanical defense system to the human tooth. Ectodermal-derived enamel provides a masticatory surface, which offers resistance to wear and acid. It faces assaults from various sources like diet, dental materials, and medical problems like reflux, which lead to loss of minerals from the enamel. The most prevalent outcome of these assaults is white spot lesions.1,2 It signifies the initiation of caries and, if not treated, may develop into dental caries. Unlike pulp, enamel cannot regenerate itself. Therefore, an agent which remineralizes enamel is the solution to prevent the loss of structure from enamel.3 Moreover, the treatment trends in dentistry have changed from invasive to minimally invasive techniques and procedures. Remineralizing agents are significant contributors to minimally invasive techniques.4
Sodium fluoride (NaF) is considered the gold standard in the field of preventive dentistry. It is one of the most efficacious and commonly used remineralizing agent.5 Fluoride helps in the formation of fluorapatite crystal, which has a larg—e lattice structure and is more acid resistant due to the replacement of calcium ions by fluoride ions in the lattice. Fluoride also participates in the demineralization-remineralization cycle by transforming the pH to alkaline, shifting the curve to remineralization.6
Recently remineralizing agents like casein phosphopeptide (CPP), casein phosphopeptide—amorphous calcium phosphate (CPP-ACP), bioactive glass, nanohydroxyapatite, NaF, etc., have gained interest in the field of dentistry. These agents alter the pH surrounding the tooth to alkaline, promoting remineralization and reversal of early lesions (white spot lesions).7 Nanohydroxyapatite (nano-HA) was first invented in Japan under the brand name Apagard and Apadent.8 It is a bioactive, biocompatible, and nonfluoride remineralizing agent composed of calcium phosphate, which acts as a reservoir for calcium and phosphate and also occludes microporosities created due to demineralization of enamel. When the pH of saliva around the tooth becomes acidic, the ions from the material leach out, transforming acidic pH to alkaline pH. It also supersaturates saliva with calcium and phosphate ions which results in remineralization.8,9 Therefore, agents containing nano-HA particles may better assist in the remineralization of early white spot lesions.
Dental caries is one of the most prevalent diseases in the 21st century.6 Fluoride has conventionally been used as a gold standard in preventing dental caries, but it is not a biocompatible and bioactive element. Considering the value and effectiveness of preventive measures against invasive approaches for the treatment of initial carious lesions and key problems associated with the use of fluoride, attempts have to be made to use nonfluoride, less studied, more advanced, and newer material containing nano-HA particles as an alternative for fluoride. Moreover, an experimental in vitro study will provide better judgment regarding the remineralizing efficacy of nano-HA dentifrices on initial carious lesions. Therefore, the study was conducted with the aim to compare the efficacy of NaF and nanohydroxyapatite-containing toothpaste in remineralizing artificial caries on extracted primary teeth using the Vickers surface microhardness test.
MATERIALS AND METHODS
This experimental in vitro study was conducted at the Department of Pediatric and Preventive Dentistry at a tertiary care dental hospital. The research proposal was reviewed and approved by University Ethics Committee (SVIEC/ON/DENT/RP/15038). Keeping the power of cα = 0.05 and 0.20, with 80% being the strength of the test, 30 sound primary anterior humans extracted or exfoliated teeth with a minimum of two-thirds root length were included as a sample of the study. Carious teeth, restored teeth, teeth with hypoplastic lesions, stains, or white spot lesions were excluded from the study sample. A total of 30 freshly extracted primary teeth from individuals requiring extractions as part of their dental treatment were collected. Collected teeth samples were cleaned ultrasonically to eliminate all the hard and soft deposits and then were stored in 10% buffered formalin till further use. A transillumination test was done to check for possible cracks and extraction damage. All the samples were stored in 0.5% chloramine-T till further use (Flowchart 1).
Flowchart 1: Schematic representation of the methodology
Specimen Preparation
Study samples were rinsed with 0.9% sodium chloride (NaCl) solution for 2 minutes before sample preparation. All the samples were mounted on cold cure resin-filled rectangular blocks, with the crown enamel surface above the resin. The enamel surface was then polished using pumice paste. These prepared samples were then stored in 0. 9% NaCl solution.
Baseline surface microhardness was recorded using the Vickers microhardness test by utilizing a 25 gm load for the duration of 5 seconds.
Demineralizing Regimen/Lesion Formation
All the samples were subjected to a demineralization regimen using a demineralizing agent consisting of 2.2 mm calcium chloride, 2.2 mm sodium phosphate, and 0.05 mm acetic acid. The pH of this agent was adjusted to 4.4 using 1 M potassium hydroxide. Each sample was immersed in 15 mL of demineralizing agent for 48 hours to generate artificial carious lesions.10
Subsequently, surface microhardness for demineralized samples was recorded using the Vickers microhardness test by utilizing a 25 gm load for a duration of 5 seconds.
Sample Randomization
The lottery method was used to randomly divide the samples into two groups. The sample was equally divided into the remineralization using the nano-HA group (coded group I or the test group) and remineralization using the sodium monofluorophosphate group (group II or the control group). A total of 15 samples in each group were included.
Dentifrice Slurry Preparation
A total of 12 gm of respective dentifrice was added to 36 mL deionized water to formulate dentifrice slurry in a dilution of 1:3. The slurry formulated was stirred using a stirring rod.
Remineralization Regimen
All the samples were then subjected to a remineralizing regime with nano-HA toothpaste and sodium monofluorophosphate toothpaste for 28 days. The prepared samples were painted with dentifrice slurry for a duration of 2 minutes twice a day. Then subsequently, they are immersed in artificial saliva for 28 days. Artificial saliva is composed of potassium chloride (KCl)—0.4 gm/L, NaCl—0.4 gm/L, CaCl2·2H2O—0.906 gm/L, NaH2PO4·2H2O—0.690 gm/L, Na2S·9H2O—0.005 gm/L, urea– 1 gm/L and pH 6.5.11
Subsequently, surface microhardness for remineralized samples was recorded using the Vickers microhardness test by utilizing a 25 gm load for a duration of 5 seconds after 28 days of remineralization (Fig. 1).
Fig. 1: Surface microhardness estimation using Vickers microhardness testing machine
Statistical Analysis
The surface microhardness scores were then entered into the computer (Microsoft Excel Office, Excel 2020) and were statistically analyzed using Statistical Package for the Social Sciences for Windows, version 24.0. Armonk, New York, IBM Corp. to compare the surface microhardness using Vickers microhardness testing machine at baseline, after demineralization, and after remineralization. The intragroup and intergroup comparisons between different remineralizing agents were statistically analyzed using the analysis of variance test and post hoc test. The level of significance was set at 5%.
RESULTS
The surface microhardness at baseline, after demineralization, and after remineralization is depicted in Table 1.
| Group I (test) (VHN) (n = 15) mean ± standard deviation | Group II (control) (VHN) (n = 15) mean ± standard deviation | Confidence interval | p-value | ||
|---|---|---|---|---|---|
| Baseline microhardness | 341.73±5.07 | 365.56±5.69 | 27.86 | 19.79 | <0.0001 |
| Microhardness after artificial carious lesion formation (demineralization) | 256.54 ± 5.10 | 275.65 ± 5.22 | 22.97 | 15.24 | <0.0001 |
| Microhardness after exposure to dentifrices (remineralization) | 321.27 ± 6.48 | 313.10 ± 8.02 | 13.62 | 2.71 | 0.005 |
Bold denotes p < 0.05.
Intragroup Comparison
The baseline microhardness expressed as Vickers hardness number (VHN) for the nano-HA group (group I) was 341.73 ± 5.07; 256.54 ± 5.10 VHN after demineralization and 321.27 ± 6.48 VHN after remineralization, respectively. For the sodium monofluorophosphate group (group II), the microhardness value was found to be 365.56 ± 5.69 VHN at baseline, 275.65 ± 5.22 VHN after demineralization, and 313.10 ± 8.02 VHN postremineralization. Intragroup comparisons revealed a statistically significant increase in microhardness in group I, and microhardness increased on exposure to dentifrice after carious lesion exposure (p-value of <0.001).
Intergroup Comparison
The intergroup comparisons as presented in Table 2 show that the difference in microhardness scores (VHN scores) after remineralization was significantly higher in both group I (20.45 ± 9.84 VHN) and group II (52.45 ± 7.79 VHN) with p-value of <0.001. Also, the difference in microhardness scores between artificial carious lesion formation and exposure to dentifrices was significantly higher in group I (−64.74 ± 8.59) and group II (−37.45 ± 5.23) with a p-value of <0.001. However, the difference in scores was higher for group I, indicating better remineralization potential with respect to nano-HA toothpaste (p-value < 0.001). In the results, a negative value indicates an increase in surface microhardness which shows better remineralizing efficacy of the nano-HA group.
| Test (group I) (n = 15) (VHN) | Control (group II) (n = 15) (VHN) | p-value | Confidence interval | ||
|---|---|---|---|---|---|
| Mean ± standard deviation | Mean ± standard deviation | Lower | Upper | ||
| Microhardness difference from baseline to artificial carious lesion formation | 85.19 ± 5.32 | 89.91 ± 6.19 | 0.033 | −09.04 | −0.396 |
| Microhardness differences from baseline to exposure to dentifrice | 20.45 ± 9.84 | 52.45 ± 7.79 | <0.001 | −38.64 | −25.35 |
| Microhardness difference from artificial carious lesion formation to the exposure to dentifrices (negative indicates increase) | −64.74 ± 8.59 | −37.45 ± 5.23 | <0.001 | −32.65 | −21.91 |
Bold denotes p < 0.05.
DISCUSSION
Fluoride has proven caries preventive effects. Fluoride ions can get incorporated into hydroxyapatite, forming fluorapatite, which is less soluble in acidic attacks.2 In this in vitro study, change in tooth surface microhardness by sodium monofluorophosphate and nano-HA toothpaste was evaluated using the Vickers microhardness score. It was found that though both remineralizing agents increased the enamel surface hardness, however, the increase was more significant for nano-HA toothpaste.
It has been well established that acidic pH affects enamel’s subsurface, causing the leaching of calcium phosphate ions from enamel, leading to demineralization of enamel. Alkaline pH causes supersaturation of calcium and phosphate ions around the tooth leading to remineralization of demineralized areas.12 Topical fluoride is a remineralizing agent that acts through the formation of fluorapatite crystals that are more resistant to acid attack than hydroxyapatite.6 For causing remineralization of an initial lesion, supersaturation of ions around lesions is essential for this agent like fluoride and nonfluoride remineralizing agents like nanohydroxyapatite, bioactive glass particles, etc. can be used.9
Calcium and phosphate ions play a significant role in remineralization of demineralized lesions. Saliva has a significant role in the demineralization and remineralization cycle as it is composed of inorganic ions and has the buffering capacity to promote remineralization. The organic content of saliva also plays a vital role in this regard. In the present research, both organic and mineral compounds were used to prepare artificial saliva to simulate the clinical setting as much as possible.
Nanohydroxyapatite (nano-HA) is a novel material that is composed of nanocrystals of hydroxyapatite. It releases calcium and phosphate ions on exposure to acidic pH, which in turn supersaturates the environment with ions and causes occlusion of microporosities and remineralization of initial demineralized lesions. It is a nonfluoride-releasing remineralizing agent. Utilization of these remineralizing agents will lead to an increase in the resistance of enamel to caries.6,7
Vickers microhardness test was used to evaluate demineralized and remineralized dental tissues, as it is a relatively simple, rapid, and nondestructive method and has been previously used.
The present study found that the microhardness of tooth samples after exposure to artificial caries decreased and application of nano-HA dentifrice increased significantly. Our results are in accordance with Juntavee et al.,13 Badiee et al.,14 Ebadifar et al.,15 and Haghgoo et al.16
Juntavee et al. compared nano-HA toothpaste with tricalcium phosphate and fluoride toothpaste for their efficacy to remineralize the lesion in terms of surface microhardness. They found that all the experimental groups have shown higher surface microhardness with nano-HA toothpaste showing a nonsignificant higher value.13
Badiee et al. evaluate and compares the clinical effects of an Iranian toothpaste containing nano-HA with F-containing on white spot lesions in orthodontics patients. They confirmed that Iranian nano-HA-containing toothpaste performed better than F-containing one in terms of the amount of remineralization and diminishing the lesion extent.14
Ebadifar et al. evaluated the effect of the combination of nano-HA toothpaste with fluoride on the microhardness level of artificially created carious lesions. They concluded that the combination of nano-hydroxyapatite (NHA) with fluoride toothpaste showed superior results in terms of surface microhardness.15
Haghgoo et al. compared the remineralizing effects of NovaMin and NHA petite on caries-like lesions in primary teeth and concluded that both NHA and NovaMin were effective for the remineralization of caries-like lesions of primary teeth.16
Sodium fluoride (NaF) varnish is a topical fluoride agent that buffers pH and causes the formation of fluorapatite crystals resistant to acid attack and supersaturates the surrounding area with calcium and phosphate ions, which results in remineralization.9 In the present study, the microhardness of the sample after exposure to artificial caries decreased, and after the application of sodium monofluorophosphate dentifrice increased but was lesser than the nano-HA group.
A similar result of NaF dentifrice application resulted in reduced microhardness was found by Juntavee et al.,13 Badiee et al.,14 and Ebadifar et al.15
In this study, Nano-HA toothpaste significantly remineralized the artificial carious lesion. Results were in accordance with a study done by Swarup and Rao, who compared the effects of synthetically processed HA particles in the remineralization of early enamel lesions compared to 2% NaF and found that the surface morphology of the nano hydroxyapatite (n-HAP) group was close to that of the baseline microhardness score of enamel.17
The current study results demonstrate that nonfluoridated novel remineralizing agents, that is, nano-HA dentifrice, enhanced the surface microhardness values after artificial demineralization, thus proving effective for remineralization. However, Nano Hydroxyapatite was proved to be better than NaF dentifrice, which could be due to its ability to gradually deposition of the mineral that precipitates and nucleates in the dark zone of demineralization, thereby offering complete biomimetic regeneration of the lost enamel crystallites.
Limitations
Possible limitations of the study are those associated with an in vitro study that has limited capability to simulate plaque, saliva pellicle, and bacterial biofilms present in the oral cavity, and thus, generalization to the clinical setting must be done with caution. Further clinical trials are needed to confirm the remineralization potential of these toothpastes.
CONCLUSION
Nanohydroxyapatite (nano-HA) causes remineralization and increases the microhardness of the tooth at the microstructural level. Therefore, it is a novel method to remineralize initial carious lesions. Prompt diagnosis and accurate treatment plans can reduce the chances of exposure of the tooth to caries and increase the tooth’s longevity. These preventive techniques are readily accepted by a child or parent and holistically treat disease.
Clinical Significance
Remineralization therapy is a contemporary approach for carious lesions in early detection and a noninvasive or minimally invasive treatment approach which is crucial forthe dental profession in shifting their therapeutic approach to a new paradigm. The present study showed that nano-HA toothpaste has the potential to remineralize the artificialcarious lesion.
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