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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 3  |  Page : 84-87

The effect difference of chitosan nanoparticles, chitosan microparticles, and casein phosphopeptide–amorphous calcium phosphate in reducing enamel demineralization


1 Department of Pediatric Dentistry, Faculty of Dental Medicine, Universitas Brawijaya, Malang, East Java, Indonesia
2 Department of Dental Material, Faculty of Dental Medicine, Universitas Brawijaya, Malang, East Java, Indonesia
3 Undergraduate Program in Dentistry, Faculty of Dental Medicine, Universitas Brawijaya, Malang, East Java, Indonesia

Date of Submission03-Jul-2020
Date of Decision06-Aug-2020
Date of Acceptance12-Sep-2020
Date of Web Publication17-Oct-2020

Correspondence Address:
Mohammad Chair Effendi
Department of Pediatric Dentistry, Faculty of Dental Medicine, Universitas Brawijaya, Jl. Veteran, Malang, East Java
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/SDJ.SDJ_41_20

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  Abstract 


Background: Chitosan has been shown to inhibit free radicals that cause tooth enamel demineralization. Nano-sized chitosan can penetrate cell membranes that larger particles cannot penetrate. Objective: This study aimed to determine the difference between chitosan nanoparticles, chitosan microparticles, and casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) in preventing tooth enamel demineralization. Methods: We used 0.2% chitosan nanoparticles. A total of 50 mL of chitosan solution was stirred and added in tripolyphosphate to prepare a nanoparticle suspension. It was then stirred for 1 h to generate crosslinking. The nanoparticles' size was 57.6 nm. The demineralization solution consisted of 2.2 mM/L CaCl2, 2.2 mM/L KH2PO4, and 50 mM of acetate buffer. Its acidity was regulated to a pH of 4.06. The sample consisted of 27 maxillary first premolar teeth post extraction due to orthodontic treatment needs divided into three groups: a chitosan microparticle treatment group, a chitosan nanoparticle treatment group, and a CPP-ACP treatment group used as a positive control. A scanning electron microscope with ×5000 magnification was used to observe the enamel surface morphology and mineral release. Results: The mean value of enamel surface microhardness in the chitosan nanoparticle group (233.39 HV) was significantly greater than those in the chitosan microparticle (153.192 HV) and CPP-ACP groups (152.626 HV) (P < 0.05). Moreover, the chitosan nanoparticle treatment resulted in the lowest enamel porosity. Conclusions: Chitosan nanoparticles are more effective than chitosan microparticles and CPP-APP in preventing enamel demineralization.

Keywords: Casein phosphopeptide–amorphous calcium phosphate, enamel demineralization, nanoparticle chitosan


How to cite this article:
Effendi MC, Fitriani D, Nurmawlidina MF. The effect difference of chitosan nanoparticles, chitosan microparticles, and casein phosphopeptide–amorphous calcium phosphate in reducing enamel demineralization. Sci Dent J 2020;4:84-7

How to cite this URL:
Effendi MC, Fitriani D, Nurmawlidina MF. The effect difference of chitosan nanoparticles, chitosan microparticles, and casein phosphopeptide–amorphous calcium phosphate in reducing enamel demineralization. Sci Dent J [serial online] 2020 [cited 2020 Oct 22];4:84-7. Available from: https://www.scidentj.com/text.asp?2020/4/3/84/298447




  Background Top


According to data published by the World Health Organization, the prevalence of caries in Indonesia is as high as 90.2% in children aged 5 years, 92.2% in people aged 35–44 years, and 95% in people aged 65 years and older.[1] One of the methods for preventing caries is using chitosan to inhibit mineral release in the teeth. Chitosan can be easily extracted from the exoskeletons of shrimp, especially the Pacific shrimp (Pandalus borealis), lobsters, crabs, especially the Dungeness crab (Cancer magister), and crawfish.[2] One of the chemical elements identified in chitosan is amino group-NH2, which exhibits high reactivity to cariogenic acids and can reduce acid dissolution in hydroxyapatite. This amino group can capture hydrogen ions, so that the charge of hydrogen ions becomes positive and it can be absorbed via the electrostatic force on surfaces with strong negative zeta potential, such as enamel.[3],[4] It triggers the formation of organic layers, especially the addition of mucins to enamel surface. Mucins can also be absorbed on surfaces with a negative charge, after which chitosan can be absorbed by mucins, forming an attached multilayer that is quite resistant to acids.[5],[6] Chitosan can inhibit the demineralization process by acting as a barrier to acid penetration and preventing the release of minerals in the enamel.[7],[8]

Nanoparticles are particles with sizes ranging from 1 to 100 nm (1 nm = 10−9 m).[9] Chitosan nanoparticles can increase their surface area by up to hundreds of times compared to micrometer-scale particles (10−6 m) and increase the ability of chitosan to bind to chemical groups. For this reason, they are used in drug delivery systems and dental tissue technology.[10],[11]

The most well-known material used in dental practice to inhibit demineralization in tooth enamel is casein phosphopeptide–amorphous calcium phosphate (CPP-ACP). In an acidic environment, CPP-ACP buffers plaque pH, inhibiting demineralization and exerting a short-term remineralization effect.[12],[13],[14],[15]

This study aimed to compare the effectiveness of chitosan nanoparticles and microparticles and CPP-ACP in inhibiting the demineralization of tooth enamel. The study hypothesis was that the three treatments would exert different effects on the microhardness of enamel surfaces.


  Materials and Methods Top


This was an experimental laboratory study with a pre–posttest group design. The study was approved by the Ethics Committee of the Faculty of Medicine of the University of Brawijaya (No. 44/EC/KEPK-S1-FKG/02/2017).

Based on Federer's formula, (n-1) (t-1) ≥15 (n: number of groups = 3 and t: the number of samples in each group), the sample consisted of 27 maxillary first premolar teeth post extraction due to orthodontic treatment needs.[16] Maxillary first premolar teeth post extraction due to orthodontic treatment needs were chosen because they were not extracted due to caries, so it was expected that their anatomy and structure would not be damaged. The inclusion criteria were as follows: (1) teeth in good condition, (2) clinically intact, and (3) with an average initial enamel hardness of 227–424 HV.[17] The exclusion criteria were as follows: (1) teeth clinically diagnosed with caries, (2) clinically abraded (noncarious lesion due to mechanical wears of tooth), and (3) clinically eroded (noncarious lesion due to chemical factors). The teeth were divided into three groups of nine: a chitosan nanoparticle (NPCh) treatment group, a chitosan microparticle (MPCh) treatment group, and a CPP-ACP (C+) treatment group used as a positive control.

We used 0.2% chitosan nanoparticle paste (Nanotech Herbal, Puspiptek, Indonesia) produced using the ionic gelation method. Chitosan was dissolved into acetic acid, then stirred using a magnetic stirrer overnight at room temperature, and filtered. Subsequently, 50 ml of chitosan solution was stirred and added in a 0.1% tripolyphosphate solution to form a nanoparticle suspension. Stirring continued for 1 h until the crosslinking process was complete.[18] Using a particle size analyzer (Beckman Coulter, Indianapolis, USA), the particle size was determined to be 57.6 nm. The demineralization solution used consisted of 2.2 mM/L CaCl2, 2.2 mM/L KH2 PO4, and 50 mM of acetate buffer. The solution's acidity was regulated by a KOH solution (a strong base solution) until it reached a pH of 4.06. This method was previously described in another research.[19]

The sample teeth were prepared to obtain a buccal surface and were then implanted in acrylic resin. The tooth roots were cut using a low-speed micromotor (Strong 207B Micro Motor; Saeshin, Daegu, South Korea) with a carborundum disc until only the crown remained. Each premolar was divided into two parts in the buccal-palatal direction. The buccal part was planted into a PVC pipe (Rucika, Jakarta, Indonesia) with a diameter of 1 cm using acrylic resin. The tooth surfaces were cleaned from debris using a brush for 3 min.

Before the treatment, the initial (pretest) hardness of the enamel surface microstructure of each sample was measured with a Vickers hardness tester (Eseway Premium Micro-Vickers Hardness Tester EW-105 Series, Bowers Group, Camberley, UK). The tip of the diamond indenter was pressed on the surface of the sample with a load of 300 g for 10 s. The hardness of each sample was measured at three different points, namely the upper-middle and lower regions (the regions close to the cervix). The average was then calculated. The three groups were treated by topical application to the tooth surface using a tip applicator for 5 min. The samples were then washed with distilled water using a syringe. Six hours after the application of the paste, each sample was immersed in 20 mL of demineralization solution (pH 4.06) for 1 h.[3],[20] The microhardness of the samples' enamel surfaces was then measured again using a Vickers hardness tester, as previously described in another study.[21] A scanning electron microscope (Phenom G2 Pro, Phenom-World, Thermo Fisher Scientific, Eindhoven, The Netherlands) with × 5000 magnification was used to observe the enamel surface morphology of the samples in each group to analyze the mineral release.

Data analysis

A paired t-test was used to evaluate differences in the enamel surfaces of the samples before and after treatment in each group. One-way analysis of variance (ANOVA) was performed to evaluate differences between the three groups after treatment. A value of P < 0.05 was considered statistically significant. The data were statistically analyzed using SPSS 17.0 Program (International Business Machines Corporate [IBM], Armonk, New York, USA).


  Results Top


[Figure 1] shows the porosity on the enamel surface in each group, as observed by scanning electron microscopy (SEM). The NPCh treatment group showed the lowest porosity compared to the MPCh treatment group and CPP-ACP group.
Figure 1: Scanning electron microscopy images of enamel surface morphology after treatment with (a) casein phosphopeptide–amorphous calcium phosphate, (b) chitosan microparticles, and (c) chitosan nanoparticles

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The paired t-test revealed significant differences in enamel microhardness before and after treatment in the C+ (P < 0.01), MPCh, and NPCh groups (P < 0.05), suggesting that demineralization occurred in all the groups, but the NPCh group showed the lowest decreased in enamel hardness [Figure 2].
Figure 2: Mean differences in enamel microhardness before and after treatment with casein phosphopeptide–amorphous calcium phosphate (C+), chitosan microparticles (MPCh), and chitosan nanoparticles (NPCh); *P < 0.05; **P < 0.01

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The mean values of enamel surface microhardness after the NPCh, MPCh, and C + treatments were 233.39, 153.192, and 152.626 HV, respectively. One-way ANOVA [Figure 3] revealed that the NPCh treatment was significantly more effective than the C+ and MPCh treatments in preventing enamel demineralization (P < 0.01). The difference between the C+ and MPCh treatments was statistically insignificant.
Figure 3: Mean differences in enamel microhardness after treatment with casein phosphopeptide–amorphous calcium phosphate (C+), chitosan microparticles (MPCh), and chitosan nanoparticles (NPCh); **P < 0.01

Click here to view



  Discussion Top


In this study, SEM revealed that the NPCh group exhibited the lowest porosity. This is in line with the statistical test results. Although demineralization still occurred in all the groups, the NPCh treatment group exhibited the highest mean microhardness value (233.39 HV), which is in line with the recommended initial enamel microhardness range of 227–424 HV. Conversely, the CPP-ACP and MPCh treatments did not differ significantly in terms of preventing enamel demineralization. Among the factors that rendered, the NPCh treatment superior to the other two is its nanoparticle size of 57.6 nm. Nano-sized particles can penetrate cell membranes that larger particles cannot penetrate, so that nanoparticles penetrate cell membranes in organisms and interact with biological systems more easily.[22],[23] Chitosan nanoparticles can be used on a subcellular scale with high accuracy in achieving target cellular and gain maximum therapeutic effect.[24]

The initial hardness range criteria of the samples that quite large (227-424 HV) could be one of the limitations of this study and may affect the final results. Another limitation is that the indentation points between initial and final microhardness tests are less precise. Further research could be undertaken to overcome certain limitations of this study. The initial hardness range of the samples needs to be reduced so that they are more homogeneous, yielding more accurate results. Moreover, the precision factor between the location of the indentation point in the final microhardness test (posttest) and that in the pretest should be taken into consideration to minimize bias and increase the accuracy of the results.


  Conclusions Top


Chitosan nanoparticles are significantly more effective than CPP-ACP and chitosan microparticles in inhibiting tooth enamel demineralization. They yield the lowest degree of enamel porosity and an enamel surface microhardness that is in line with the recommended microhardness of 227–424 HV.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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