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 Table of Contents  
ORIGINAL RESEARCH
Year : 2022  |  Volume : 14  |  Issue : 5  |  Page : 509-517

Effect of chitosan and hydroxyapatite nanocomposite on dentin erosion: An in-vitro study


1 Department of Conservative Dentistry and Endodontics, SRM Dental College, SRM Institute of Science and Technology, Ramapuram, Chennai, India
2 Department of Metallurgical and Material Engineering, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India

Date of Submission22-Feb-2022
Date of Decision08-Aug-2022
Date of Acceptance09-Aug-2022
Date of Web Publication31-Oct-2022

Correspondence Address:
Dr. M Madhana Madhubala
Department of Conservative Dentistry and Endodontics, SRM Dental College, SRM Institute of Science and Technology, Bharathi Salai, Ramapuram, Chennai 600089, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_50_22

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  Abstract 

Aim: To evaluate the dentinal tubule occlusion and collagen stabilization potential of nanohydroxyapatite (nHAp) and nanochitosan (nCH) combination paste on eroded dentin surface. Materials and Methods: In this in-vitro study, nHAp was prepared using the microwave-accelerated wet chemical synthesis method and nCH was made by the ionic gelation technique. The particles were characterized separately under dynamic light scattering and made into a paste by mixing them at a ratio of 1:1, which was further analyzed using Fourier-transform infrared spectroscopy (FTIR). Dentin slabs were prepared from 32 extracted human molars and subjected to erosion by exposing to 3% citric acid for 5 min. They were divided into four groups by convenience sampling method (n = 15): group I-control (no treatment); group II-nHAp; group III-nCH; group IV-nHA–nCH paste. All dentin samples were treated according to their respective groups by the active application of pastes using microbrushes for 1 min everyday for 14 days. Later, the samples were subjected to FTIR and scanning electron microscopy and energy dispersive X-ray spectroscopy (SEM-EDX) analysis. Statistical analysis was done using one-way analysis of variance (P < 0.05). Results: SEM-EDX revealed complete occlusion of dentinal tubules in the nHA–nCH group with HAp deposits on the surface. The Ca/P ratio of various groups was significantly different from each other (control group-1.05, nHAp-1.64, nCH-1.14, and nHA–nCH-1.71) (P < 0.05). The FTIR spectra marked the presence of amide I peak in nCH and nHA–nCH groups, indicating collagen stabilization. Conclusion: The nHA–nCH paste shows a potential for tubular occlusion and stabilizes both the inorganic and organic components of eroded dentin, respectively.

Keywords: Chitosan Nanoparticles, Dentin Collagen, Erosion, Hypersensitivity, Nanohydroxyapatite


How to cite this article:
Gurucharan I, Derick Isaac D R, Madhubala M M, Vijay Amirtharaj L, Mahalaxmi S, Jayasree R, Sampath Kumar T S. Effect of chitosan and hydroxyapatite nanocomposite on dentin erosion: An in-vitro study. J Int Oral Health 2022;14:509-17

How to cite this URL:
Gurucharan I, Derick Isaac D R, Madhubala M M, Vijay Amirtharaj L, Mahalaxmi S, Jayasree R, Sampath Kumar T S. Effect of chitosan and hydroxyapatite nanocomposite on dentin erosion: An in-vitro study. J Int Oral Health [serial online] 2022 [cited 2022 Dec 4];14:509-17. Available from: https://www.jioh.org/text.asp?2022/14/5/509/359968


  Introduction Top


Dentin erosion can lead to exposure of an outer layer of completely demineralized inorganic zone and partly denatured inner organic matrix.[1],[2] Desensitizing and mineralizing agents such as fluoride varnishes, sodium nitrate, potassium nitrate, casein phosphopeptide-amorphous calcium phosphate-based pastes, beta-tricalcium phosphate, bioactive glass, and so on have been employed for treating hypersensitivity caused by denuded or exposed dentin.[3],[4],[5] Most of these materials have shown to exhibit a superior degree of surface mineralization and tubular occlusion, rendering least importance to stabilization of the lost dentin collagen architecture.[6]

The major structural protein in dentin is a highly crosslinked type I collagen and around 10% of organic dentin.[7] It is also composed of non-collagenous proteins and proteoglycan molecules which form the backbone of collagen network incorporating hydroxyapatite (HAp) crystals within.[7] During dentin erosion, denaturation of these organic proteins and proteoglycan molecules exposes the collagen fibrils. The exposed collagen network could further be susceptible to degradation by bacteria and matrix metalloproteinases (MMPs), which progressively weaken the dentin matrix and compromise the bonding ability of future adhesive restorations.[8],[9] An attempt to stabilize this collagen ultrastructure can significantly enhance the mechanical integrity of dentin, preventing further degradation in order to accomplish desensitization and durable adhesive restorations.

Synthetic nanohydroxyapatite (nHAp) is more similar to the basic inorganic component of dentin. Due to their nano-size, nHAp particles can penetrate deep into the dentinal and aid in blocking the external stimuli by interacting with the nerves.[10] Also, the application of nHAp on eroded dentin has been shown to exhibit a superior degree of surface re-mineralization and tubular occlusion.[11],[12],[13] Besides its action on exposed dentin, nHAp particles can also precipitate onto the remaining enamel surface and remineralize ion-deficit areas, resulting in the formation of a new apatite layer. This layer possesses increased surface hardness and hence increased resistance to further insults.[14] Recently, Coelho et al.[15] have confirmed that nHAP does not pose any safety concerns and is non-cytotoxic in addition to desensitizing tooth pastes.

Incorporation of biopolymers such as chitosan (β 1→4 N-acetyl glucosamine) to the inorganic component has been attempted to crosslink and/or stabilize the collagen fibrils. It is structurally similar to that of glycosoaminoglycans, a ground substance of dentin.[16] Nanochitosan (nCH) can penetrate and crosslink the collagen network by chemical interaction of its free reactive hydroxyl and amino groups.[17],[18],[19] Also, studies have proved that this crosslinking resists further bacterial collagenolytic activity on dentin.[20],[21] In addition, it has also been shown to have an excellent MMP inhibitory activity, which helps to inhibit MMPs activated at low pH caused by erosion.[20]

In this study, an attempt was made to prepare biopolymeric paste using nHAp and nCH to serve as a scaffold for the bio-remineralization of dentin. A careful review of the literature revealed that no studies were evidenced on simultaneous therapeutic management of both organic and inorganic breakdown of dental erosion. Hence, the study aimed to evaluate the surface remineralization and collagen stabilization potential of nHA–nCH paste on eroded dentin. The null hypothesis was that nHA–nCH paste does not have influence on dentin erosion.


  Materials and Methods Top


The Institutional Review Board approved the use of extracted human teeth for the experiments conducted in this study. All the materials obtained were in their analytical grade. Calcium nitrate [Ca3(NO4)2.4H2O], diammonium hydrogen phosphate [(NH4)2HPO4], ammonia (30% GR), 1% acetic acid, sodium tripoly-phosphate, 0.1% thymol, 3% citric acid, and distilled water were purchased from MERCK, India. Chitosan powder was procured from Panvo Organics, India.

Preparation of chitosan nanoparticles

Chitosan powder was prepared according to the methodology followed by Zhao et al.,[22] in which 1% acetic acid was added to chitosan and kept under magnetic stirring at room temperature for 10 min. To this, 1 mg/mL sodium tripolyphosphate solution was added drop-wise, while mixing the solution in a homogenizer (Remi Homogenizer, Mumbai, India) at 5000 rpm. The nanoparticles were obtained on formation of visible zone of opalescent suspension. The concentration of tripolyphosphate solution in the final solution was 0.3–0.6 mg/mL.

Preparation of nHAp powder

nHAp was synthesized according to Niu et al.,[23] using the microwave-accelerated wet chemical synthesis method. Ca3(NO4)2.4H2O and (NH4)2HPO4 were used as precursor solutions which were mixed at a Ca/P ratio of 1.67. During synthesis, pH was maintained above 10 by neutralizing with ammonia. Following complete mixing, the solution was kept under irradiation in a microwave oven (BPL, India) at 800 W for about 30 min using 60% of the power. The resultant precipitate was then washed thrice with distilled water to remove NH4+ and NO2−3 ion and oven-dried at 100°C. Finally, it was ground to a fine powder using an agate mortar and pestle.

Particle size analysis

The particle size of resultant nHAp powder and nCH suspension was measured by the dynamic light scattering (DLS) method. Measurements were done in a particle size analyzer (Horiba Partica LA 950 Instrument, Horiba Scientific, USA) using an aqueous suspension containing Tween-80 as a dispersion aid.

Preparation of nHAp and nCH paste

About 180 mg of nHAp powder was blended in 30 mL of nCH suspension in a graduated beaker at a ratio of 1:6 using a magnetic stirrer (C-MAG HS 10 Digital Magnetic Stirrer, IKA India Pvt. Ltd). The obtained white-colored suspension was characterized by Fourier transform infrared spectroscopy (FTIR, Perkin Elmer Spectrum FTIR, Ohio, USA) to evaluate the elemental components of the nHA–nCH paste before subjecting the paste to experimental procedures. The analysis was done using the spectrum spotlight FTIR system that provides 25 and 6.25 µm pixel resolution and can generate chemical image size up to 1.64 cm2.

Specimen preparation

This in-vitro experimental study was carried out in the Department of Conservative Dentistry and Endodontics at our institution for a period of 1 year from March 2018 to March 2019. Sample size calculation was done by the “Resource Equation” method in which E (sample size) was measured, which is the degree of freedom of analysis of variance (ANOVA). A total of 32 unerupted caries-free, sound mandibular third molars extracted from patients reporting to outpatient department were used in this study. Teeth with caries, cracks, white spots, and hypoplastic appearance were excluded from the study. The extracted teeth were stored in 0.1% thymol at 4oC until use. The specimens were utilized within 1 month of their extraction. The teeth were sectioned at the level of cement–enamel junction followed by mesiodistal sectioning so that two dentin slabs were obtained from each tooth. These specimens were mounted on resin blocks and sectioned to 3 mm thick dentin slabs using hard tissue microtome (Yushuoda Hard Tissue Microtome, Liaoning, China). Markings were made on the pulpal side so as to identify the coronal surface after sectioning. The specimens were then polished using 600 grit silicon-carbide papers. Following this, the slabs were subjected to ultrasonication in distilled water (Maxsell MS300SH-9LQ Ultrasonicator, Chennai, Tamil Nadu, India) to remove the smear layer for 10 min. The dentin specimens were then coated with nail varnish leaving a window of 3 × 3 mm at the center. In order to remove the smear plugs and to expose the dentinal tubules like erosive dentin, all the dentin samples were pre-treated with 3% citric acid for 5 min and then rinsed thoroughly with distilled water. Four specimens were randomly selected and subjected for scanning electron microscopic (SEM) examination to confirm the eroded and open dentinal tubules. Sampling was done using the convenience sampling method. Then, all the dentin samples were divided randomly into four groups and allocated as follows (n = 15): group I-no treatment (negative control); group II-nHAp; group III-nCH; and group IV-nHA–nCH paste.

Application of experimental agents

The experimental agents were applied on the specimens using microbrush continuously for 1 min every 24 h. All the specimens including the control group were stored in artificial saliva (KCl 11.2 g, (HOCH2)3CNH2 2.42 g, CaCl2.2H2O 0.167 g, KH2PO4 0.123 g, distilled water; pH adjusted to 7.0) for a period of 14 days. The artificial saliva was replenished daily.

Evaluation of tubule occlusion and calcium/phosphorus (Ca/P) ratio

Ten dentin discs were taken from each group and dried in a desiccator for 24 h and mounted on aluminum stubs. The discs were sputter-coated with gold and examined under SEM and energy dispersive X-ray spectroscopy (SEM-EDX, Hitachi S 3400 N SEM-EDX, Tokyo, Japan) to evaluate dentinal tubule occlusion and surface elemental analysis, respectively. Qualitative assessment of the photomicrographs was done based on the surface characteristics and patency of the dentinal tubules at 3000× magnification. Quantitative analysis of the Ca and P deposits was made using EDX. The analytic points were made at three places over the surface of specimens, and then the obtained Ca/P values were averaged.

Evaluation of collagen stability

Five dentin slabs from each group were subjected to FTIR in the bandwidth of 400–4000 cm−1 at 8 cm−1. Analysis was done using the spectrum spotlight FTIR system that provides 25 and 6.25 µm pixel resolution and can generate chemical image size up to 1.64 cm2. The specimen was placed on germanium crystal and the pointed tip of the standard pressure was positioned at the center of the mark. The acquisition accuracy was 4 cm−1. The image size was approximately 100 µm by 150 µm. An atmospheric correction was applied to the raw image subtracting the contribution of atmosphere absorbance, i.e., water and carbon dioxide.

Statistical analysis

Statistical analysis was done using SPSS software (Version 22, IBM Corp., USA) by applying one-way ANOVA followed by post hoc test on the Ca/P ratio. P < 0.05 was considered significant at a confidence interval of 95% and degree of freedom 3 between the groups [Table 1].
Table 1: Results of one way ANOVA to determine the calcium/phophorus ratio of the deposits on the dentin surface as measured by SEM-EDX

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  Results Top


Determination of particle size

DLS analysis of nHAp and nCH showed a clear particle size distribution in the range of 70–100 and 80–210 nm, respectively. Based on the intensities of particles, it can be observed that about 40–50% of the particles are distributed with a particle size of this range.

FTIR analysis of nHAp and nCH paste

As shown in [Figure 1], FTIR spectra reveal a slight CH bend at 2883 cm−1, CC stretch at 2361 and 2113 cm−1, NH2 peak at 1638 cm−1, NH peak at 1556 cm−1, and CO stretch at 1022 cm−1, which correspond to chitosan moieties. The presence of HAp can be conferred to the appearance of OH stretch at 3212 cm−1 and phosphate contours at 654, 601, and 558 cm−1.
Figure 1: Fourier transform infrared spectra of nHA–nCH paste

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Evaluation of tubule occlusion and calcium/phosphorus (Ca/P) ratio

As shown in [Figure 2], SEM evaluation revealed completely open dentinal tubules in the control group and good occlusion of dentinal tubules in the nHAp group. There was partial occlusion in the nCH group and a further better occlusion of dentinal tubules on using nHA–nCH compared with other experimental groups. nHA–nCH also exhibited the orderly deposition of crystallites. EDX values of Ca/P ratio indicate a statistically significant difference between control and nCH groups and the nHAp-containing groups. [Table 1] and [Table 2] represent the mean Ca/P and standard deviation values of the four groups as obtained by the one-way ANOVA test and post hoc test. The Ca/P ratio of the untreated control group was 1.05 (P = 0.000), the nHAp group was 1.64 (P = 0.000), the nCH group was 1.14 (P = 0.000), and the nHA–nCH group was 1.71 (P = 0.000), with high statistical significant difference between all the groups.
Figure 2: Scanning electron microscopic representative images of dentin samples from each experimental group, (A): Group I no treatment (negative control); (B): Group II-nHAp; (C): Group III-nCH; (D): Group IV-nHA–nCH paste. Red arrows indicate nHAp particles

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Table 2: Mean and standard deviation (SD) of the various groups as measured by post hoc test

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Evaluation of collagen stability

The results revealed the emergence of the low intensity amide I peak in the region of 1632.40 and 1619.40 cm−1 in control and nHAp groups, respectively. Higher intensity peaks in the region of 1641.80 cm−1 in the nCH group and 1643.05 cm−1 in the nHA-nCH group are shown in [Figure 3].
Figure 3: Fourier transform infrared spectroscopic representative image from the respective groups. Red arrows indicate amide I peak depicting dentinal collagen. The amide peaks were seen in the region of 1632.40 cm−1 in group I-no treatment (negative control); 1619.40 cm−1 in group II-nHAp; 1641.80 cm−1 in group III-nCH; 1643.05 cm−1 in group IV-nHA–nCH paste

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  Discussion Top


The nHA–nCH paste was chosen to provide a synergistic effect on tubular occlusion along with biomimetic remineralization of collagen fibrils. In this study, coronal dentin sections were used to analyze the effectiveness of desensitizing agents. Though erosion manifests more commonly in the cervical regions, it is more pertinent to perform in the coronal area as critical variables such as surface characteristics, surface area, and dentin thickness and can be better standardized.[24]

Determination of particle size confirmed the non-agglomeration of particles during preparation which could attribute to its effectiveness on nano-delivery. On FTIR analysis, individual peaks of NH2, NH, CO stretch, OH stretch, and phosphate contours clearly showed that the characteristic feature of both the materials without influence on each other to exhibit their potential bioactivity. Ibrahim et al.[25] also showed the strong interaction between the two components, suggesting the originality of components being maintained in the composite paste. Ren et al.[26] also showed the stretching vibration absorption of –NH2 and –OH from chitosan, and the peak of PO3−4 in nHA appeared at 567, 604, 961, and 1037–1093 cm−1, demonstrating the successful synthesis of chitosan nHA/methyl vinyl ether-alt-maleic anhydride complexes with electrostatic interaction. Nanocrystalline composites with various hydroxyapatite/chitosan ratios were successfully synthesized by Nikpour et al.[27] In this study, chitosan exhibits strong adsorption interaction with calcium phosphate material by well pervading nHA particles in the matrix forming homogeneous aggregations around 40–100 nm. nHA aligned along the chitosan molecules showed strong interfacial interaction between the nanoparticles and the polymer matrix. Thus incorporation of HA nanoparticles in biocompatible chitosan matrix could be attributed for reinforcement of the eroded dentin.

Demineralization of dentin disks with open dentinal tubules was achieved by treating with citric acid so as to simulate eroded dentin with exposed, denatured collagen. Erosion was evidenced by SEM-EDX results, which showed open dentinal tubules and a calcium phosphate ratio of 1.05, predicting calcium-deficient hydroxyapatite deposits, respectively. The amide I peak in an infrared spectrum corresponds to dentin collagen and is usually present in the region of 1655–1667 cm−1 in healthy dentin. This peak corresponds to C = O stretching of the amide I band.[28],[29] A decrease in the intensity or loss of this peak indicates instability/absence of collagen fibrils. Likewise, the intensity of amide peak was low in the eroded dentin samples indicating denatured collagen.

The complete occlusion of dentinal tubules by nHAp could be attributed to the greater surface area and surface charge of nHAp, which confer increased bioactivity and better ion penetration into the dentinal tubules culminating in greater occlusion.[10],[30] The presence of HAp deposits was further confirmed by the EDX results, showing a Ca/P ratio of 1.64 which is close to the HA molar atomic ratio of 1.67. Our findings were in accordance with Amaechi et al.[12] and Ohta et al.[13] and other studies in which nHAp has the ability to interact with the apatite of dentin, forming stronger bonds resistant to removal.[30] However, studies showed that nHAp alone did not effectively block the dentinal tubules, and a combination with fluoride was required. The deposited nHAp acts as nucleation sites attracting more and more Ca and P ions from the artificial saliva, resulting in progressive mineral deposition and tubular occlusion.[31],[32] However, the results of the FTIR analysis showed low-intensity amide I peak at the band region of 1619.46 cm−1, which implies the inability of nHAp to stabilize the collapsed collagen. Most of the traditional remineralizing agents work only on the basis of epitaxial deposition of calcium and phosphate ions over existing apatite seed crystallites.[6] Remineralization cannot occur in locations where seed crystallites are absent. This could be the reason for diminished expression of stabilized collagen.

Effective remineralization requires the use of various biomimetic analogs of non-collagenous proteins along with calcium phosphate materials.[6] One of the most commonly investigated non-collagenous analogs being chitosan; SEM analysis of the nCH group showed masking of the dentinal tubules partially, characterized by amorphous deposition of nCH with few apatite crystals formed on the dentin surface. Ca2+ ions and hydroxyapatite crystals could be bound by nCH, which might act as a binder between them. The long chains of chitosan interact with dentin by winding around the triple helix of dentin collagen.[33] As collagen has hydroxyl groups that form hydrogen bonds within the chains and functional side groups, they are free to interact with other molecules. Thus, side groups of collagen molecules interact with the amine (NH2) and hydroxyl groups of chitosan to form hydrogen bonds. Additionally, the carboxyl groups (-COOH) and the amine groups present in collagen can also react with -OH and -NH2 groups of chitosan via hydrogen bonds.[33],[34] Besides this dipole interaction, there exists an electrostatic attraction between the cationic polysaccharide chitosan and polyanionic -COOH of the collagen molecule.[19],[34] Thus, these interactions can lead to the intertwining of chitosan onto collagen, forming an insoluble entangled amorphous deposit macromolecule with a higher viscosity than their individual components. All these interactions could have contributed to the expression of the amide peak at the band region of 1641.80 cm−1 with greater intensity by nCH. However, the EDX analysis revealed a 1:1 ratio of calcium-deficient HAp, indicating the active effect of chitosan on collagen stabilization with compromised surface mineralization. Therefore, bioactive materials can be combined as a complex biocomposite to make it as a reservoir for ions for remineralization.

Due to the active regions present in the structure of chitosan, various assemblies of chitosan complexes have been tried to achieve native mineralization. Santoso et al.[35] showed that the carboxymethyl chitosan and amorphous calcium phosphate (CMC/ACP) complex significantly strengthened dentin by serving as a reservoir of mineral ions that promote remineralization. Similarly, Zhang et al.[36] demonstrated subsurface remineralization efficacy of the chitosan–bioglass complex on artificial white spot lesion, which enhanced subsurface remineralization under the presence of salivary pellicle. The combination of the nHAp and nCH composite displayed a complete and higher degree of dentin occlusion, which was depicted as precipitation of crystals in the most orderly manner on the dentin surface. The Ca/P ratio of 1.71 indicates the presence of Hap suggesting of surface remineralization. The higher expression amide I peak could be a validating factor of higher reactivity and suggestive of the presence of highly cross-linked and stabilized collagen compared with the nCH group. This might also be attributed to the formation of coordination (covalent) bonds with the N atom of amine in nCH, one by Ca and another bond by P in nHAp.[19] There might be no transfer of charge between the molecules; instead, there could be an increase in the dipole moment due to asymmetric distribution of electrons between nHAp and nCH.[25] Zhang et al.[37] also proved that chemical modification of collagen fibers with phosphorylated chitosan can accelerate the rate of crystallization by its ion chelation mechanism and their ability to cause a high degree of conglutination. Similarly, in the CMC/ACP complex, CMC can enhance the formation of ACP nano-complexes to accomplish mineralization of intrafibrillar collagen by employing bottom-up approach.[38] The structural explanation of mechanism of nHA and nCH mechanism has been depicted in [Figure 4].
Figure 4: Pictorial representation of methodology and the mechanism of action of nHA–nCH biocomposite paste

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Moreover, Ibrahim et al.[25] have also demonstrated that when nHAp was added to chitosan, the hydrophobicity of the composite paste is increased much further. This might prove to be useful in treating areas of desensitization as the components will not be easily washed away by oral fluids when incorporated into toothpastes.

This is only a preliminary study showing that a combination of HAp and chitosan nanoparticles has the potential to restabilize the dentinal collagen with native remineralization. However, the limitation of the study includes the lack of pH cycling regimen and an evaluation at various time periods. Also, future studies on the influence of this composite on mechanical properties and detailed intrafibrillar biomodification of dentin after surface treatment have to be carried out before clinical experiments.


  Conclusion Top


Within the limitations of this study, it can be concluded that nHA–nCH paste has a dual effect of mineralization and collagen stabilization potential on eroded dentin. Together, they can serve as a promising therapeutic component to treat dentin hypersensitivity and to mechanically integrate dentin to deliver durable future adhesive restorations by giving better protection to the underlying pulp–dentin complex.

Acknowledgements

Nil.

Financial support and sponsorship

No funding was received for this study.

Conflicts of interest

The authors declare no conflict of interest.

Authors’ contributions

All the authors have equally contributed to conception, design, data acquisition and interpretation, drafted and critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.

Ethical policy and Institutional Review Board statement

This in-vitro study has been approved by our Institutional Review Board. The human teeth samples were collected according to provisions of Declaration of Helsinki.

Patient declaration of consent

Not applicable for in-vitro studies.

Data availability statement

All the data are available within the article.

 
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