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ORIGINAL ARTICLE
Year :   |  Volume :   |  Issue :   |  Page :  

Evaluation of dentin remineralization with zinc oxide and calcium fluoride nanoparticles – An In vitro study


1 Department of Pedodontics and Preventive Dentistry, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India
2 Department of Dental Materials, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India
3 Department of Oral Pathology, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India

Date of Submission13-Oct-2021
Date of Decision28-Jan-2022
Date of Acceptance30-Mar-2022
Date of Web Publication03-Nov-2022

Correspondence Address:
Kakarla Sri RojaRamya,
Department of Pedodontics and Preventive Dentistry, Vishnu Dental College, Bhimavaram - 534 202, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ccd.ccd_701_21

   Abstract 


Background: Partially demineralized dentin is remineralizable when mineral ions are made available in the near vicinity. Nanoparticles (NPs) have wide applications in remineralization process. Zinc promotes remineralization and has a synergistic effect when combined with fluoride. Hence, zinc oxide and calcium fluoride NPs were considered for dentin remineralization. Aim: The aim of this study was to evaluate the remineralizing ability of zinc oxide and calcium fluoride NPs on demineralized dentin individually and in combined form. Settings and Design: This was an in vitro study. Materials and Methods: Forty-eight dentin disks were prepared from the crowns of 12 extracted human molars and were allocated into four groups of 12 each, i.e., Group I – nano zinc oxide (nZnO), Group II – nano calcium fluoride (nCaF2), Group III – combined group (nZnO + nCaF2), and Group IV – artificial saliva (AS, control group). The specimens were demineralized with 37% phosphoric acid and placed in the respective remineralizing solutions for 24 h and 1 month. Calcium (Ca) and phosphorous (P) uptake was measured using energy-dispersive X-ray spectrometry, and structural changes were analyzed using scanning electron microscopy (SEM). Statistical Analysis: One-way analysis of variance, Student's t-test, and post hoc Tukey's test were used for statistical analysis. Results: At 1-month interval, all the groups showed an increase in Ca/P ratio, with highest being the combined group (4.24), followed by nCaF2 (3.30), nZnO (1.71), and AS (1.31) groups, and these differences were statistically significant (P = 0.000). On SEM analysis of dentinal samples at 1 month, depositions were evident in intertubular regions, wherein the highest deposits were observed in the nZnO group, followed by nZnO + nCaF2 and nCaF2 groups. Conclusion: Dentin samples subjected to remineralization with aqueous solutions of nZnO and nCaF2 showed an increase in calcium and phosphorous uptake and also dense granular depositions were evident in intertubular regions of dentin.

Keywords: Calcium fluoride, demineralization, nanoparticles, remineralization, scanning electron microscopy, spectrometry, zinc oxide



How to cite this URL:
Ahalya P, Uloopi K S, Vinay C, RojaRamya KS, Alla R, RangaRaju P. Evaluation of dentin remineralization with zinc oxide and calcium fluoride nanoparticles – An In vitro study. Contemp Clin Dent [Epub ahead of print] [cited 2022 Nov 29]. Available from: https://www.contempclindent.org/preprintarticle.asp?id=360380




   Introduction Top


Contemporary caries management is based on minimally invasive approach of preserving partially demineralized dentin, which is considered to serve as a bioactive matrix for remineralization. The partially demineralized layer at tooth–resin interface is susceptible to endogenous matrix metalloproteinases (MMPs) and therefore acts as a site for further caries progression affecting the durability of the restoration.[1],[2] Remineralization will be facilitated when these endogenous MMPs are inhibited and a milieu of minerals made available, such that a more acid-resistant layer is formed.[3]

Fluoride is a well-known remineralizing agent used in various forms and at different concentrations for decades. Conventional NaF results in the formation of low amounts of CaF2 and CaF2-like deposits because of the availability of low calcium concentrations in the saliva.[4] Calcium concentration acts as a driving force for the formation of CaF2 deposits.[5] Hence, calcium fluoride particles (reservoir of both calcium and fluoride ions) were considered for remineralization in the present study.

Zinc and fluoride have shown a synergistic effect on remineralization of the body of the lesion.[6],[7] The inherent properties of zinc which promote remineralization are as follows: it serves as an MMP inhibitor at resin–tooth interface;[8] ionic radius of zinc is smaller than calcium and therefore replaces calcium from hydroxyapatite;[9] zinc maintains surface porosity for the ingress of minerals;[10] and the availability of zinc is more in acidic medium,[11] etc. Hence, ZnO particles were tested for remineralization.

Nanoparticles (NPs) have enhanced properties such as increased surface–volume ratio and higher bioactivity compared to regular particles.[12] Therefore assuming that nanoform may have a greater impact on remineralization ability, a study was carried out to evaluate zinc oxide and calcium fluoride NPs individually and in combined form.


   Materials and Methods Top


The in vitro study was approved by the institutional review board (IRB/VDC/14/5). Sample size was calculated using G*Power 3.1 software. At a level of significance 5%, power of the study 80%, and for an expected effect size of 0.50 (obtained from a pilot study), it was derived that 12 samples per group are required to perform the study.

Sample preparation

Twelve noncarious human third molars which were extracted for periodontal reasons were disinfected according to the Centers for Disease Control and Prevention guidelines.[13] For dentin disk preparation, the teeth were embedded in clear acrylic using modeling wax cylindrical molds of dimension 1.5″ height and 1″ diameter so that only the cusp tips were exposed. Samples were then processed in hard tissue microtome to obtain dentin disks of dimension 1–1.5 mm thickness from the middle third of the coronal portion, such that four disks were prepared from each tooth. On each dentin disk, a window of 3 mm × 3 mm was prepared by painting the remaining area with an acid-resistant nail varnish. Then, the prepared window is etched using 37% phosphoric acid for 15 s, rinsed with distilled water for 30 s, and dried.

Group allocation

Forty-eight dentin disks were allocated into 4 groups of 12 each, namely Group I – nano zinc oxide (nZnO), Group II – nano calcium fluoride (nCaF2), Group III – combined group (nZnO + nCaF2), and Group IV – artificial saliva (AS, control group).

Preparation of remineralizing solutions

Artificial saliva was prepared as per the composition given by Sato et al., and Hepes buffer of 50 mM concentration was added to this AS to maintain a pH of 7.2.[14] Penicillin (100 U/ml) and streptomycin (1000 μg/l) were added to the prepared AS to prevent the bacterial and fungal growth.

Nano ZnO remineralizing solution was prepared by adding zinc oxide NPs of 153 μmol/L concentration to the AS, and pH was set at 6.7 for solubility of NPs. To prepare nano CaF2 solution, calcium fluoride NPs of 52.6 μmol/L concentration were added to AS and pH was set at 6.8 to ensure solubility. For the combined group, nZnO particles of 153 μmol/L concentration and nCaF2 of 52.6 μmol/L concentration were added to AS and pH was set at 6.7.

Fourier transform infrared spectroscopy (FTIR) analysis was done in transmittance mode for characterization and to confirm the availability of functional groups nZn and nF in the prepared solutions [Figure 1].
Figure 1: Fourier transform infrared spectra; (a) Fourier transform infrared spectrum of nano zinc oxide solution; (b) Fourier transform infrared spectrum of nano calcium fluoride solution; (c) Fourier transform infrared spectrum of combined group (nano zinc oxide + nano calcium fluoride) solution

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Scanning electron microscopy (SEM) analysis was done to qualitatively check for mineral depositions in inter- and intratubular dentinal regions. EDX analysis was done to quantitatively assess the uptake of Ca and P in wt%. Initially, for the baseline data, the etched samples were analyzed using SEM and EDX. The samples were then immersed in their respective solutions and analyzed after 24 h and 1 month for evaluating the mineral uptake by partially demineralized dentin.

Statistical analysis

The obtained data were statistically analyzed using SPSS software (version 16, Armonk, NY, USA: IBM Corp.). One-way analysis of variance followed by post hoc were done for intergroup comparisons and Student's paired t-test for intragroup comparisons. P ≤ 0.05 is considered statistically significant and P ≤ 0.001 as highly significant.


   Results Top


EDX analysis

The Ca/P ratios were calculated from Ca and P wt% values obtained from EDX analysis. On intragroup comparison of mean differences of Ca/P ratio from baseline to 24 h and 1 month, statistically significant changes were observed in all the groups (P = 0.00). In the nZnO group, the gain in Ca/P ratio was observed at both 24 h (0.94) and 1 month (1.71). Similar trend was observed in the AS group also. In the nCaF2 group, Ca/P ratio was decreased at 24 h (−0.32) and thereafter an increase was noticed at 1-month interval (3.30). The combined group also exhibited the same trend of decrease at 24 h (−2.00) and an increase at 1 month (4.24) [Table 1].
Table 1: Intragroup comparison of mean differences of calcium/phosphorous ratio between different time intervals

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On intergroup comparison of mean differences of Ca/P ratios, at 24 h interval, an increase was observed in the nZnO (0.94) and AS (0.71) groups, whereas the nCaF2 (−0.32) and combined groups (−2.00) showed a decrease in Ca/P ratio, and these differences were statistically significant in all the groups (P = 0.00). At 1-month interval, all the groups showed an increase in Ca/P ratio, highest being the combined group (4.24), followed by nCaF2 (3.30), nZnO (1.71), and AS (1.31) groups, and these differences were statistically significant (P = 0.000) [Table 2].
Table 2: Intergroup comparison of mean differences of calcium/phosphorous ratio between different time intervals

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On pairwise comparison, a high statistically significant difference was observed in the mean differences of Ca/P ratios between all the groups at both the intervals except for the nZnO and AS groups (P = 0.000) [Table 3].
Table 3: Pairwise comparison of intergroup calcium/phosphorous ratio between different time intervals

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Scanning electron microscopy analysis

Dense granular mineral deposits partially occluding the dentinal tubules were noticed in both inter- and intratubular dentinal regions in the nZnO group, whereas in the combined group, fewer specks of mineral precipitates were observed in inter- and intratubular regions. In the nCaF2 group, sparse mineral deposits were evident only in intertubular regions of dentin, and in the AS group, no appreciable deposits were noticed.


   Discussion Top


The advantages of nanotechnology are well reaped in dentistry, especially in material sciences. Nanomaterials have wide applications in preventive dentistry either to inhibit demineralization or to enhance remineralization processes.[15] The NPs can thus be used either for replacement of lost minerals or to act as carriers for ions, for example, nCaF2 and nano-hydroxyapatite.[16],[17]

Literature evidences reported a synergistic effect of zinc and fluoride at 153 μmol/L and 52.6 μmol/L concentrations, respectively, on dentin remineralization.[6],[7] Hence, the same concentrations of ZnO and CaF2 were prepared in nanoparticulate form and tested for their remineralization efficacy individually and in combined form. FTIR analysis of the prepared test solutions confirmed the presence of functional groups with wave number of 433 cm− 1 for nZn and 483 cm− 1 for nF in transmittance mode [Figure 1].

The samples treated with nZnO and AS showed a greater uptake of Ca and P (Ca/P ratio) at 24 h. In samples subjected to the nCaF2 and combined groups, the Ca and P uptake was less compared to baseline. The concentration gradient of calcium between demineralized dentin and remineralizing solution might have resulted in efflux of Ca ions from dentin samples. The samples of the combined group showed a greater reduction in Ca/P ratio compared to the nCaF2 group, and this might be due to the availability of Zn which maintained the surface zone porosity for the movement of the ions.

There was a concomitant gain in both Ca and P ions in nZnO-treated samples at 1-month interval. This might be attributed to the remineralizing properties of zinc, supplementation of zinc in nanoform, and the availability of other minerals in the AS. Osorio et al. have also reported that the zinc oxide group showed higher Ca/P ratio and nanohardness values.[18] Higher zinc concentrations in nanoform as well as greater reactivity, high surface energy, and smaller dimension of NPs might be the reason for increased uptake of Ca and P ions.[12] Lynch et al. noticed an increased Ca/P ratio of 2.14 when macro zinc particles were used.[6] Whereas, in the present study, higher Ca/P ratio (2.56) was evident and this difference might be because of using zinc in nanoform and its remineralizing properties.

In the nCaF2 group, calcium and fluoride ions were available at a higher concentration of 1.1526 mM each which might have resulted in supersaturated state, thereby causing precipitation of Ca and F ions on the demineralized dentin. The combined group showed higher gain than the nCaF2 group since zinc is also available along with the nCaF2 which might have facilitated greater ingress of mineral ions. The formation of fluorapatite diffusion barrier in the nCaF2 group might have prevented further uptake of mineral ions, hence the Ca/P ratio was lesser than the combined group at 1-month time interval.

SEM analysis showed opening of dentinal tubules after etching at baseline [Figure 2]. At 24 h, although there is an increase in Ca/P ratios in the nZnO and AS groups, noticeable structural changes in inter- and intratubular dentinal regions were not evident, since the time available for mineral uptake was short [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d.
Figure 2: Scanning electron micrograph (×1000) of dentin sample treated with 37% phosphoric acid showing the opening of dentinal tubules

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Figure 3: Scanning electron micrograph (×5000) of dentin samples; (a-d) Images of samples treated with nano zinc oxide, nano calcium fluoride, combined group (nano zinc oxide + nano calcium fluoride), and artificial saliva, respectively, at 24 h showing no appreciable deposits; (e) Sample treated with nano zinc oxide at 1 month demonstrating dense granular mineral deposits partially occluding the dentinal tubules in both inter- and intratubular dentinal regions; (f) Sample treated with nano calcium fluoride at 1-month interval showing specks of deposits in intertubular dentin; (g) Sample treated with nano zinc oxide + nano calcium fluoride at 1 month demonstrating fewer mineral deposits in intertubular dentin; (h) Sample treated with artificial saliva at 1 month showing no appreciable deposits

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At 1 month, the nZnO and combined groups showed noticeable mineral depositions than at 24 h, suggesting a definite long-term effect of tested NPs on dentin remineralization. In the nZnO group, dense granular mineral deposits partially occluding the dentinal tubules were noticed in both inter- and intratubular dentinal regions which may be due to the concomitant gain in Ca as well as P; while in the combined group, fewer specks of mineral precipitates were observed only in intertubular regions which could be due to a relatively greater increase in Ca only without much gain in P (Ca/P ratio: 4.24). In the nCaF2 group, the Ca/P ratio was lesser (3.30) than the combined group, hence sparse mineral depositions were evident only in intertubular regions of dentin in SEM images [Figure 3]e, [Figure 3]f, [Figure 3]g, [Figure 3]h.

Although the pH of the prepared nano remineralizing solutions was below 7, there was an increase in Ca/P ratio suggestive of remineralizing ability even under acidic conditions. The Ca/P ratio of sound/healthy dentin varies from 1.7 to 2.14.[19] Since the observed ratio in dentinal samples treated with the test nanogroups was greater than these values, the formation of acid-resistant hypermineralized zone is possible with these solutions. Therefore, these NPs can be incorporated into restorative materials to promote remineralization by release of ions at the tooth–resin interface. However, studies evaluating the sustained release of the ions from ZnO and CaF2 NPs are yet to be carried out for its clinical implication.

Limitations of the study are as follows: remineralization was carried out under static conditions without pH cycling since the pH above 6.7 reduces the solubility of NPs. Being an in vitro study, it could not mimic the in vivo conditions wherein the mineral concentrations in saliva and their bioavailability are pH and time dependent.


   Conclusion Top


The nanoformulations of ZnO and CaF2 promoted remineralization of partially demineralized dentin. The nZnO group was found to be more effective for dentin remineralization followed by combined group (nZnO + nCaF2). The zinc and fluoride showed a synergistic effect at the considered concentrations in their nanoforms.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Chaussain-Miller C, Fioretti F, Goldberg M, Menashi S. The role of matrix metalloproteinases (MMPs) in human caries. J Dent Res 2006;85:22-32.  Back to cited text no. 1
    
2.
Tjäderhane L, Nascimento FD, Breschi L, Mazzoni A, Tersariol IL, Geraldeli S, et al. Optimizing dentin bond durability: Control of collagen degradation by matrix metalloproteinases and cysteine cathepsins. Dent Mater 2013;29:116-35.  Back to cited text no. 2
    
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Gu H, Ling J, LeGeros JP, LeGeros RZ. Calcium phosphate-based solutions promote dentin tubule occlusions less susceptible to acid dissolution. Am J Dent 2011;24:169-75.  Back to cited text no. 3
    
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Vogel GL, Mao Y, Carey CM, Chow LC, Takagi S. In vivo fluoride concentrations measured for two hours after a NaF or a novel two-solution rinse. J Dent Res 1992;71:448-52.  Back to cited text no. 4
    
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Saxegaard E, Rølla G. Kinetics of acquisition and loss of calcium fluoride by enamel in vivo. Caries Res 1989;23:406-11.  Back to cited text no. 5
    
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Lynch RJ, Churchley D, Butler A, Kearns S, Thomas GV, Badrock TC, et al. Effects of zinc and fluoride on the remineralisation of artificial carious lesions under simulated plaque fluid conditions. Caries Res 2011;45:313-22.  Back to cited text no. 6
    
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Lippert F. Dose-response effects of zinc and fluoride on caries lesion remineralization. Caries Res 2012;46:62-8.  Back to cited text no. 7
    
8.
Osorio R, Yamauti M, Sauro S, Watson TF, Toledano M. Zinc incorporation improves biological activity of beta-tricalcium silicate resin-based cement. J Endod 2014;40:1840-5.  Back to cited text no. 8
    
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Masala O, O'Brien P, Rafeletos G. Formation of spherical granules of calcium pyrophosphate. Cryst Growth Des 2003;3:431-4.  Back to cited text no. 9
    
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Spero JM, Devito B, Theodore L. Regulatory Chemicals Handbook. New York: Marcel Dekker Inc.; 2000. p. 79.  Back to cited text no. 10
    
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Akbari B, Tavandashti MP, Zandrahimi M. Particle size characterization of nanoparticles – A practical approach. Iran J Mater Sci Eng 2011;89:48-56.  Back to cited text no. 11
    
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Holec D, Dumitraschkewitz P, Vollath D, Fischer FD. Surface energy of au nanoparticles depending on their size and shape. Nanomaterials (Basel) 2020;10:484.  Back to cited text no. 12
    
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Centers for Disease Control and Prevention. Guidelines for infection control in dental health-care settings-2003. MMWR Morb Mortal Wkly Rep 2003;52:33.  Back to cited text no. 13
    
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Sato Y, Sato T, Niwa M, Aoki H. Precipitation of octacalcium phosphates on artificial enamel in artificial saliva. J Mater Sci Mater Med 2006;17:1173-7.  Back to cited text no. 14
    
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Sharan J, Singh S, Lale SV, Mishra M, Koul V, Kharbanda P. Applications of nanomaterials in dental science: A review. J Nanosci Nanotechnol 2017;17:2235-255.  Back to cited text no. 15
    
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Sun L, Chow LC. Preparation and properties of nano-sized calcium fluoride for dental applications. Dent Mater 2008;24:111-6.  Back to cited text no. 16
    
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Chow LC, Sun L, Hockey B. Properties of nanostructured hydroxyapatite prepared by a spray drying technique. J Res Natl Inst Stand Technol 2004;109:543-51.  Back to cited text no. 17
    
18.
Osorio R, Osorio E, Cabello I, Toledano M. Zinc induces apatite and scholzite formation during dentin remineralization. Caries Res 2014;48:276-90.  Back to cited text no. 18
    
19.
Arnold WH, Konopka S, Gaengler P. Qualitative and quantitative assessment of intratubular dentin formation in human natural carious lesions. Calcif Tissue Int 2001;69:268-73.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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