Evaluation of zinc oxide nanoparticle coated elastomeric modules and it’s effect on Streptococcus mutans concentration and enamel mineralization - A randomized split mouth trial

Abstract

To assess the long term effects of zinc oxide (ZnO) nanoparticle coated elastomeric modules on S. mutans concentration and enamel mineralization among orthodontic patients over a period of one year. A total of 16 patients were recruited for this study. Either the left or right maxillary lateral incisor received the coated module. Group I : ZnO coated elastomeric modules (N = 16); Group II: Non-coated elastomeric modules (N = 16). S. mutans in plaque was assessed at the end of 3 months and one year using Rt -PCR. Enamel mineralization was assessed by laser fluorescence at the end of one year. Integrity of the ZnO nanoparticle coating over a period of one month on a weekly basis was also assessed. Independent t-test was done to find the significant difference between the bivariate samples and intra-group comparison was done using paired t-test. Pearson’s correlation was done to determine the relationship between the variables. For all the statistical tests, p < 0.05 was considered to be statistically significant. S. mutans concentration at the end of three months and one year was higher in the non-coated group compared to the coated group but it was statistically significant only at the end of one year (p value = 0.032). Laser fluorescence values was higher for the non-coated group compared to the coated group which showed statistical significance (p = 0.020). ZnO coating of the modules showed evidence of disintegration of the coating after two weeks, with further deterioration of the coating at the end of one month. As elastomeric modules are changed periodically, this appears to be a viable option, especially since the nanoparticle release would be around the most common area of plaque retention.

Introduction

Orthodontic appliances create a nidus for plaque accumulation and make the cleaning process more difficult. The plaque accumulated forms a biofilm over the tooth surface which harbors among others, cariogenic bacteria such as Streptococcus mutans and Lactobacilli¹. There is a change in the amount, structure and composition of plaque during orthodontic treatment which leads to increased colonization of microorganisms that affects the ecosystem². S. mutans, a Gram-positive cocci, a common commensal of the oral cavity is one of the first to colonize and can be seen as early as 6 weeks after orthodontic treatment³. It degrades fermentable carbohydrates leading to increased lactic acid production and lowering of pH which in turn leads to release of calcium and phosphate ions from the hydroxyapatite crystals causing enamel demineralisation⁴.

Decalcification of tooth surfaces can present as early enamel caries which are manifested clinically as white spot lesions (WSLs)⁵. WSLs are defined as subsurface enamel porosity from carious demineralization that presents itself as a milky white opacity when located on smooth surfaces⁵. Prevalence of WSLs among orthodontic patients was reported to be about 55.06%⁶. It can appear within 1 month of bracket placement⁷ around the brackets and commonly noted on the maxillary anterior tooth surface⁸.

Common methods to prevent and manage WSLs are dependent on patient compliance and pose a challenge since orthodontic treatment is of a longer duration. In recent times, attempts have been made to modify orthodontic appliances using nanotechnology by either coating the surface or by incorporating nanoparticles into bonding systems and cements. Inorganic nanomaterials such as metal oxides have been extensively researched for their antibacterial properties when integrated with orthodontic appliances⁹. Among these metal oxides, Zinc oxide has gained considerable attention owing to its antimicrobial properties, low-cost and stability¹⁰. Other advantages include an esthetic outlook due its comparable transmittance to glass¹¹ and reduced cytotoxicity^12,13.

Another factor that accounts for plaque retention is the type of ligation of orthodontic arch wires. Elastomeric modules are preferred over stainless steel ligatures due to their easy application, decreased chair side time and patient comfort. However, they exhibit a greater number of microorganisms in the plaque around the brackets when compared with steel ligatures¹⁴. A recent in vitro study evaluated silver nanoparticles synthesized in situ on modules and suggested that nanocoated modules have the potential to combat the dental biofilm¹⁵.

In the past, research has shown that nanoparticle coated brackets and wires do offer antibacterial advantages over microbial colonization. However most of these studies are in-vitro or short term clinical studies⁹. A recent in-vivo study by Venkatesan et al.¹⁶, evaluated the integrity of TiO2 coating on NiTi wires and reported that there was delamination and deterioration of the nanocoating after one month of intra-oral usage. Since modules are changed every appointment, coating them with nanoparticles might still offer antibacterial efficacy. No study has evaluated the efficacy of zinc oxide nanoparticle coated elastomeric modules in vivo and on a long term basis and therefore this study sought to evaluate the long term effects of ZnO nanoparticle coated modules on S. mutans concentration in plaque of orthodontic patients and on enamel mineralization over a period of one year.The objectives of this study included: (1) To evaluate the S. mutans concentration in plaque surrounding orthodontic brackets with ZnO coated modules and non-coated modules at 3 months and one year after appliance placement. (2) To compare the changes in enamel mineralization around the coated and non-coated modules at the end of one year. (3) To assess the integrity of the ZnO nanoparticle coating over a period of one month.

Materials and methods

This randomized split mouth trial was performed in accordance with Consolidated Standards of Reporting Trials (CONSORT) 2010 guidelines. Orthodontic patients who were to undergo fixed appliance mechanotherapy were recruited for this study. The inclusion criteria were: age 15–30 years, patients undergoing orthodontic treatment (extraction/non-extraction cases), patients with good oral hygiene (OHI-S score of 0-1.2)¹⁷, patients with no relevant medical history and good gingival and periodontal health. Exclusion criteria were: patients with missing lateral incisors or any morphological variations in lateral incisors (peg laterals), patients with poor oral hygiene and extensive dental caries, periodontitis and bone loss. Also, patients who were on use of antibiotics were excluded from the study.

Maxillary lateral incisors (both right and left) were included in the study. Sample size calculation was performed using Stata statistical software, version 17 (Statacorp., college station, Texas, USA). Based on data from the study by Zeidan et al.¹⁸, (power, 0.80; α = 0.05) the effective sample size was computed to be 16 per group using one way ANOVA F test. Thus, 16 lateral incisors would receive ZnO coated modules (GROUP I) and 16 lateral incisors would receive non-coated modules (GROUP II).

Allocation concealment for this study was done by simple randomization protocol. The operator placing the orthodontic appliances was asked to pick up lots that determined equal allocation of either coated or non-coated modules to the right/left maxillary lateral incisors. The operator and the participants were blinded until the completion of the study. However, the blinding of the investigator who delivered the modules to the operator who did orthodontic bonding was not possible. Since the investigator who delivered the modules was not blinded, it was ensured that the same sequence was followed in subsequent reviews. Plaque specimens were coded and sent for RT - PCR. The final data assessment for statistical analysis was blinded.

Orthodontic modules (American Orthodontics, Uni-Stick Silver Ligature sticks, Washington Ave, USA) were coated with ZnO nanoparticles using radio frequency magnetron sputtering method. ZnO coatings were achieved in a cathodic sputtering unit (Anelva Sputtering Unit Model SPF-332 H; Canon Anelva Corp, Kawasaki, Japan) equipped with a radiofrequency generator operating at 13.56 MHz, ZnO target of 100 mm diameter and 99.9% purity fixed on an equilibrated magnetron cathode. The deposition temperature used for sputter coating was standardized at 200 °C. Before deposition, the substrates were cleaned with deionized water and mounted carefully on the substrate holder inside a circular chamber for coating. The substrates were rotated at a constant 30 rpm with a distance of 50 mm between the target and substrate to get uniform and quality films. Once one side of the substrate was coated, it was carefully inverted to coat the other side¹⁹. Representative sample from the coated modules was evaluated using field- emission scanning electron microscopy (FESEM) with energy dispersive X-ray spectroscopy (EDX) (Carl Zeiss, Thornwood, NY) to determine the uniformity of the coated surface¹⁹.

Patients were recalled every month for their routine review and the modules were changed during every visit ensuring that group I always received coated modules. Time points assessed:

T0- Immediately after bonding of fixed appliances.

T1- 3 months after appliance placement.

T2- 1 year after appliance placement.

In each group, plaque surrounding the maxillary lateral incisors were collected at T1 and T2. Plaque specimens were collected using the 4-pass technique as suggested by Pellegrini et al.²⁰S.mutans concentration was evaluated using real-time polymerase chain reaction (Applied Biosystems, Foster City, California) (RT-PCR)²¹.

Enamel mineral content was assesed using a laser device DIAGNOdent^® (KAVO Dental Corporation, Lake, Zurich, IL, USA). Labial surface of the maxillary lateral incisor was divided into four quadrants - gingival, mesial, occlusal and distal²². The peak fluorescence on DIAGNOdent pen was assessed on the mesial, gingival, distal and incisal surfaces and averaged to obtain the mean overall score for each tooth. Laser fluorescence assessment was done at T0 and T2. After completion of one year evaluation, coated modules were retrieved from two patients each, at the end of 1 st week, 2nd week, 3rd week and 4th week for evaluation of integrity of the nanoparticle coating.

The collected data was analyzed with SPSS statistical software (version 23.0; IBM Corp, Armonk, New York, USA). Normality of the data was assessed by Shapiro-Wilk test. To find the significant difference between the bi-variate samples in paired groups, independent t-test was used. Intra-group comparison was done using paired t-test. Pearson’s correlation was done to determine the relationship between the variables. For all tests, p < 0.05 was considered to be statistically significant.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of University’s Institutional Ethics Committee [Number CSP/22/OCT/117/526 dated 02/05/2023]. The study was conducted in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The trial was registered prospectively in the Indian Council of Medical Research (ICMR)-Clinical Trials Registry-India (CTRI) [CTRI/2023/11/060313] .

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent. The patient has given his/her consent for his/her images and other clinical information to be reported in the journal. All the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Informed consent statement

Informed consent was obtained from all the subjects who participated in this study. For patients below age group of 16, the informed consent was obtained from their respective parents/guardians.

Results

Thirty patients were assessed for eligibility. Based on the inclusion criteria, 16 patients were selected. To assess the outcomes, 16 patients patients were randomized in a 1:1 ratio, so that 16 lateral incisors would receive ZnO coated modules and 16 lateral incisors would receive non-coated modules. There were no exclusions after randomization and no patients were lost to follow- up. The Consolidated Standards of Reporting Trials (CONSORT) flowchart demonstrating participant flow has been described in Fig. 1.

[See PDF for image]

Fig. 1

CONSORT flow diagram of the study selection process

S. Mutans concentration

Real time - PCR performed in this study revealed the relative quantification of S.mutans by evaluating the Ct (Cycle Threshold) values. This value is inversely proportional to the amount of bacterial genome present, which means a higher Ct value indicates a lower S.mutans count and vice versa.

Intra-group comparison of Ct values between T1 and T2 indicated that bacterial concentration increased in the non-coated group and decreased in the coated group at the end of one year. However, this difference was not statistically significant in both the groups. Inter-group comparison showed that there was no significant difference in Ct values between both the groups at T1. At T2, S. mutans concentration was less in the coated group and this was statistically significant (p value = 0.032). Effect size (Cohen’s d) for Ct values among the coated group was found to be moderate (Table 1).

Table 1. Ct values of coated and non-coated modules at T1 and T2

Group	T0	P value	T2	P value	Intra-group comparison between T0 and T2	Effect size
Coated group	2.87 ± 0.56	0.285	2.28 ± 0.98	0.020*	0.665	0.201
Non-coated group	3.01 ± 0.65		3.75 ± 0.76		0.014*	0.321

*Statistical significance: p<0.05, values indicated in terms of Mean ± Standard Deviation

Assessment of Enamel Mineralization

Enamel mineral content was assessed by the DIAGNOdent pen in terms of laser fluorescence values. As the values increase, it indicates enamel demineralization occurred and signifies development of WSLs. Measurements from 2 to 9 signify incipient decalcification²³.

Intragroup comparison of laser fluorescence in the coated group showed that values had decreased from T2 to T0 indicating remineralization however this was not statistically significant. In the non-coated group, laser fluorescence values increased at T2 compared to T0 indicating demineralization and this was statistically significant (p=0.014). At T0, there were no significant differences between both the groups (p=0.285) .However at T2, values was higher for the non-coated group compared to the coated group and this was statistically significant (p=0.020). Effect size (Cohen’s d) for laser fluorescence among the coated group was found to be minimal (Table 2).

Table 2. Laser fluorescence values of coated and non-coated modules at T0 and T2

Group	T1	P value	T2	P value	Intra-group comparison between T1 and T2	Effect size
Coated group	21.60 ± 4.68	0.412	22.14 ± 4.16	0.032*	0.785	0.665
Non-coated group	19.96 ± 4.35		18.09 ± 2.90		0.154	0.244

*Statistical significance: p<0.05, values indicated in terms of Mean ± Standard Deviation

Pearson’s correlation

The S. mutans concentration in plaque was correlated to the enamel mineral content at the end of one year (T2) for both the coated and non-coated group. Pearson’s correlation revealed a weak positive correlation between Ct values and laser fluorescence for the coated module group. This correlation was not statistically significant (p=0.492). Moderate negative correlation was noted between Ct values and laser fluorescence for the non-coated module group with no statistical significance (p=0.336).

Integrity of coating

The surface was noted to have uniform distribution of ZnO nanoparticles immediately after coating (Fig. 2). After completion of the one year follow up period, coated modules were retrieved from two patients after one, two, three and four weeks and subjected to FESEM with EDX . The scans revealed integrity of the coating at the end of one week and EDX analysis showed the presence of zinc oxide nanoparticles with mild deposits (food debris and plaque) present on the surface of the coated module as shown in figure 3. At the end of two weeks, the coating was intact. However, decrease in the ZnO nanoparticles was noted with increased deposits over the surface of the module (Fig. 4). At the end of three weeks ,the coating showed disintegration with further decrease in the ZnO nanoparticle content (Fig. 5). At the end of one month ,there was disintegration of the coating with very low quantity of ZnO nanoparticles which was confirmed by FESEM and EDX analysis as shown in figure 6.

[See PDF for image]

Fig. 2

2A: FESEM photograph of surface of the ZnO coated module immediately after coating. 2B, 2C, 2D: EDX Images showing uniform distribution of ZnO particles.

[See PDF for image]

Fig. 3

3A: FESEM photograph of ZnO coated module retrieved after one week. 3B, 3C, 3D: EDX Images showing distribution of ZnO particles.

[See PDF for image]

Fig. 4

4A: FESEM photograph of ZnO coated module retrieved after two weeks. 4B, 4C, 4D: EDX images showing distribution of ZnO particles..

[See PDF for image]

Fig. 5

5A: FESEM photograph of ZnO coated module retrieved after three weeks. 5B, 5C, 5D: EDX images showing decreased distribution of Zn particles.

[See PDF for image]

Fig. 6

6A: FESEM photograph of ZnO coated module retrieved after 4 weeks . 6B, 6C, 6D: EDX images showing reduced distribution of Zn particles.

Discussion

Demineralization of enamel is considered one of the most inevitable and yet avoidable complications of fixed mechanotherapy.Since modules are changed every appointment and nanocoatings on modules have not been studied in a clinical setting we decided to evaluate the efficacy of nanocoated modules on S.mutans concentration. Modules were coated through a process of magnetron sputtering which is a type of physical vapor deposition method. It’s exemplary properties include low temperature, thin film deposition, inherent versatility and uniform coating over the surface of the material and better coating adhesion^24,25. A split mouth technique was employed as it has the advantages of reduced inter-subject variability and lesser sample size requirement. Lateral incisors were evaluated since they are the teeth that are most susceptible for WSL formation⁹.

We evaluated the S.mutans concentration at the end of 3 months because a study by Baka et al²⁶ revealed concentration of S. mutans was highest at the end of three months. Our results showed that at the end of three months, bacterial concentration in the non-coated group was higher compared to the coated group, although this difference was not statistically significant. However at the end of one year, there was further decrease in bacterial concentration in the coated group and increase in the non-coated group and the difference between the groups was statistically significant. Hammad et al²⁷ who evaluated the antibacterial efficacy of ZnO nanoparticle coated NiTi wires reported that the coating was effective against gram positive and gram negative microorganisms. Anita et al¹⁹ reported that ZnO coated aligners had a negative effect on S. mutans colonies and the effect lasted for only a week.

The laser fluorescence values evaluated at T0 were similar in both the groups ensuring parity at the start of the study. Over a period of one year laser fluorescence values in the coated group reduced although this was not statistically significant. In the non-coated group the laser fluorescence values increased indicating progressive demineralization and this was statistically significant. Correlation of Ct values to the laser fluorescence at the end of one year revealed a weak positive correlation in the coated group and a moderately negative correlation for the non-coated group. However, neither of them was statistically significant.

Behnaz et al²⁸ reported that addition of ZnO nanoparticles to orthodontic bonding agent reduced the incidence of WSL formation at the end of 4 weeks and our findings were similar at the end of one year. A study by Nagasaki et al²⁹ reported that materials containing ZnO and bioactive glass nanoparticles promotes remineralization of tooth surfaces due to their acid buffering nature. Also, zeolite-ZnO nanoparticles were found effective for remineralization by improving the micro-hardness of enamel surface and by reducing the biofilm as reported by Pourhajibagher and Bahador³⁰. ZnO nanoparticles are biocompatible and research has shown no harmful effects on the human cells^13,31., Further, a recent study found that ZnO -Tin oxide coating of ceramic brackets reduces their surface roughness which could further reduce biofilm formation³².

Representative samples of coated modules were evaluated with FESEM & EDX at the end of one year of the study in order to not confound the results of these evaluations. It was noted that the inner halo region of the module that was in contact with the bracket wings demonstrated the presence of ZnO coating without much disintegration as compared to the other regions. ZnO coating rendered the module more shiny with minimal colour change making it extremely acceptable (Fig. 7). Future studies could focus on strengthening the longevity of the nano coating which appeared to lose its integrity after two weeks.

Besides modifying orthodontic appliances with nanocoatings, recent research has focused on producing new materials such as biomimetic zinc-carbonate hydroxyapatite³³, Rennou (theobromine calcium and phosphate)³⁴, and calcium sodium phosphosilicate³⁵ which are added to oral dentrifices to control demineralization and promote remineralization. Their use in orthodontic patients prone to WSL need to be explored in the future and researched in a clinical setting.

[See PDF for image]

Fig. 7

7A: Modules before coating, 7B: Modules after coating.

Limitations

Laser fluorescence values were not evaluated at 3 months and thus it was not possible to correlate the S.Mutans concentration with this. Cytotoxicity testing of ZnO coated modules was not studied although no adverse reactions were reported. Though the study showed statistically significant reduction in both S. mutans concentration and laser fluorescence values validating the effect of the ZnO coating, the difference between the two groups was not large and therefore the clinical significance of these findings needs further exploration.

Conclusion

ZnO coated modules reduced S. mutans concentration and prevented enamel demineralization over a period of one year. Integrity of the coating was stable over two weeks of intraoral usage after which it it displayed delamination and discontinuity.

Author contributions

HJ-Data curation; Formal analysis; Investigation; Writing - original draft; and Writing - review & editing. SP - Conceptualization and design; Validation; Supervision; Writing - review & editing and final approval.

Funding

Not applicable.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1. Lucchese, A; Bondemark, L; Marcolina, M; Manuelli, M. Changes in oral microbiota due to orthodontic appliances: a systematic review. J. Oral Microbiol.; 2018; 10, 1 1476645. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29988826][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032020]

2. Kharbanda, O. P. & Orthodontics Diagnosis and Management of Malocclusion and Dentofacial Deformities, E-Book (Elsevier Health Sciences, 2019). Chap. 79.

3. Peros, K; Mestrovic, S; Anic-Milosevic, S; Slaj, M. Salivary microbial and nonmicrobial parameters in children with fixed orthodontic appliances. Angle Orthod.; 2011; 81, 5 pp. 901-906. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21534725][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8916182]

4. Bishara, S; Ostby,. Adam. White spot lesions: formation, prevention, and treatment. Semin. Orthod.; 2008; 14, pp. 174-182.

5. Summitt, J. B., Robbins, J. W. & Schwartz, R. S. Fundamentals of Operative Dentistry: A Contemporary Approach, 3rd ed. Hanover Park, IL, Quintessence Publishing, Chap. 1, pp 2–4. (2006).

6. Hussain, U et al. Prevalence, incidence and risk factors of white spot lesions associated with orthodontic Treatment – A systematic review and Meta-Analysis. Orthod. Craniofac. Res.; 2025; 28, pp. 379-399. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/39717964]

7. Øgaard, B; Rolla, G; Arends, J. Orthodontic appliances and enamel demineralization. Part 1. Lesion development. Am. J. Orthod. Dentofac. Orthop.; 1988; 94, pp. 68-73.

8. Julien, KC; Buschang, PH; Campbell, PM. Prevalence of white spot lesion formation during orthodontic treatment. Angle Orthod.; 2013; 83, 4 pp. 641-647. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23289733][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8754044]

9. Padmanabhan, S. Nanotechnology in orthodontics. Semin. Orthod.; 2023; 29, pp. 79-84.

10. Moradpoor, H et al. An overview of recent progress in dental applications of zinc oxide nanoparticles. RSC Adv.; 2021; 11, 34 pp. 21189-21206.1:CAS:528:DC%2BB3MXhtlWhu7vF [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35479373][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9034121]

11. Choi, HJ et al. Enhanced transparency, mechanical durability, and antibacterial activity of zinc nanoparticles on glass substrate. Sci. Rep.; 2014; 4, 6271.1:CAS:528:DC%2BC2MXktlSgsrk%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25183360][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4152755]

12. Bahrami, R; Pourhajibagher, M; Badiei, A; Masaeli, R; Tanbakuchi, B. Evaluation of the cell viability and antimicrobial effects of orthodontic bands coated with silver or zinc oxide nanoparticles: an in vitro study. Korean J. Orthod.; 2023; 53, 1 pp. 16-25. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36696956][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9877365]

13. Cierech, M et al. Zinc oxide nanoparticles cytotoxicity and release from newly formed PMMA-ZnO nanocomposites designed for denture bases. Nanomaterials (Basel); 2019; 9, 1318.1:CAS:528:DC%2BC1MXitVWis7fL [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31540147]

14. Türkkahraman, H et al. Archwire ligation techniques, microbial colonization, and periodontal status in orthodontically treated patients. Angle Orthod.; 2005; 75, 2 pp. 231-236. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15825788]

15. Hernández-Gómora, AE et al. Biosynthesis of silver nanoparticles on orthodontic elastomeric modules: evaluation of mechanical and antibacterial properties. Molecules; 2017; 22, 9 1407. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28841178][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6151712]

16. Venkatesan, K; Kailasam, V; Padmanabhan, S. Evaluation of titanium dioxide coating on surface roughness of nickel-titanium archwires and its influence on Streptococcus mutans adhesion and enamel mineralization: A prospective clinical study. Am. J. Orthod. Dentofac. Orthop.; 2020; 158, 2 pp. 199-208.

17. Greene, JC; Vermillion, JR. The simplified oral hygiene index. J. Am. Dent. Assoc.; 1964; 68, pp. 7-13.1:STN:280:DyaF2c%2Fjtl2kuw%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/14076341]

18. Zeidan, NK; Enany, NM; Mohamed, GG; Marzouk, ES. The antibacterial effect of silver, zinc-oxide and combination of silver/ zinc oxide nanoparticles coating of orthodontic brackets (an in vitro study). BMC Oral Health; 2022; 22, 1 230.1:CAS:528:DC%2BB38XitFCqsb3K [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35681128][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9185939]

19. Anita, P; Sathyanarayana, HP; Kumar, K; Ramanathan, K; Kailasam, V. Antimicrobial efficacy of zinc oxide nanoparticle-coated aligners on Streptococcus mutans and Candida albicans. Am. J. Orthod. Dentofac. Orthop.; 2023; 163, 3 pp. 338-346.

20. Pellegrini, P et al. Plaque retention by self- ligating vs elastomeric orthodontic brackets: quantitative comparison of oral bacteria and detection with adenosine triphosphate-driven bioluminescence. Am. J. Orthod. Dentofac. Orthop.; 2009; 135, 4 pp. 426e1-42427.

21. Childers, NK et al. Real-time quantitative polymerase chain reaction for enumeration of Streptococcus mutans from oral samples. Eur. J. Oral Sci.; 2011; 119, 6 pp. 447-454.1:CAS:528:DC%2BC38XhsVKgt7Y%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22112030][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517291]

22. Banks, PA; Richmond, S. Enamel sealants: a clinical evaluation of their value during fixed appliance therapy. Eur. J. Orthod.; 1994; 16, 1 pp. 19-25.1:STN:280:DyaK2c3jslamsw%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/8181546]

23. Demito, CF; Rodrigues, GV; Ramos, AL; Bowman, SJ. Efficacy of a fluoride varnish in preventing white-spot lesions as measured with laser fluorescence. J. Clin. Orthod.; 2011; 45, 1 pp. 25-29. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21874779]

24. Arango, S; Peláez-Vargas, A; García, C. Coating and surface treatments on orthodontic metallic materials. Coatings; 2013; 3, 1 pp. 1-15.1:CAS:528:DC%2BC3sXhsVGls7Y%3D

25. Garrocho-Rangel, A et al. Graphene-Based coatings for surface modification and their applications in fixed orthodontics: A scoping review. Dentistry J.; 2023; 11, 12 285.

26. Baka, ZM; Basciftci, FA; Arslan, U. Effects of 2 bracket and ligation types on plaque retention: a quantitative microbiologic analysis with real-time polymerase chain reaction. Am. J. Orthod. Dentofac. Orthop.; 2013; 144, 2 pp. 260-267.

27. Hammad, SM; El-Wassefy, NA; Shamaa, MS; Fathy, A. Evaluation of zinc- oxide Nanocoating on the characteristics and antibacterial behavior of nickel- titanium alloy. Dent. Press. J. Orthod.; 2020; 21, pp. 51-58.

28. Behnaz, M; Kasraei, S; Yadegari, Z; Zare, F; Nahvi, G. Effects of orthodontic bonding containing TiO2 and ZnO nanoparticles on prevention of white spot lesions: an in vitro study. Biointerface Res. Appl. Chem.; 2021; 12, pp. 431-440.

29. Nagasaki, R; Nagano, K; Nezu, T; Iijima, M. Synthesis and characterization of bioactive glass and zinc oxide nanoparticles with enamel remineralization and antimicrobial capabilities. Materials; 2023; 16, 21 6878.1:CAS:528:DC%2BB3sXitlCks7zE [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37959475][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10647288]

30. Pourhajibagher, M; Bahador, A. Enhanced reduction of polymicrobial biofilms on the orthodontic brackets and enamel surface remineralization using zeolite-zinc oxide nanoparticles-based antimicrobial photodynamic therapy. BMC Microbiol.; 2021; 21, 1 273.1:CAS:528:DC%2BB3MXisVeitb%2FE [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34620084][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8499451]

31. Rosenberg, M et al. Selective antibiofilm properties and biocompatibility of nano-ZnO and nano-ZnO/Ag coated surfaces. Sci. Rep.; 2020; 10, 13478.1:CAS:528:DC%2BB3cXhsF2isrvO [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32778787][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417576]

32. Kumar, A; Jain, RK. Evaluation of surface roughness and frictional resistance of zinc oxide–Tin oxide nanocoated ceramic orthodontic brackets: an In vitro study. World J. Dent.; 2025; 16, 3 pp. 223-228.

33. Andrea, B et al. Biomimetic action of zinc hydroxyapatite on remineralization of enamel and dentin: A review. Biomimetics (Basel); 2023; 8, 1 71. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36810402]

34. Gokce, A.N., Kelesoglu, E., Sagır, K., Kargul, B. Remineralization potential of a novel varnish: an in vitro comparative evaluation. J. Clin. Pediatr. Dentistry48, 173–180 (2024).

35. Scribante, A et al. Evaluation of two different remineralising toothpastes in children with drug-controlled asthma and allergic rhinitis: a randomised clinical trial. Eur. J. Pediatr. Dentistry; 2024; 25, 2 pp. 137-142.1:STN:280:DC%2BB1cvgt12nsg%3D%3D