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Enamel Remineralization of Mesoporous Bioactive Glass - In Vitro Study
J Dent Hyg Sci 2024;24:343-9
Published online December 31, 2024;  https://doi.org/10.17135/jdhs.2024.24.4.343
© 2024 Korean Society of Dental Hygiene Science.

Se-Young Jeon1 ,*, Young-Seok Kim2 ,*, and Ji-Hyun Min3,†

1Sejong Star Dental Clinic, Sejong 30128, 2Department of Dental Hygiene, College of Science & Technology, Kyungpook University, Sangju 37224, 3Department of Dental Hygiene, College of Medical and Health Sciences, Cheongju University, Cheongju 28503, Korea
Correspondence to: Ji-Hyun Min, https://orcid.org/0000-0001-5177-7600
Department of Dental Hygiene, College of Medical and Health Sciences, Cheongju University, 298 Daesung-ro, Cheongwon-gu, Cheongju 28503, Korea
Tel: +82-43-229-8373, Fax: +82-43-229-8969, E-mail: jhmin@cju.ac.kr
*These authors contributed equally to this work.
Received November 25, 2024; Revised December 8, 2024; Accepted December 11, 2024.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: This study compared and analyzed various concentrations of mesoporous bioactive glass (MBG) gel, commercial acidulated phosphate fluoride gel (APF-gel), and Tooth MousseTM (TM) to evaluate the enamel remineralization effect of early dental caries.
Methods: The samples prepared using sound bovine teeth were subjected to MBG gel, APF-gel, and TM to measure changes in surface hardness (Vickers hardness number, VHN) and fluorescence loss values compared to those of sound teeth (ΔF). Repeated measures analysis of variance (ANOVA), Friedman’s ANOVA, Wilcoxon signed-rank test, one-way ANOVA, and Kruskal-Wallis test were used for statistical analysis.
Results: The VHN was significantly increased after the two treatments compared to the initial VHN in all other groups, with the exception of the APF gel group (p<0.05). Compared to the initial ΔF, the APF-gel group significantly decreased ΔF after two treatments, and the remaining groups exhibited significantly increased ΔF (p<0.05). The MBG group exhibited significantly higher ΔF values than those of the APF gel and TM groups (p<0.05).
Conclusion: The MBG group exerted a significantly higher remineralization effect than that of the APF gel group and exhibited a similar or slightly higher remineralization effect compared to that of the TM group.
Keywords : Acidulated phosphate fluoride, Casein phosphopeptide- amorphous calcium phosphate nanocomplex, Mesoporous silicate bioactive glass nanoparticles, Tooth remineralization
Introduction

1. Background

Fluorine has been widely used to induce enamel remineralization in teeth with early dental caries1). Fluoride is effective for inhibiting mineral dissolution, and in particular, fluoride ions promote mineral deposition by increasing the diffusivity of calcium and phosphate, thus remineralizing the demineralized enamel2). Fluorohydroxyapatite is formed on this remineralized surface, and this makes it more resistant to acid2,3). Fluorine is an economical substance that efficiently induces tooth remineralization and is used in various forms such as toothpaste and acidulated phosphate fluoride gels (APF-gel).

Tooth MousseTM (TM) (GC International AG, Luzern, Swiss) is a product for tooth remineralization containing 10% casein phosphopeptide (CPP)-amorphous calcium phosphate. CPP is a bioactive polypeptide that stabilizes calcium and phosphorus, can prevent tooth demineralization, and promotes remineralization by increasing calcium phosphate levels4,5).

Bioactive glass (BG) is a reactive substance with high biocompatibility and bioactivity characteristics and possesses the ability to combine with mineralized bone tissue in the human body6,7). BG has been used in bone regeneration, tissue engineering, medicine and dentistry due to its bone-forming capability, degradability, and antibiotic properties8,9). Similar effects of BG on the bones have been observed in dental tissues. It forms an apatite layer on the enamel surface and exhibits antibacterial properties against caries bacteria10-12). Mesoporous BG (MBG) possesses a larger surface area than that of BG, and this increases its reactivity and bioactivity13,14). Therefore, it is expected to be effective for remineralization of tooth tissue.

2. Objectives

The purpose of this study was to compare and evaluate the remineralization effect of various concentrations of MBG gel and commercial APF-gel, TM on early enamel caries.

Materials and Methods

1. Manufacturing of the tooth specimen

Sound bovine anterior teeth without caries or cracks were cut into 5×5 mm pieces using a low-speed diamond disc (NTI-KAHLA GmbH, Kahla, Germany). After coating the teeth with acrylic resin (curing acrylic denture repair material; Vertex Dental, Soesterberg, Netherlands), 220p, 400p, 800p, and 1,200p abrasive paper (Allied High Tech Products Inc., Compton, CA, USA) were installed in a polishing machine (RB 209 MINIPOL; R&B Inc., Daejeon, Korea) to expose and flatten the enamel surface, and the teeth were polished step-by-step. A transparent nail varnish was applied to 1/2 of the specimens.

2. Measurement of specimen hardness

The Vickers hardness number (VHN) of the tooth specimen surfaces was measured using a microhardness tester (HM-200; Mitutoyo, Kanagawa, Japan). The hardness was obtained by measuring the horizontal and vertical lengths of the rhombus indentation caused by applying a load of 200 g to three parts (center, left, and right) for 10 seconds for each specimen. Fifty-five sound specimens with an average VHN of greater than 290 were used in this experiment.

3. Demineralization of the specimen

To prepare the demineralization solution, tribasic calcium phosphate was added to 1 M lactic acid to prepare a saturated solution, and 1% Carbopol was then added to bring the final pH to 4.815). Five specimens were distributed in 50 ml of demineralization solution and deposited in a 37°C incubator for 36 hours. The VHN values of the specimens were measured again.

4. Manufacture of artificial saliva

Artificial saliva of pH 6.8 consisted of KCl (14.93 mM), KH2PO4 (5.42 mM), NaCl (6.51 mM), CaCl2⋅2H2O (1.45 mM), and gastric mucin (0.22%; Sigma-Aldrich, Saint Louis, MO, USA).

5. Application of experimental materials

Fifty-five specimens with a VHN of 170 or less were randomized and divided into five groups, including the 0% MBG gel (negative control group), 0.5% MBG gel (experimental group 1), 1% MBG gel (experimental group 2), 1.23% APF-gel (positive control group 1) (Eazigel [strawberry scent]; Vericom, Chuncheon, Korea), and TM (positive control group 2) (GC Korea, Seoul, Korea) groups.

The MBG formulations, including the 0% MBG, 0.5% MBG, and 1.0% MBG groups, were in a non-commercialized gel form based on silicate glass synthesized using the sol-gel method and provided by MEDICLUS Co., Ltd. (Cheongju, Korea). The material from each group was applied to the specimen for 30 seconds using a microbrush. Three specimens were deposited in 45 ml of artificial saliva and stored in a 37°C incubator for 5 days (1st treatment). To simulate a situation in which the remineralization material is not wiped after application in the dental clinical field, the material from each group was deposited in artificial saliva without wiping it off. The specimens were removed from artificial saliva, washed with water, and analyzed for VHN measurement and minimal loss value (ΔF). After analysis, the above process was repeated one more time in the same manner. Specifically, the material was reapplied for 30 seconds, deposited in artificial saliva, and stored in a 37°C incubator for 5 days (2nd treatment). The VHN and ΔF were also re-analyzed.

6. Analysis of ΔF

The surface of the specimen was photographed using a quantitative light-induced fluorescence-digital camera (2+ BiluminatorTM; Inspektor Research Systems B.V., Amsterdam, Netherlands). The camera shooting conditions were ISO 1600, shutter speed of 1/160 seconds, and aperture value of 8.0. The ΔF value of the teeth that were treated with the material after demineralizing was compared to that of the sound enamel tooth that was treated with transparent nail varnish, and the results were analyzed using a dedicated program (QA2 version 1.15, Inspektor Research Systems B.V.).

7. Statistical analysis

IBM SPSS Statistics 29.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses, and a p-value of less than 0.05 was judged to be statistically significant. Normal distribution was confirmed through Shapiro-Wilk analysis. The remineralization effect of enamel on the initial caries was confirmed by VHN and ΔF values of baseline (before treatment) and the 1st and 2nd treatments. Repeated-measures analysis of variance (ANOVA) was performed, and Tukey’s post-hoc test was utilized to assess whether there was a change in VHN according to the number of treatments. Differences between the VHN groups were confirmed using one-way ANOVA and post-hoc tests. The difference according to the number of treatments of ΔF was confirmed by Friedman’s ANOVA and Wilcoxon signed-rank post-hoc test. The difference in ΔF between groups was confirmed by Kruskal-Wallis and Bonferroni correction post-hoc tests.

Results

1. Change in Vickers hardness number

There was no statistically significant difference in the baseline VHN of early carious dental specimens between the groups (p>0.999). The 0% MBG gel exhibited no significant difference in the VHN over time (p=0.06). In the 0.5% MBG gel, 1% MBG gel, and TM groups, the VHN was significantly increased compared to baseline after the 1st treatment (p<0.05). However, in the APF gel group, VHN decreased significantly after the 1st treatment, and after the 2nd treatment, the VHN recovered to approximately baseline.

The VHN of the APF gel group was significantly lower than that of the other groups after the 2nd treatment (p<0.05) (Table 1).

Changes in Vickers Hardness Number Values (n=11)

Group Baseline After 1st treatment After 2nd treatment p-value
0% MBG gel 141.62±20.10A 156.23±15.75aA 173.83±28.43abB 0.06
0.5% MBG gel 141.15±21.95A 170.82±27.90aB 172.10±31.99abB <0.001
1% MBG gel 140.77±22.31A 172.65±32.33aB 186.57±31.00aB <0.001
APF-gel 141.46±22.22A 94.35±17.69b,B 139.08±31.67bA 0.001
TM 141.87±22.90A 160.65±31.55aB 173.33±30.16abC <0.001
p-value >0.999 <0.001 0.011

Values are presented as mean±standard deviation.

MBG: mesoporous bioactive glass, APF-gel: acidulated phosphate fluoride gel, TM: Tooth mousseTM.

The p-value is obtained through repeated measured ANOVA.

The p-value is obtained through one-way ANOVA.

A,B,COther letters indicate a significant difference in Tukey’s post-hoc test of repeated measures ANOVA.

a,bOther letters indicate a significant difference in Tukey’s post-hoc analysis of one-way ANOVA.



2. Change in ΔF

There was no statistically significant difference in ΔF values of baseline between each group (p=0.937). However, there were significant differences in ΔF values between groups in both the 1st (p=0.042) and 2nd (p<0.001) treatments. After the 2nd treatment, there was no significant difference in ΔF values between 0% MBG gel, 0.5% MBG gel, and 1% MBG gel groups, and there was no significant difference in between APF-gel and TM groups. There were significant differences in ΔF in all groups according to the time changes for baseline, 1st treatment, and 2nd treatment (p<0.05). The 0% MBG gel, 0.5% MBG gel, 1% MBG gel, and TM groups exhibited significant increases in ΔF after the 1st treatment compared to baseline (p<0.05). However, in the APF-gel group, there was no significant difference in ΔF between baseline and the 1st treatment, and the ΔF value decreased significantly at the 2nd treatment (p<0.05) (Table 2).

Changes in ΔF Values (n=11)

Group Baseline After 1st treatment After 2nd treatment p-value
0% MBG gel –9.40 (–14.90, –6.80)A –6.20 (–8.30, –5.10 )B –6.50 (–8.10, –5.50)aB 0.004
0.5% MBG gel –9.30 (–13.30, –7.50)A –6.00 (–8.30, –5.40)B –6.20 (–7.80, 0.00)aB <0.001
1% MBG gel –8.90 (–14.60, –7.70)A –6.20 (–9.10, –5.00)B –5.70 (–7.80, 0.00)aB <0.001
APF-gel –8.60 (–19.00, –6.60)A –6.90 (–14.20, –6.50)A –17.20 (–39.80, –7.10)bB <0.001
TM –9.10 (–18.30, –7.80)A –6.80 (–11.40, –5.20)B –7.50 (–12.20, –5.80)bAB 0.024
p-value 0.937 0.042 <0.001

Values are presented as median (range).

MBG: mesoporous bioactive glass, APF-gel: acidulated phosphate fluoride gel, TM: Tooth mousseTM.

The p-value was obtained using Friedman’s ANOVA.

The p-value is obtained through Kruskal-Wallis test.

A,BOther letters indicate significant differences in the Wilcoxon signed-rank post-hoc test of Friedman’s ANOVA.

a,bOther letters indicate a significant difference in the Bonferroni correction post-hoc Kruskal-Wallis test.


Discussion

1. Interpretation and comparison to previous studies

White spot lesion is a disease that has not yet been resolved in the dental field and is caused by desorption of the enamel surface by acid16). White spot lesion can recover to normal teeth through remineralization. Remineralization occurs due to salivary ions or an increase in pH, and the remineralization rate can be accelerated using auxiliary materials17,18). This study evaluated the enamel remineralization effect of MBG compared to that of APF-gel and TM that are commercial remineralization products previously used for remineralization of white spot lesion in clinical practice.

In this study, in the group in which the APF gel was applied, the surface hardness decreased after the 1st treatment and recovered after the 2nd treatment. This pattern was consistently observed in both the VHN and the ΔF values. This is the same as the result for the decrease in surface hardness when pH cycling was performed without wiping after APF gel application in a previous study19). It can be observed that this is due to the APF application method and pH. In this study, as in previous studies, to maximize the effectiveness of each agent and simulate the situation of not wiping off the remineralization material after application in actual clinical practice, the sample was deposited in the remineralization solution without wiping after application of the experimental material19). In this process, the surface hardness appears to have decreased due to the corrosion of the tooth surface, as the acidic gel was immersed in saliva while covering the tooth surface, preventing remineralization of the tooth surface19).

Unlike the mechanism of action of fluorine, MBG and TM induce remineralization by forming a new hydroxyapatite layer on the tooth surface through calcium and phosphate glass11). BG upon contact with body fluids rapidly releases ions such as Ca2+ and PO43– that elevate the surrounding pH and promote osteogenesis12). Additionally, calcium and phosphate ions precipitate onto the silica gel layer formed on the surface of the BG, resulting in the formation of an apatite layer with a composition similar to that of natural hydroxyapatite present in bone tissue12,20,21). Furthermore, MBG features a unique porous structure compared to that of BG, and this significantly enhances its reaction kinetics21). In this study, MBG and TM exhibited a degree of remineralization similar to that reported in previous studies12). Previous studies have demonstrated that when MBG and TM were applied to the demineralized tooth specimen on days 0 and 10 and deposited in saliva for 15 days, the VHN of the demineralized tooth specimen was restored to the baseline value, with no significant difference between the two agents12). This is consistent with previous study that also demonstrated that the application of TM to demineralized teeth significantly increased ΔF values, confirming its remineralization effect22). In this study, both MBG and TM exhibited improved surface hardness and fluorescence loss after 10 days, confirming the remineralization effect. However, as a result of this study, remineralization occurred even at 0% MBG, so it seems that there will be a remineralization effect by the saliva deposited on the specimen during the experiment.

2. Limitation

This study possessed limitations in terms of the sample size, and there were differences between the actual oral environment and the in vitro laboratory setting that may lead to outcomes that differ from those observed in the oral cavity. Therefore, it is necessary to confirm the treatment effect in an actual oral cavity in the future. In this study, artificial saliva was replaced once after the first and second treatments. As the bioactive ability of MBG is increased in simulated body fluid, follow-up studies are needed to confirm the degree of remineralization when artificial saliva is frequently replaced. In this study, remineralization agents were applied for a relatively short duration of 30 seconds, and their effects were observed over a limited period of 10 days. In future studies, it will be necessary to investigate the effects of various application times and the outcomes of longer and more sustained applications.

3. Suggestion

MBG exhibits high biocompatibility and promotes the remineralization process by releasing calcium and phosphate ions to form a new hydroxyapatite layer. Furthermore, this study confirmed that MBG exerts a remineralization effect comparable to that of TM, as evidenced by VHN and ΔF values. Therefore, with optimized application methods, MBG possesses significant potential as a remineralization agent in dental clinical practice.

4. Conclusion

In this study, we attempted to confirm the enamel remineralization effect of MBG by comparing it to the existing commercial remineralization products APF gel and TM. In the MBG and TM groups, a significant increase in hardness was observed compared to that of the APF gel. Additionally, the MBG group exhibited significantly higher ΔF values than those of the APF gel and TM groups.

Acknowledgements

MBG used in this study was supplied by Mediclus Co., Ltd.

Notes

Conflict of interest

Ji-Hyun Min has been journal manager of the Journal of Dental Hygiene Science since January 2023. Ji-Hyun Min was not involved in the review process of this editorial. Otherwise, no potential conflict of interest relevant to this article was reported. Se-Young Jeon and Young-Seok Kim declare that she has no conflicts of interest.

Ethical approval

This study did not involve human subjects; therefore, review and approval by the Institutional Review Board (IRB) were not required.

Author contributions

Conceptualization: Se-Young Jeon and Ji-Hyun Min. Data acquisition: Se-Young Jeon. Formal analysis: Se-Young Jeon, Young-Seok Kim, and Ji-Hyun Min. Writing-original draft: Se-Young Jeon, Young-Seok Kim, and Ji-Hyun Min. Writing-review & editing: Se-Young Jeon, Young-Seok Kim, and Ji-Hyun Min.

Funding

None.

Data availability

Raw data is provided at the request of the corresponding author for reasonable reason.

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