Keywords
Remineralization, agarose, enamel, microhardness, surface roughness.
Remineralization, agarose, enamel, microhardness, surface roughness.
Biomimetic remineralization is a non-invasive therapeutic approach that has received great attention in the last decades. It aims to restore the dental tissues to its normal biological function and esthetics1. Although several studies have proposed different methods to remineralize enamel lesions, their clinical applications are limited because they require difficult application conditions2–5. Agarose is a natural biocompatible polysaccharide that has been proposed as a matrix for crystal formation6–9. Therefore, the purpose of this study was to investigate the possible biomimetic effect of agarose hydrogel in remineralizing a human demineralized enamel model.
The experiment was done according to the recommendations and approval of the Ethics Committee of the Faculty of Dentistry, Cairo University for working on extracted human teeth (Approval no.18766). Mandibular third molars were collected after being surgically extracted due to impaction with patients' written consents. The roots of 47 tooth were removed using diamond disk (Komet, Rock Hill, USA, K6974) in low speed under water cooling. The crowns were divided mesio-distally and each half was embedded in self-cured acrylic resin (Acrostone Co. Cairo, Egypt, 01CCP50) exposing the uncovered enamel surface. Specimens were examined under stereomicroscope (Leica S8 APO, Leica Microsystems, Switzerland) and specimens with defects (erosions, cracks, visible stains, hypo-calcification) were excluded. Specimens were distributed into three groups (n = 31/ group), according to follow up time (Table 1). Specimens were demineralized using 37% phosphoric acid gel (Super Etch, SDI Limited, Australia, 8100040) for 1 min and rinsed with de-ionized water for 60 seconds.
Agarose (Vivantis, USA, PC0701) hydrogel and phosphate solution were prepared as previously mentioned by Cao et al.,7. Agarose hydrogel was applied on the specimen using acrylic template of 2mm thickness to adjust the thickness of the applied hydrogel. After gelation of the applied hydrogels each specimen was placed into a container filled with 20 mL of phosphate solution and placed in an incubator at 37°C. The phosphate solution and the hydrogel were changed every 24 and 48 h respectively.
Thirteen specimens from each group were mounted on the SEM plate with electro-conductor glue (Electron Microscopy Sciences, PA, USA, 12660) to examine their surfaces. The used SEM Model was Quanta FEG 250 (Field Emission Gun) with accelerating voltage 30 K.V.
SMH of 9 specimens from each group was measured using microhardness tester with Vickers diamond indenter in different areas of the specimens (Vickers diamond, 100 g, 5 s, HMV 2; Shimadzu Corporation, Tokyo, Japan). SMH was measured at baseline, after demineralization and after remineralization.
SR of 9 specimens from each group was measured using digital microscope equipped with a built-in camera (Digital Microscope U500X, Guangdong, China). The microscope is connected to IBM compatible computer. WSxM software (Version 5 develop 4.1, Nanotec, Electronica, SL) was used to analyze the photos and to create a 3D image of the specimen surface. The average SR was estimated using WSxM software and expressed in µm. SR was measured at baseline, after demineralization and after remineralization.
The mean SMH values and the mean SR values were statistically analyzed. One-way ANOVA followed by Tukey's post hoc test were performed to compare remineralizing potential at different time intervals (2,4,6 days). Furthermore, the same tests were used to compare enamel surfaces within the same group. The significant level was set at 0.05. Statistical analysis was performed with SPSS 18.0 for Windows (Statistical Package for Scientific Studies, SPSS, Inc., Chicago, IL, USA).
Sound enamel has a smooth surface with some pits and scratches (Figure 1A, Figure 2A & Figure 3A). After acid etching different etching patterns were seen, most commonly type I and type II with scattered areas of type III (Figure 1B, Figure 2B & Figure 3B). After remineralization, G1 revealed partial occlusion of some rod cores with clearly thickened interprismatic substance (Figure 1C) while in G2 prismatic enamel configurations became hidden by mineral depositions (Figure 2C). G3 revealed a relatively smooth surface with less clearly seen rod ends. Some rods’ peripheries showed complete remineralization while others were still empty (Figure 3C).
The mean SMH values of enamel at different intervals (2,4,6 days) are presented in Table 2. In G1, significant differences were revealed between baseline, demineralized and remineralized enamel (p<0.05) with the highest SMH at baseline. While in G2 and G3, there was a significant difference between the baseline and the demineralized enamel (p<0.05), however there wasn’t a significant difference between baseline and remineralized enamel. Furthermore, there were significant differences among the remineralized enamel surfaces of different groups (p<0.05) with the highest SMH at G3.
Baseline (B), after demineralization (D), after remineralization (R).
Different upper and lower-case superscript letters indicate significant difference between tested groups at P<0.05. Lower case superscript letters are used for comparison within the same row and upper case letters are used for comparison within each column.
The mean SR values of enamel at different intervals (2,4,6 days) are presented in Table 3. In G1, there were significant differences between baseline, demineralized and remineralized enamel (p<0.05) with the highest SR at the demineralized enamel. While in G2 and G3, there was a significant difference between the baseline and the demineralized enamel (p<0.05), however there wasn’t a significant difference between baseline and the remineralized enamel. Furthermore, there were significant differences among the remineralized enamel surfaces of different groups (p<0.05) with the highest SR in G1. The differences in SR at baseline, demineralized enamel and after remineralization in different groups were obvious when inspecting the 3D images in Figure 4.
Baseline (B), after demineralization (D), after remineralization (R).
Different upper and lower-case superscript letters indicate significant difference between tested groups at P<0.05. Lower case superscript letters are used for comparison within the same row and upper case letters are used for comparison within each column.
Biomimetic synthesis of enamel like apatite structures under a physiological condition is an alternative restorative pathway10. Acid etching technique was used to mimic early enamel lesions because of the simplicity and reproducibility of this technique11. SEM results of the present study are in agreement with previous studies6–9. Agarose hydrogel acted as enamel organic matrix to control the size and form of the formed hydroxyapatite crystals through the interaction between hydroxyl group of agarose and calcium. In addition, it acts as a mineral reservoir for continuing remineralization7. The SR analysis results confirmed the SEM results, as the SR values were gradually decreased between different groups which revealed a smoother enamel surface. SMH results are in accordance with previous studies7,9. In the current work, the lower SMH than sound enamel could be attributed to incomplete compaction of formed crystals on enamel surface12.
Agarose hydrogel model have a remineralizing potential to treat early carious lesion. Further studies are required to clarify the stability of agarose hydrogels in clinical application.
Dataset 1: Raw surface microhardness (SMH) and surface roughness (SR) 10.5256/f1000research.16050.d21739813
Dataset 2: Raw scanning electron microscope (SEM) images 10.5256/f1000research.16050.d21739914
Views | Downloads | |
---|---|---|
F1000Research | - | - |
PubMed Central
Data from PMC are received and updated monthly.
|
- | - |
Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Alongside their report, reviewers assign a status to the article:
Invited Reviewers | |||||
---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | |
Version 1 17 Sep 18 |
read | read | read | read | read |
Click here to access the data.
Spreadsheet data files may not format correctly if your computer is using different default delimiters (symbols used to separate values into separate cells) - a spreadsheet created in one region is sometimes misinterpreted by computers in other regions. You can change the regional settings on your computer so that the spreadsheet can be interpreted correctly.
Click here to access the data.
Spreadsheet data files may not format correctly if your computer is using different default delimiters (symbols used to separate values into separate cells) - a spreadsheet created in one region is sometimes misinterpreted by computers in other regions. You can change the regional settings on your computer so that the spreadsheet can be interpreted correctly.
Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list:
Sign up for content alerts and receive a weekly or monthly email with all newly published articles
Already registered? Sign in
The email address should be the one you originally registered with F1000.
You registered with F1000 via Google, so we cannot reset your password.
To sign in, please click here.
If you still need help with your Google account password, please click here.
You registered with F1000 via Facebook, so we cannot reset your password.
To sign in, please click here.
If you still need help with your Facebook account password, please click here.
If your email address is registered with us, we will email you instructions to reset your password.
If you think you should have received this email but it has not arrived, please check your spam filters and/or contact for further assistance.
Comments on this article Comments (1)