Biomimetic remineralization of acid etched enamel using agarose hydrogel model

Background: Minimally invasive dentistry aims to prevent progression of caries and treats non-cavitated lesions through non-invasive approaches to preserve the integrity of tooth structure. The aim of this research was to investigate the possible biomimetic effect of agarose hydrogel in remineralizing a human demineralized enamel model. Methods: Mandibular third molars were distributed into three groups (G1, G2 and G3) according to the follow up time (2, 4 and 6 days respectively). Caries like lesion was prepared by applying 37% phosphoric acid gel for 1 minute and then remineralization was performed through applying agarose hydrogel on the demineralized surfaces. The specimens were placed in phosphate solution at 37˚C for 2, 4 & 6 days. Scanning electron microscope (SEM), surface microhardness (SMH) and surface roughness analysis (SR) were performed to assess the regenerated tissue. Results: SEM revealed mineral depositions on the demineralized enamel surface that increased in density by time resulting in a relatively smooth surface in G3. SR and SMH analysis revealed significant differences between the remineralized enamel surfaces of different groups (p< 0.00001) with the highest SR in G1 and the highest SMH in G3. Conclusions: Agarose hydrogel application is a promising approach to treat early carious lesion. Further studies are needed to clarify the stability of agarose hydrogels in clinical application.


Introduction
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 esthetics 1 . Although several studies have proposed different methods to remineralize enamel lesions, their clinical applications are limited because they require difficult application conditions 2-5 . Agarose is a natural biocompatible polysaccharide that has been proposed as a matrix for crystal formation 6-9 . Therefore, the purpose of this study was to investigate the possible biomimetic effect of agarose hydrogel in remineralizing a human demineralized enamel model.

Specimens preparation
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.

Remineralization
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.

Scanning electron microscope (SEM) examination
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.
Surface microhardness (SMH) analysis 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.
Surface roughness (SR) analysis 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.

Statistical analysis
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).

SEM examination
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).

SMH analysis
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.

SR analysis
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.

Discussion
Biomimetic synthesis of enamel like apatite structures under a physiological condition is an alternative restorative pathway 10 . Acid etching technique was used to mimic early enamel lesions because of the simplicity and reproducibility of this technique 11 . SEM results of the present study are in agreement with previous studies 6-9 . 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 remineralization 7 . 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 studies 7,9 . In the current work, the lower SMH than sound enamel could be attributed to incomplete compaction of formed crystals on enamel surface 12 .

Conclusions
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.

Grant information
The author(s) declared that no grants were involved in supporting this work. 1.

2.
3. However, there are a few issues that need to be addressed to improve the quality of the manuscript:

Open Peer Review
Introduction and discussion sections are extremely short and do not cite enough literature on the topic.
Discussion section besides being short is basically a repetition of some results without discussing technical aspects of the study and comparing it to previous similar studies.
The quality of some of the images could be improved.
The statistical analysis is not a true representation of the results and could be inaccurate. The effect of time is neglected in this case. Ideally a two way ANOVA should be used to illustrate the interaction between time and treatment. If no interaction was observed then the single main effects of time and/or treatment could be reported then. Using the post hoc analysis is not the best practice as it neglects the effect of time and will most likely lead to false-positive results.
Some images for illustration of the techniques used in the materials and methods section would be helpful.

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
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Minor comments would help to improve the impact of this paper:

Methods
Specimens preparation: It is better to use premolars extracted for orthodontic treatment, the surface of enamel is usually intact and standard in enamel rods orientation, crystallization and size rather than mandibular third molars with the anatomical varieties, surgical instrumentation and surface irregulars.
Remineralization: (Agarose hydrogel and phosphate solution were prepared as previously mentioned) there is no previously mentioned information about hydrogels and Phosphate Solution Preparation.
Surface roughness (SR) analysis: The average SR was expressed in 1 μm only. how can you be sure that you measure SR at baseline, after demineralization and after remineralization of the same 1 μm every time. It is recommended to use another type of surface roughness analysis with wider surface area at least 25 μm like AFM.

Results
SEM examination: Figures x3000 is not good enough to show baseline, after demineralization and after remineralization changes also x5000 is not so clear please do not minimize them to save the magnification benefits. It is recommended to use 20000 to 50000 to show the crystals, regenerated mineralized tissue and crystal orientation.

Discussion
Needs more details about mechanism of growth of the enamel crystals and the mechanical properties of the regeneration tissue.

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 The current study performed by El Moshy el al, is an interesting one that aims to investigate the possible biomimetic effect of agarose hydrogel in remineralizing a human demineralized enamel model.
Overall, the study is well constructed and presented with the results efficiently supporting the discussion and conclusions.
However some minor points might be helpful in adding to the study: SEM images used were croped from the original images which caused some blurriness in the images. A more detailed statistical analysis needs to be performed on the relatively large amount of data obtained. The discussion section needs to be more detailed as it is too short in comparison to the results obtained.

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 1.

2.
Finally, I preferred to see a figure plate comparing the 3 states together to be easy to compare (i.e.: a plate of Figures 1A, 2A and 3A, a plate of Figures 1B, 2B and 3B, and a plate of Figures 1C, 2C  and 3C).
3. The discussion is too short to clarify the findings of the study.

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

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
No competing interests were disclosed. In the Results: In Figure 2 you mention acid etch type? Where is the reference of this classification and arrows to show the different type? Picture C is hazy, please change it. Figure 3: please put arrows to show us rod complete and empty one in Picture C.
Is the work clearly and accurately presented and does it cite the current literature?

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
No competing interests were disclosed.

Competing Interests:
We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Interesting topic, here are some points to be considered:

Discuss this Article
In the methodology: specimens with defects (erosions, cracks, visible stains, hypo-calcification) were excluded and you mentioned that teeth are surgically extracted third molar, erosion and visible stains detected in erupted teeth not impacted ones. Etching pattern in SEM results with no reference. Discussion is too short.
No competing interests were disclosed.

Competing Interests:
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