Chemical Bonding of Nanorod Hydroxyapatite to the Surface of Calciumfluoroaluminosilicate Particles for Improving the Histocompatibility of Glass Ionomer Cement
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe author presents a study aimed at enhancing the compatibility of glass ionomer cement (GIC) with tooth tissue by chemically modifying its primary component, calciumfluoroaluminosilicate (CFAS), through the incorporation of nanorod hydroxyapatite (nHA). The process involved binding L-glutamic acid to nHA, followed by activation and binding of albumin to create nHA immobilized with albumin. This modified nHA was then used to surface-modify CFAS particles, producing nHA-CFAS powder. The modified powder was mixed with poly(acrylic acid) and UV-cured to form GIC containing nHA-CFAS (GIC-nHA). Fourier transform infrared spectroscopy and scanning electron microscopy were used to confirm the transformation of GIC into GIC-nHA. Cytocompatibility tests with osteoblasts showed that GIC-nHA had superior cell viability and bone formation capabilities compared to the control GIC. The improved histocompatibility is attributed to nHA enhancing the biological activity of osteoblasts, indicating that the surface modification method significantly improves the functional integration of GIC with tooth tissue. However, there are some aspects where revisions are recommended:
1. Provide more detailed mechanistic insights into how the chemical bonding of nHA to CFAS improves histocompatibility and osteoblast activity.
2. How does the presence of albumin as a spacer influence the chemical bonding efficiency and stability of nHA on CFAS particles?
3. What specific molecular pathways are activated in osteoblasts that lead to enhanced cell viability and bone formation on GIC-nHA compared to GIC?
4. Have any in vivo studies been planned to validate the improved histocompatibility and functionality of GIC-nHA observed in vitro?
5. What are the long-term stability and durability of the GIC-nHA in oral environments, considering factors like hydrolysis and wear resistance?
6. What challenges might arise when scaling up the synthesis and application of GIC-nHA for clinical use, and how can they be addressed?
Author Response
Dear Reviewer
Thank you very much for taking the time out of your busy schedule to read our manuscript and provide good comments and guidance.
We did our best to revise and respond to each of the reviewers' comments to raise the quality of the research paper. Corrections to the points reviewers have pointed out are indicated in red in the main text, and the response has been summarized as follows. In accordance with the reviewer's comments, English editing was thoroughly carried out.
We hope that the revised paper meets the comments of the reviewers. We also hope to quickly publish this study in "Coatings".
Reviewer 1
Comments 1-1
- Provide more detailed mechanistic insights into how the chemical bonding of nHA
to CFAS improves histocompatibility and osteoblast activity.
Answer: Thanks for the great point. We added consideration to explain how chemically combining nHA with CFAS improves the activity of osteoblasts. First, we explained a research example showing that HA promotes the activity of osteoblasts, and second, we explained that nHA can be dispersed and combined on the CFAS surface through chemical bonding rather than simple physical mixing.(See page 12, line 380).
Comments 1-2
- How does the presence of albumin as a spacer influence the chemical bonding efficiency and stability of nHA on CFAS particles?
Answer: Thank you for asking this very important and penetrating question. We have supplemented the explanation as follows. (See page 12, line 361)
CFAS are inorganic particles tens of micrometers in size. nHA is an inorganic particle that is several tens of nanometers in size. These two types of high-density inorganic parti-cles easily precipitate in aqueous solution; therefore, no reaction occurs between the two particles. First, we introduced primary amino groups by reacting 3-aminopropyltriethoxysilane on the CFAS surface. In addition, L-glutamic acid was chemically bonded to the nHA surface to introduce an organic spacer on the CFAS surface. By binding L-glutamic acid to nHA, the water dispersibility of nHA-G was slightly im-proved, but the water dispersibility of nHA-G was still low [39]; therefore, no reaction oc-curred between the two particles. After trial and error, albumin was selected as the second linker. When albumin was combined with nHA-G, the water dispersibility of nHA-Alb significantly improved. Thus, the reaction between CFAS-A and nHA-Alb was successful [21].
Comment 1-3
- What specific molecular pathways are activated in osteoblasts that lead to enhanced cell viability and bone formation on GIC-nHA compared to GIC?
Answer: Thank you for your comments regarding the specific molecular pathways related to cell viability and bone formation of GIC-nHA. We attempted to explain the activation of osteoblasts in GIC-nHA by citing previously published research papers. (See page 11, line 351)
Comment 1-4
- Have any in vivo studies been planned to validate the improved histocompatibility and functionality of GIC-nHA observed in vitro?
Answer: We are deeply grateful for your interest in this study. We consider in vivo research to be the final goal of this study. As a follow-up, we would like to share the results of future in vivo research. Thank you.
Comment 1-5
- What are the long-term stability and durability of the GIC-nHA in oral environments, considering factors like hydrolysis and wear resistance?
Answer: Thank you for asking a question based on the assumption that GIC-nHA will actually be used as a tooth filling material. In fact, hydrolysis and wear resistance experiments of GIC-nHA were not conducted in this study. However, we supplemented the discussion related to hydrolysis using references. (See page 12, line 375)
Comment 1-6
- What challenges might arise when scaling up the synthesis and application of GIC-nHA for clinical use, and how can they be addressed?
Answer: Thank you for your comments regarding clinical use. Since nano HA and silicate-based inorganic powder are mass-produced and commercialized, it is believed that it will be possible to manufacture GIC-nHA for clinical use if GIC manufacturers are interested. These details are briefly included in the conclusion. (See page 13, line 428)
Reviewer 2 Report
Comments and Suggestions for AuthorsIn this study, the cytocompatibility and bone formation ability of CFAS-nHA GIC were investigated. Some issues need to be addressed.
Materials and Methods
1. For the sol-gel synthesis of CFAS, did you need to adjust the pH of the reaction?
2. Did you measure the setting time of CFAS-nHA and liquid component of GIC-nHA?
3. Did you examine the CFAS phase pattern by XRD? It is a single phase or multi-phases?
4. Line 126, “… no precipitate was observed with the naked ...”. Did you mean the aggregation of nHA-G nanoparticles?
Results
1. The nano-size of nHA is 30 – 150 nm (Fig.2). What is the particle nano-size of CFAS?
2. Please check the resolution of Figs. 9, 10, 13. It is not clear to see the scale bar.
3. In Fig. 6, there are adsorption peaks for albumin-bound nHA (nHA-Alb) around 2800 – 3000 cm-1.
Discussion
1. Lines 345 – 359, you discussed the implant surface modification with nHA. Why? Do you mean the nHA applications in other areas?
2. Please discuss briefly the CFAS compared with conventional GIC powder, e.g., the setting time is shorter for CFAS, it is more biocompatible, etc.
Other comments
1. Please check the reference format.
2. English editing is recommended.
Comments on the Quality of English Language1. Minor English editing is recommended.
Author Response
Dear Reviewer
Thank you very much for taking the time out of your busy schedule to read our manuscript and provide good comments and guidance.
We did our best to revise and respond to each of the reviewers' comments to raise the quality of the research paper. Corrections to the points reviewers have pointed out are indicated in red in the main text, and the response has been summarized as follows. In accordance with the reviewer's comments, English editing was thoroughly carried out.
We hope that the revised paper meets the comments of the reviewers. We also hope to quickly publish this study in "Coatings".
Reviewer 2
Comment 2-1
- For the sol-gel synthesis of CFAS, did you need to adjust the pH of the reaction?
Answer: Thank you for your sharp point. No, we did not adjust the pH to obtain CFAS material.
Comment 2-2
- Did you measure the setting time of CFAS-nHA and liquid component of GIC-nHA?
Answer: Thank you for your important comments. We measured the setting time of the CFAS-nHA and the liquid component of the GIC-nHA. We added an explanation of the setting time to the text. (See page 5, line 178)
Comment 2-3
- Did you examine the CFAS phase pattern by XRD? It is a single phase or multi-phases?
Answer: Thank you for your deep interest in our research. We examined the CFAS phase pattern and we added information about the XRD pattern to the content. (See page 2, line 82)
Comment 2-4
- Line 126, “… no precipitate was observed with the naked ...”. Did you mean the aggregation of nHA-G nanoparticles?
Answer: Thank you for your comments, we rewrite the sentences. (See page 4, line 136)
Comment 2-5
Results
- The nano-size of nHA is 30 – 150 nm (Fig.2). What is the particle nano-size of CFAS?
Answer: Thank you for your deep interest in our research. As shown in Fig. 1 and line 94, the particle size of CFAS was 20 ~ 50 μm. (See page 3 , Fig. 1)
Comment 2-6
- Please check the resolution of Figs. 9, 10, 13. It is not clear to see the scale bar.
Answer: Thank you for pointing out the resolution of the scale bar. As pointed out, the scale bars in Figs 9, 10, and 13 were rewritten with higher resolution (See Figs 9, 10, 13).
Comment 2-7
- In Fig. 6, there are adsorption peaks for albumin-bound nHA (nHA-Alb) around 2800 – 3000 cm-1.
Answer: We agree with the reviewer's point that nHA-Alb's absorption peak appears at 2880 - 2000 cm-1. In the case of Silicate-A, an absorption peak appears at 2800 - 3000 cm-1 due to the alkyl group of aminopropyltriethoxysilane introduced. These peaks overlap with the absorption peak at 2800 - 3000 cm-1 of nHA-Alb. Therefore, in this study, the range of waver number was limited to 2000 - 400 cm-1 to focus on identifying amide I and II peaks caused by albumin. Thank you again for the reviewer's comments.
Comment 2-8
Discussion
- Lines 345 – 359, you discussed the implant surface modification with nHA. Why? Do you mean the nHA applications in other areas?
Answer: We sincerely thank the reviewers for their comments. The discussion in Lines 345 - 359 introduces applications for different types of ceramic implants, different from the content of this study. We deleted the discussion content in lines 345 - 359 following the reviewer's comments. Thanks for the good point.
Comment 2-9
- Please discuss briefly the CFAS compared with conventional GIC powder, e.g., the setting time is shorter for CFAS, it is more biocompatible, etc.
Answer: We sincerely respect the sharp comments of the reviewers. We have modified and added paragraphs as shown below. Thanks for the good advice. (see page 12, line 411).
The use of CFAS-nHA in GIC-nHA significantly enhanced its biocompatibility com-pared to conventional GIC. The introduction of nanohydroxyapatite (nHA) effectively supported cell compatibility and proliferation, as demonstrated by the MTT assays and von Kossa staining. The chemical bonding between CFAS and nHA improves the water dispersibility and ensures uniform particle distribution, indicating potential improve-ments in the mechanical properties. This study aimed to overcome the long working time and poor physical properties of conventional GIC, as well as the limited use of light-cured GIC due to their lower biocompatibility. Future research should explore the long-term tis-sue compatibility of GIC-nHA in vivo to validate these findings and optimize the proper-ties of GIC by adjusting the nHA concentration and exploring other potential linkers. The development of GIC formulations tailored for specific dental applications may lead to broader clinical use and improved patient outcomes.
Comment 2-10
Other comments
- Please check the reference format.
Answer: Thank you for pointing out how to write references. We rewrote the reference format to unify it.
Comment 2-11
- English editing is recommended.
Answer: In accordance with the reviewer's comments, English editing was thoroughly carried out. Thank you again.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
First of all, please let me congratulate for the nice work.
I would like only to raise a few, minor issues:
In the first sentence it is somewhat misleading to state, that “GIC... being used as a restorative material for primary teeth”. Please rephrase, as GIC is an accepted restorative for permanent teeth also.
Lines 36-37: "there is a need for further improvement in terms of brittleness, abrasion”. Please add, a short part introducing the alternatives available for this improvement, for example coating.
Line 41: "many reports focused on..” but only one reference, which is not a review article. Please either add more references, or rephrase the sentence.
Please add aim, and if possible hypotheses at the end of introduction, I cannot see any there only in the abstract.
Line 71: nHA was synthesized according to the details provided in a previous study
But the reference used is actually not the original one, where it was first described. Please add original reference also.
The sentence in line 71-72: "The mouse pre-osteoblast cells… is incorrect, please rephrase.
Although TEM is a well know microscopy, it shall be added to the text also to explain the abbreviation. (you explain SEM, but not TEM).
Figure 5. seems to be an image from the net or book. Please double check, you have copyright or change it for an own drawing…
Also MTT abbreviation is not explained. Please check all abbreviations.
For the Cytotoxicity part, you only have one reference at the middle of the description. Please clarify if the rest of the method was own protocol, or it is a referenced protocol already. If it is a known protocol, please add references!
Statistical analysis - please add the details of te software used.
It would be good to add details in the discussion, why the whole experiment was done with LC material, and not a pure GIC cement?
Comments on the Quality of English Language
Please correct minor mistakes and double check abbreviations - some are missing!
Author Response
Dear Reviewer
Thank you very much for taking the time out of your busy schedule to read our manuscript and provide good comments and guidance.
We did our best to revise and respond to each of the reviewers' comments to raise the quality of the research paper. Corrections to the points reviewers have pointed out are indicated in red in the main text, and the response has been summarized as follows. In accordance with the reviewer's comments, English editing was thoroughly carried out.
We hope that the revised paper meets the comments of the reviewers. We also hope to quickly publish this study in "Coatings".
Reviewer 3
We sincerely thank you for reading our manuscript and providing appropriate comments and helpful suggestions. The paper was revised to reflect the reviewer's comments, and the contents are as follows.
Comment (1): First of all, please let me congratulate for the nice work.
Answer: Thank you for acknowledging our research.
Comment 3-1
Comment (2): In the first sentence it is somewhat misleading to state, that “GIC... being used as a restorative material for primary teeth”. Please rephrase, as GIC is an accepted restorative for permanent teeth also.
Answer: We completely agree with the reviewer's comments. Thank you for your important point. We have revised the explanation about the scope of GIC usage as follows. (See page 1, line 31)
Glass ionomer cement (GIC) has various applications in dentistry, including use as a bonding agent for restorative materials, liners, fissure sealants, and orthodontic brackets for permanent and primary teeth.
Comment 3-2
Comment (3): Lines 36-37: "there is a need for further improvement in terms of brittleness, abrasion”. Please add, a short part introducing the alternatives available for this improvement, for example coating.
Answer: Thanks for the good point. The sentence was revised by adding references according to the reviewer's comments. (See page 1, line 41)
Comment 3-3
Comment (4): Line 41: "many reports focused on..” but only one reference, which is not a review article. Please either add more references, or rephrase the sentence.
Answer: Thank you for pointing this out. We added reference [8] in line with the reviewer's comments. (See page 2, line 46)
Comment 3-4
Comment (5): Please add aim, and if possible hypotheses at the end of introduction, I cannot see any there only in the abstract.
Answer: Thank you for your positive comments on the abstract. We rewrote the abstract in accordance with the reviewer's comments (See page 1, abstract). The rewritten abstract is below.
Glass ionomer cement (GIC) is composed of anionic polyacrylic acid and a silica-based inorganic powder. GIC is used as a filling material in the decayed cavity of the tooth; therefore, compati-bility with the tooth tissue is essential. In the present study, we aimed to improve the histo-compatibility of GIC by introducing nano-hydroxyapatite(nHA), a component of teeth, into a silica-based inorganic powder. CFAS-nHA was prepared by chemically bonding nanorod hy-droxyapatite (nHA) to the surface of calciumfluoroaluminosilicate (CFAS). The synthesis of CFAS-nHA was confirmed using Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The prepared CFAS-nHA was mixed with polyacrylic acid and cured to prepare GIC containing nHA (GIC-nHA). Cytocompatibility tests of GIC-nHA and GIC were performed using osteoblasts. Osteoblast activity and bone formation ability were superior after GIC-nHA treatment than after control GIC treatment. This enhanced histocompatibility is be-lieved to be due to the improvement of the biological activity of osteoblasts induced by the HA introduced into the GIC. Therefore, to enhance its compatibility with dental tissues, GIC could be manufactured by chemically bonding nHA to the surface of GI inorganic powder.
Comment 3-5
Comment (6): Line 71: nHA was synthesized according to the details provided in a previous study. But the reference used is actually not the original one, where it was first described. Please add original reference also.
Answer: Thank you for your useful point. We have added the original references in line with the reviewer's comments (See page 2, line 76, ref. 10).
Comment 3-6
Comment (7): The sentence in line 71-72: "The mouse pre-osteoblast cells… is incorrect, please rephrase.
Answer: Thank you for pointing this out. The description of the cell lines used in this study has been revised. (See page 2, line 76)
Comment 3-7
Comment (8): Although TEM is a well know microscopy, it shall be added to the text also to explain the abbreviation. (you explain SEM, but not TEM).
Answer: Thank you for pointing out the abbreviations. We rewrote to use the full names of terms like Transmission Electron Microscopy (TEM) and other abbreviations the first time they appear in the text. (See page 3, line 116)
Comment 3-8
Comment (9): Figure 5. seems to be an image from the net or book. Please double check, you have copyright or change it for an own drawing…
Answer: Thank you for pointing out Figure 5. We rewrote Figure 5. Thank you again. (See Figure 5)
Comment 3-9
Comment (10): Also MTT abbreviation is not explained. Please check all abbreviations.
Answer: Thanks for pointing out the abbreviations. In the manuscript, we provided the full names and abbreviations of MTT and TRICT when they were first mentioned. (See page 2, line 89)
Comment 3-10
Comment (11): For the Cytotoxicity part, you only have one reference at the middle of the description. Please clarify if the rest of the method was own protocol, or it is a referenced protocol already. If it is a known protocol, please add references!
Answer: As pointed out by the reviewer, the referenced protocol has been added to section 2.6.3. Cytoxicity. (See page 6, line 224, reference [23])
Comment 3-11
Comment (12): Statistical analysis - please add the details of the software used.
Answer: Thank you for pointing out the detail that we missed. We rewrote the name of software. (see page 7, line 257)
Comment 3-12
Comment (13): It would be good to add details in the discussion, why the whole experiment was done with LC material, and not a pure GIC cement?
Answer: Thank you for pointing out the discussion that explains the entire study. We have added new explanations to clarify why we used the liquid components of LC GIC (see page 11, line 339)
Comment 3-13
Comment (13): Please correct minor mistakes and double check abbreviations - some are missing!
Answer: We thoroughly revised the English text of the manuscript again, and especially described the full name when using the abbreviation for the first time. Thank you again.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors addressed all comments. I have a minor comment.
1. Please check if you added this information "We measured the setting time of the CFAS-nHA and the liquid component of the GIC-nHA. We added an explanation of the setting time to the text. (See page 5, line 178)".
Author Response
Dear Reviewer,
Thank you for your important feedback, and we apologize for the misunderstanding.
In our study, we conducted light curing for 20 seconds as the initial step. After preparing the experimental specimens, we left them for 2 days under the UV clean benches before the cell culture experiment. However, we did not measure the complete setting time of the light-cured glass ionomer cement by evaluating its physical properties over time. This represents a limitation of our study.
According to the literature, light-cured glass ionomer cements undergo a rapid initial set within minutes due to light curing, but the complete chemical curing process can take significantly longer. A study by Sidhu and Nicholson (2016) suggests that while the initial curing phase occurs quickly, full mechanical strength and stability may not be reached until 24 to 48 hours post-light curing.
We have added this information to the materials and methods section and discussed the limitation in the discussion part. (See page 5, line 181, and page 12, line 414).
Thank you for your understanding.
