Investigating the Cytotoxicity of Dual-Cure Bulk-Fill Resin Materials on L929 Cells
, Nazmiye Donmez 2, Vildan Betul Yenigun 3, Fatemeh Bahadori 4 and Abdurrahim Kocyigit 3
Round 1
Reviewer 1 Report
The manuscript titled "Investigating the Cytotoxicity of Dual-Cure Bulk-Fill Resin Materials on L929 Cells" is well written and adapted to the journal's prosthesis, however, some minor revisions are required :
1)The authors should explain why they chose to place the uncured material directly on the cells and then light cure directly in situ.
In this way, the concentration of monomers that reaches the cells is certainly higher than that released after polymerization.
2)The bibliography should be enriched with more recent works. Most of the works indicated, although pertinent, are prior to 2015.
3) The discussion is too dispersed and deals with aspects not dealt with in the experimental part, it should be reduced and more targeted.
Author Response
Point 1: The authors should explain why they chose to place the uncured material directly on the cells and then light cure directly in situ. In this way, the concentration of monomers that reaches the cells is certainly higher than that released after polymerization.
Response 1: In clinical restoration, the uncured material is polymerized after it is placed in the cavity. In this study, this method was chosen to imitate this situation. The information of our reference in which this method is used is below and it is specified as reference number 11 in the report (Kazak, M.; Donmez, N.; Bahadori, F., Yenigun, V.B.; Kocyigit, A. A preliminary research study on the cytotoxicity of expired and non-expired composite resins: In vitro study. Odovtos 2020, 22(3), 123-134).
Point 2: The bibliography should be enriched with more recent works. Most of the works indicated, although pertinent, are prior to 2015.
Response 2: Since the materials are new, we could not find many up-to-date cytotoxicity articles about these materials. Therefore, we were able to write the discussion part with the content of studies made with similar materials.
Point 3: The discussion is too dispersed and deals with aspects not dealt with in the experimental part, it should be reduced and more targeted.
Response 3: According to your suggestions the discussion rearranged.
Reviewer 2 Report
The manuscript can be accepted
Author Response
Point 1: The manuscript can be accepted
Response 1: Dear referee, thank you for evaluating our manuscript.
Reviewer 3 Report
The manuscript prosthesis-1808430 report about the cytocompatibility of Dual-Cure Bulk-Fill Resin Materials in respect to L929 cells; accordingly, after direct contact and activation the morphology and the metabolic activity of the cells were evaluated and compared to the controls.
Despite the topic of the manuscript can be of interest for the Journal’s readers, I regret to say that the manuscript in the present form can’t be accepted for publication in my opinion.
Here my major comments:
- Most of the results obtained by the Authors are definitely in contradiction with previous literature as also reported in the Discussion section; however, some of the cited literature deal with in vitro 3D models or animal models that in my opinion resemble much more the physiological/pathological scenario thus providing results more consistent than those obtained by the Authors using fibroblasts monolayers. The provided explanation that such differences are due to different experimental conditions is quite obvious, but it is not sufficient to justify the results (especially considering the best experimental conditions of the other works).
- The choice of the L929 cells is questionable; despite the experimental conditions claimed by the ISO standard suggesting for the use of those cells, the biological target of the materials should be taken into account. In fact, fillers are not aimed for soft tissues and so their composition can be well tolerated by the cells in direct contact: for example the hydroxyapatite from the HyperFil and the zinc from the FillUp will be probably well tolerated by osteoblasts or odontoblasts growing in a mineralized environment but not by fibroblasts requiring other conditions. In my opinion this aspect may have penalized the Authors in the results.
- There is a general lack of rationale in the discussion section. The Authors mostly refer to previous works (often not in line with their results) but they do not provide any results/data to justify their own results. Why they obtained such toxicity? Is due to the release of toxic compounds? Or due to unfavorable environment (acidic ph or other9? Those evidences are necessary to provide a proper rationale to the obtained results, the present data are too preliminary to justify the claimed conclusions.
Minor comments:
- Please provide the bar scale in the Images
- Figure 2: I suggest adding the control cells to better appreciate the changes in cells’ morphology in the test groups
Author Response
Point 1: Most of the results obtained by the Authors are definitely in contradiction with previous literature as also reported in the Discussion section; however, some of the cited literature deal with in vitro 3D models or animal models that in my opinion resemble much more the physiological/pathological scenario thus providing results more consistent than those obtained by the Authors using fibroblasts monolayers. The provided explanation that such differences are due to different experimental conditions is quite obvious, but it is not sufficient to justify the results (especially considering the best experimental conditions of the other works).
Response 1: Since there is no previous cytotoxicity study with the material and method used in the study, cytotoxicity studies performed with different test methods using these materials were compared.
Point 2: The choice of the L929 cells is questionable; despite the experimental conditions claimed by the ISO standard suggesting for the use of those cells, the biological target of the materials should be taken into account. In fact, fillers are not aimed for soft tissues and so their composition can be well tolerated by the cells in direct contact: for example the hydroxyapatite from the HyperFil and the zinc from the FillUp will be probably well tolerated by osteoblasts or odontoblasts growing in a mineralized environment but not by fibroblasts requiring other conditions. In my opinion this aspect may have penalized the Authors in the results.
Response 2: In this study, L929 fibroblast cells were used because they are easy to produce and have a low cost of production. It is also stated in ISO standards that these cells can be used. And there are many studies in the literature using these cells.
Point 3: There is a general lack of rationale in the discussion section. The Authors mostly refer to previous works (often not in line with their results) but they do not provide any results/data to justify their own results. Why they obtained such toxicity? Is due to the release of toxic compounds? Or due to unfavorable environment (acidic ph or other9? Those evidences are necessary to provide a proper rationale to the obtained results, the present data are too preliminary to justify the claimed conclusions.
Response 3: In the discussion section, conditions that may cause cytotoxicity are specified in the last sentences of the paragraphs where the results of the study are discussed.
Point 4: Please provide the bar scale in the Images
Response 4: The optical microscope (Novel, China) used in the study does not have the ability to display a bar scale.
Point 5: Figure 2: I suggest adding the control cells to better appreciate the changes in cells’ morphology in the test groups
Response 5: The morphologies of the control groups are shown in Figure 3.
Round 2
Reviewer 3 Report
The Authors provided a revised version of their previous manuscript prosthesis-1808430.
I thank the Authors for considering my revision, but the provided improvements are marginal and most of my comments were not addressed. Therefore, I regret to say that the manuscript is not acceptable for publication.
They did not provide the requested evidences related to materials (for example degradation, release of toxic compounds) or experimental conditions (for example pro-inflammatory environment due to the light activation) that are of crucial importance to justify the observed toxic effects towards cells; therefore is still not clear why they obtained those results beyond the selected experimental conditions.
As minor concerns, the use of L929 is acceptable but it must be supported by a proper rationale (for example due to the possible spread of resins to the surrounding soft tissue) and not by the evidence that they are cells easy to be handled (with “produced” I guess the Authors means “cultivated”) and poor expensive. Finally, the bar scale on images is a basic requirements and it can be easily deduced by free software (such as ImageJ) if the microscope software is not displaying it.
Author Response
Point:
I thank the Authors for considering my revision, but the provided improvements are marginal and most of my comments were not addressed. Therefore, I regret to say that the manuscript is not acceptable for publication.
They did not provide the requested evidences related to materials (for example degradation, release of toxic compounds) or experimental conditions (for example pro-inflammatory environment due to the light activation) that are of crucial importance to justify the observed toxic effects towards cells; therefore is still not clear why they obtained those results beyond the selected experimental conditions.
As minor concerns, the use of L929 is acceptable but it must be supported by a proper rationale (for example due to the possible spread of resins to the surrounding soft tissue) and not by the evidence that they are cells easy to be handled (with “produced” I guess the Authors means “cultivated”) and poor expensive. Finally, the bar scale on images is a basic requirements and it can be easily deduced by free software (such as ImageJ) if the microscope software is not displaying it.
Response:
- Thank you very much for your valuable contribution. According to your advice, the lack of evaluating the degradation of the experimental materials exposed to the medium and the determination of the cytotoxic effects of the test materials not according to the release of toxic compounds but only according to the content were added as the limition of the study at the end of the manuscript. The experimental conditions (for example pro-inflammatory environment due to the light activation) which has crucial importance to justify the observed toxic effects towards cells was mentioned in the manuscript. You can follow the non-inflammatory effect of the light curing device applied directly on the cells in terms of the cell morphology (Figure 3) and cell viability percentage (100%, Figure 4).
- In the below, you can find the reason of choosing L929 Fibroblast cell line in the experiment is details.
According to ISO 10993-5: 2009, various cell culture testing models can evaluate the cytotoxicity of dental restorative materials. These cell culture testing models are direct contact (direct method), indirect contact with a barrier (indirect method), and the extract method. Therefore, in this study one of the cell culture testing models, direct contact test method, was choosen. During cell culture tests, different types of cells can be used. L929 is one of the cells which are generally preferred. L929 mouse fibroblast cell lines are the most widely used cell lines to evaluate the in vitro cytotoxicity of dental materials. From this point of view, this type of cell line was preferred safely in this study.
This paragraph was included in the related part of the manuscript.
Dental materials usually interact with the oral epithelial cells, and they can also interact with the underlying fibroblasts [25]. Besides in gingival connective tissue, fibroblasts are also the predominant cell type in the pulp and might be affected by eluted substances from dental restorative materials if the odontoblastic layer is destroyed [26].
- According to your recommendation the pictures of the control groups and experimental groups that were in the manuscript converted regarding Image J programme (Figure 2, Figure 3).
Round 3
Reviewer 3 Report
The Authors clarified the required aspects and now the work can be accepted for publication.
