Angiogenic Effects of Secreted Factors from Periodontal Ligament Stem Cells

Round 1

Reviewer 1 Report

The present manuscript aims to investigate the angiogenic effects of secreted factors from periodontal ligament stem cells. The Introduction is adequate and presents the background of the study, as well as recent progress in the field. Materials and methods are well presented and executed, the study design is very good and capable of yielding valuable results. Results are very clear and persuasive, well presented and accompanied by relevant tables and figures. Discussions are adequate, they present the main outcomes of the study and compare them to other related papers, also pointing out the importance of this study and the added value that it brings. Conclusion is in accordance with the results. The present manuscript is well written overall, the English language is suitable, and the amount of originality as well as added value is sufficient to support its publication. I recommend to accept the manuscript as it is.

Author Response

Reviewer 1

Thank you very much for reviewing our manuscript. We are very delighted to know that you recommended to accept our manuscript in Dentistry Journal.

Reviewer 2 Report

Material and method section must be improved.

How many volunteers were included? Average age? Hoy many men/women? Also, authors must explain which teeth were used in the study (molars, incisors...): % of each one? Have the samples been differentiated according to the degree of periodontal disease? If not, justify why authors consider that it does not alter the results of the study. If yes, explain the differences that could be considered and how this bias has been corrected. Authors must present the sample size. 

About the statistical analysis, what is the significance? and the confidence interval? Was the sample size determined before starting the study? Justify the answer. 

Line 133, reference is noted as author-year. Please, review the manuscript because sometimes double citation appears. Other new references must be included in the manuscript. 

Why was the ELISA technique chosen to measure growth factors? Justify its use against other alternatives.

What implications does it have at the clinical level?

Author Response

Reviewer 2

We would like to thank you for your insightful comments. We revised our manuscript in accordance with the precious inputs from the reviewer. Revisions in the revised manuscript are shown in red font. Our point-by-point responses to reviewer’s comments are shown below.

COMMENT: Material and method section must be improved. How many volunteers were included? Average age? Hoy many men/women?

REPLY: Thank you very much for your valuable comment about volunteers participated in this study. We apologize for the lacking of important information, regarding sample collection in the original manuscript. We used total 8 premolars from 8 systemically healthy volunteers (14-28 years of age, average 18 years old, 1 male and 7 female). All these premolars were free from any symptoms of periodontal disease. We added above information to the Materials and Methods section in revised manuscript. (Line 89 to 91)

COMMENT: Also, authors must explain which teeth were used in the study (molars, incisors...): % of each one? Have the samples been differentiated according to the degree of periodontal disease? If not, justify why authors consider that it does not alter the results of the study. If yes, explain the differences that could be considered and how this bias has been corrected.

REPLY: As the reviewer commented, the characteristics of PDLSCs are affected by the periodontal conditions of the extracted tooth. Previously, it has been demonstrated that inflammation around donor tooth and aging of the donors affected the stem cell characteristics of obtained PDLSCs (Liu et al., 2011 and Zhang et al., 2012). We completely agree with the reviewer on this point.

We collected PDLSCs from young and healthy donors and used them at lower passage numbers to minimize the variation of characteristics of primary cells. Considering the differences among lines of primary cells, we performed similar experiments and confirmed the same tendency of the results using different PDLSC lines to prove the reproducibility of obtained results. Additionally, the multiple differentiation capacity of PDLSCs, which is the one of the most important characteristics of stem cells, were checked prior to experimental use in this study. We added this information, regarding how we considered the differences among primary cell lines and its influence on experimental data. (Line 89 to 101) We appreciate this important query by the reviewer.

Reference

Liu, N.; Shi, S.; Deng, M.; Tang, L.; Zhang, G.; Liu, N.; Ding, B.; Liu, W.; Liu, Y.; Shi, H. The effect of aging on the pluripotential capacity and regenerative potential of human periodontal ligament stem cells. Biomaterials. 2012, 33, 6974–6986. doi: 10.1016/j.biomaterials.2012.06.032

Zhang. J.; An, Y.; Gao, L.N.; Zhang, Y.J.; Jin, Y.; Chen, F.M. High levels of β-catenin signaling reduce osteogenic differentiation of stem cells in inflammatory microenvironments through inhibition of the noncanonical Wnt pathway. J Bone Miner Res. 2011 Sep;26(9):2082-95. doi: 10.1002/jbmr.440.

COMMENT: Authors must present the sample size.

REPLY: We appreciate the indication by the reviewer about sample size of each experiment in this study. We added the number of samples in captions of each figure in revised manuscript. Thank you for your important comment.

COMMENT: About the statistical analysis, what is the significance? and the confidence interval? Was the sample size determined before starting the study? Justify the answer.

REPLY: Thank you for your comments regarding the statistical analysis used in this study and apologize for the lacking of the information in the original manuscript.

As for significance, “significance” means that difference between hypothesis and experimental finding is not derived from error. We used “significance” when the null hypothesis that two experimental groups are not different is rejected as a result of statistical analysis. Confidence interval is an estimated range of values which is likely to include an unknown parameter. We chose confidential level as 95% in this study. This means the population mean falls in this range 95% of times when we collect samples from statistical population and estimate confidence interval. We did not perform sample size estimation before the start of this study. Instead, we confirmed the reproducibility of results by producing both biological and technical replicates. We agree with the reviewer that sample size estimation gives adequate number of samples based on the statistical analysis, however, many in vitro studies have been performed using biological and technical replicates for the confirmation of reproducibility. We presented the representative data from the same experiments performed more than three times in each figure. We believe that our data is reliable based on this conventional method. According to the precious input from the reviewer, we added sentences regarding sample size and data presentation in each caption. Thank you very much for your valuable comments.

COMMENT: Line 133, reference is noted as author-year. Please, review the manuscript because sometimes double citation appears. Other new references must be included in the manuscript.

REPLY: Thank you very much for your indication. References were not properly described as the reviewer pointed out in Line 133. We also found the same mistake in Line 125 and corrected both of them. We again checked the presentation of references including the order and double citation throughout the revised manuscript.

COMMENT: Why was the ELISA technique chosen to measure growth factors? Justify its use against other alternatives.

REPLY: We used ELISA method to measure VEGF amount in conditioned medium from PDLSCs and fibroblasts. In general, western blot, dot blot, and ELISA are used to quantify the protein amount in liquid samples. ELISA is accepted as the most sensitive method among them. The method is also useful for protein measurement in liquid samples. These are the reasons why we used ELISA for the comparison of VEGF amount between conditioned medium from PDLSCs and fibroblasts. We added this explanation in revised manuscript. (Line 139 to 140) We would like to appreciate the valuable comment from the reviewer.

COMMENT: What implications does it have at the clinical level?

REPLY: We appreciate the precious query by the reviewer about the clinical relevance of new findings of this study. This study demonstrated that PDLSC contained greater amount of VEGF than Fibroblast-CM and enhanced angiogenesis in network formation assay. These results suggested that PDLSC-CM may enhance the angiogenesis when transplanted into periodontal defects and this may explain the enhanced periodontal regeneration by PDLSC-CM. Additionally, clinical trials for periodontal regeneration by PDLSC transplantation is now underway and new findings of this study may provide the information regarding the regenerative mechanism by PDLSC transplantation. Responding to the precious comment from the reviewer, we added new sentence in the Discussion section about the clinical importance of this study. (Line 289 to 291)

Round 2

Reviewer 2 Report

The authors have made improvements to the manuscript, but I consider it isnecessary to clarify several things. Thank you very much for your effort. 

First, statistical significance is a number expressed as a percentage. I suggest that when in doubt consult an experienced health science statistician for guidance. This is determined, among others, by the sample size, so being the sample of only 8 premolars, I consider essential to know the statistical power of the study in order to assess it correctly. In any experimental study, the first step should be to determine the sample size (this will give an idea of the real value of our study)

On the other hand, I miss more current bibliographic references.

Kind regards

Author Response

Reviewer 2

Thank you very much for reviewing our manuscript. We re-revised our manuscript in accordance with the precious inputs from the reviewer. Our point-by-point responses to reviewer’s comments are shown below.

COMMENT: First, statistical significance is a number expressed as a percentage.

I suggest that when in doubt consult an experienced health science statistician for guidance. This is

determined, among others, by the sample size, so being the sample of only 8 premolars, I consider essential to know the statistical power of the study in order to assess it correctly. In any experimental study, the first step should be to determine the sample size (this will give an idea of the real value of our study)

REPLY: Thank you very much for your valuable comment. We understood your query with regards to the meaning of statistical significance. We also consulted an experienced health science statistician at our university for guidance according to your suggestion, and have revised our manuscript as follows.

As you pointed, the first step of experimental design is to determine the sample size. It was expected that each PDLSC line demonstrated some variations in their characteristics, even though PDLSCs were isolated through same procedures. Therefore, we needed to confirm the reproducibility of the effect of conditioned medium from multiple PDLSCs lines. We decided to determine the sample of 3 premolars for the following reason. When we started this project, we searched for previous studies in which similar experiments were performed, and found that the following reliable papers used 3 extracted teeth for the examination of the effect of PDLSCs in vitro.

1. Wang, Z.S.; Feng, Z.H.; Wu, G.F;, Bai, S.Z,; Dong, Y.; Chen, F.M.; Zhao, Y.M. The use of platelet-rich fibrin combined with periodontal ligament and jaw bone mesenchymal stem cell sheets for periodontal tissue engineering. Sci Rep. 2016, 6, 28126. doi: 10.1038/srep28126.
2. Huang, L.; Li, Z.; Tian, H.; Wang, W.; Cui, D.; Zhou, Z.; Chen, X.; Cheung, H.S,; Xu, G.T.; Chen, Y. Adult human periodontal ligament-derived stem cells delay retinal degeneration and maintain retinal function in RCS rats. Stem Cell Res Ther. 2017, 8, 290. doi: 10.1186/s13287-017-0731-y.
3. Albuquerque-Souza, E.; Schulte, F.; Chen, T.; Hardt, M.; Hasturk, H.; Van Dyke, T.E.; Holzhausen, M.; Kantarci, A. Maresin-1 and Resolvin E1 Promote Regenerative Properties of Periodontal Ligament Stem Cells Under Inflammatory Conditions. Front Immunol. 2020, 11, 585530. doi: 10.3389/fimmu.2020.585530. eCollection 2020.

Upon this revision, we asked the experienced health science statistician about this experimental design and had a reply that the sample size of 3 premolars is adequate. When we conducted the experiments, we used PDLSC-CMs taken from three PDLSC lines. However, due to the limitation of collecting of clinical samples and limited passage number of PDLSC for research use, considering cell aging during cell culture, PDLSCs from total of 8 premolars were used in this study. We confirmed the reproducibility of obtained data using CMs form several PDLSC lines.

              As the reviewer pointed out, we understand that the higher the number of PDLSC is needed for the higher the power of statistical analysis. However, considering the previous studies and limitation of stem cells from clinical samples, we greatly appreciate it if the reviewer kindly understands our method and reliability of results in this study.

              For the detailed explanation of sample size, we added above information in the Materials and Methods section in the revised manuscript. (Line 90- 92) and added the three papers in Reference. We again appreciate your precious comment which made our manuscript improved.

Comment：On the other hand, I miss more current bibliographic references.

REPLY: Thank you for your comment. According to the reviewer’s comment, we checked our reference list and changed two references (Reference #1 and #3) to recent publications as listed below. Reference #7, #8, and #9 were also old papers. However, we would like to keep them in revised manuscript, because they are important classic papers, regarding the discovery of mesenchymal stem cells from bone marrow. We hope the reviewer to understand our decision. Thank you for your precious suggestion.

1.Loos. B.-G.; Van Dyke T.-E.The role of inflammation and genetics in periodontal disease. Periodontol 2000. 2020 ,83, 26-39.doi: 10.1111/prd.12297.

3.Hajishengallis.G.; Chavakis. T.; Lambris J.-D. Current understanding of periodontal disease pathogenesis and targets for host‐modulation therapy. Periodontol 2000.  2020,81, 14-34. doi: 10.1111/prd.12331.

Round 3

Reviewer 2 Report

Thank you very much for your changes.