Comparison of 2- and 3-Dimensional Cultured Periodontal Ligament Stem Cells; a Pilot Study

Round 1

Reviewer 1 Report

Thank you very much for the interesting manuscript. I have the following remarks.

1. line 56/57: pellet not pallet. (line 277, too)
2. line 64: Similarly, not similarly
3. line 77: 2.1 2D culture, not 2.1.2 D culture
4. line 78: please give more detailed information about the purchased PDL stem cells (if possible age, gender), ethics committee consent (should be mentioned)
5. line 79: missing space between value and unit (37_°C)
6. line 80: please add more information about the growth media. DMEM high or low glucose? FCS addition? Company of FCS, antibiotics, etc.
7. line 80: Why was not always used the same passage number of usual 3?
8. line 121: filtered – please explain more.
9. line 122: missing concentration of Dexa
10. line 157: statistical analysis, method for analysis the morphology? (line 167?).
11. In general, please provide exact P-values. Please add significance asterisk’s in figures.
12. Fig. 1: the image could be bigger in size.
13. Fig. 2: ordinate title could be enhanced by the measured wavelength (in other figures, too)
14. Fig 3: Staining of death cells could be enhanced (red/black)
15. line 195: Stemness testing: FACS analysis and usual gene markers should be added. 3-lineage differentiation is missing to proof stemness.
16. line 211: A Supplement with some additional material to the genes with a changed expression would be very interesting.

Author Response

Reviewer 1

Thank you very much for the interesting manuscript. I have the following remarks.

1. line 56/57: pellet not pallet. (line 277, too)

1. Thank you. We corrected it.

 

1. line 64: Similarly, not similarly

1. Thank you. We corrected it.

 

1. line 77: 2.1 2D culture, not 2.1.2 D culture

1. Thank you. We corrected it.

 

1. line 78: please give more detailed information about the purchased PDL stem cells (if possible age, gender), ethics committee consent (should be mentioned)

1. The company did not provide personal information of the cell donor. In addition, IRB was waived because cells that could not verify personal information were used.

 

1. line 79: missing space between value and unit (37_°C)

1. Thank you. We corrected it.

 

1. line 80: please add more information about the growth media. DMEM high or low glucose? FCS addition? Company of FCS, antibiotics, etc.

1. We had used low glucose DMEM with FBS and antibiotics and corrected the manuscript.

 

1. line 80: Why was not always used the same passage number of usual 3?

1. We tried to use cells no more than passage 4. Cultured cell were used according to the experimental schedule and the required amount of cells.

 

1. line 121: filtered – please explain more.

1. The word ‘filtered’ is used to mean purified. We deleted ‘filtered’ to avoid misunderstanding.

 

1. line 122: missing concentration of Dexa

1. Thank you. We corrected it.

 

1. line 157: statistical analysis, method for analysis the morphology? (line 167?).

1. Since a simple arithmetric mean was used, the p-value was deleted.

 

1. In general, please provide exact P-values. Please add significance asterisk’s in figures.

1. Thank you. We corrected it.

 

 

1. Fig. 1: the image could be bigger in size.

1. Thank you. We will ask it to the editorial board.

 

1. Fig. 2: ordinate title could be enhanced by the measured wavelength (in other figures, too)

1. Thank you. We corrected it.

 

 

1. Fig 3: Staining of death cells could be enhanced (red/black)

1. Thank you. We corrected it.

 

1. line 195: Stemness testing: FACS analysis and usual gene markers should be added. 3-lineage differentiation is missing to proof stemness.

1. Your comment is right. We tried FACS analysis, but failed for the big size of 3D cultured cells. This is the weak point of this research and why we call this paper a pilot study. We will find out how to do FACS analysis with 3D cultured cells in future.

 

1. line 211: A Supplement with some additional material to the genes with a changed expression would be very interesting.

1. We added it as a supplemetal file.

 

Reviewer 2 Report

In the manuscript entitled: “Comparison of 2- and 3-dimensional cultured periodontal ligament stem cells; a pilot study” the authors investigated compared the characteristics of periodontal ligament stem cells (PDLSCs) cultured using 3-dimensional (3D) versus conventional 2-dimensional (2D) methods.

The authors concluded that the viability of the 3D-cultured cells was decreased, but they showed superior osteogenic differentiation compared to 2D-cultured cells. Within the limitations of this study, the results demonstrate that the structure and function of PDLSCs are influenced by the cell culture method.

 

Major comments:

In general, the idea and innovation of this study, regards the analysis of periodontal stem cells is interesting, because the role of periodontitis and systemic diseases released during wound healing are validated, but further studies on this topic could be an innovative issue in this field could be open a creative matter of debate in literature by adding new information. Moreover, there are few reports in the literature that studied this exciting topic with this kind of study design.

The study was well conducted by the authors; However, there are some concerns to revise that are described below.

The introduction section and the manuscript resume the existing knowledge regarding the important factor linked with periodontal cells.

However, as the importance of the topic, the reviewer strongly recommends, before a further re-evaluation of the manuscript, to update the literature through read, by must discuss and cites in the references with great attention all of those recent interesting articles, that helps the authors to better introduce and discuss the aim of the study in light of stem cells and others mediators released following periodontitis: 1) Isola G, Lo Giudice A, Polizzi A, Alibrandi A, Murabito P, Indelicato F. Identification of the different salivary Interleukin-6 profiles in patients with periodontitis: A cross-sectional study. Arch Oral Biol. 2020 Nov 30;122:104997. doi: 10.1016/j.archoralbio.2020.104997. 2) Isola G, Polizzi A, Alibrandi A, Williams RC, Leonardi R. Independent impact of periodontitis and cardiovascular disease on elevated soluble urokinase-type plasminogen activator receptor (suPAR) levels. J Periodontol. 2020 Oct 22. doi: 10.1002/JPER.20-0242. 3) Isola G, Polizzi A, Patini R, Ferlito S, Alibrandi A, Palazzo G. Association among serum and salivary A. actinomycetemcomitans specific immunoglobulin antibodies and periodontitis. BMC Oral Health. 2020 Oct 15;20(1):283. doi: 10.1186/s12903-020-01258-5.

The authors should be better specified at the end of the introduction section, the rationale of the study. In the materials and methods, should better clarify the 2d and 3d morphology, and the LIVE/DEAD assay.

The conclusion should be added with the main findings of the study and reinforce in light of the future directions.

In conclusion, I am sure that the authors are excellent clinicians who achieve very nice results with their adopted protocol. However, this study, in my view, does not in its current form satisfy a very high scientific requirement for publication in this journal and requests a revision before a further re-evaluation of the manuscript.

 

Minor Comments:

 

 

Introduction:

• Please refer to major comments

 

Discussion

• Please add a specific sentence that clarifies the results obtained in the first part of the discussion

Author Response

Reviewer 2

In the manuscript entitled: “Comparison of 2- and 3-dimensional cultured periodontal ligament stem cells; a pilot study” the authors investigated compared the characteristics of periodontal ligament stem cells (PDLSCs) cultured using 3-dimensional (3D) versus conventional 2-dimensional (2D) methods.

The authors concluded that the viability of the 3D-cultured cells was decreased, but they showed superior osteogenic differentiation compared to 2D-cultured cells. Within the limitations of this study, the results demonstrate that the structure and function of PDLSCs are influenced by the cell culture method.

 

Major comments:

In general, the idea and innovation of this study, regards the analysis of periodontal stem cells is interesting, because the role of periodontitis and systemic diseases released during wound healing are validated, but further studies on this topic could be an innovative issue in this field could be open a creative matter of debate in literature by adding new information. Moreover, there are few reports in the literature that studied this exciting topic with this kind of study design.

The study was well conducted by the authors; However, there are some concerns to revise that are described below.

The introduction section and the manuscript resume the existing knowledge regarding the important factor linked with periodontal cells.

However, as the importance of the topic, the reviewer strongly recommends, before a further re-evaluation of the manuscript, to update the literature through read, by must discuss and cites in the references with great attention all of those recent interesting articles, that helps the authors to better introduce and discuss the aim of the study in light of stem cells and others mediators released following periodontitis: 1) Isola G, Lo Giudice A, Polizzi A, Alibrandi A, Murabito P, Indelicato F. Identification of the different salivary Interleukin-6 profiles in patients with periodontitis: A cross-sectional study. Arch Oral Biol. 2020 Nov 30;122:104997. doi: 10.1016/j.archoralbio.2020.104997. 2) Isola G, Polizzi A, Alibrandi A, Williams RC, Leonardi R. Independent impact of periodontitis and cardiovascular disease on elevated soluble urokinase-type plasminogen activator receptor (suPAR) levels. J Periodontol. 2020 Oct 22. doi: 10.1002/JPER.20-0242. 3) Isola G, Polizzi A, Patini R, Ferlito S, Alibrandi A, Palazzo G. Association among serum and salivary A. actinomycetemcomitans specific immunoglobulin antibodies and periodontitis. BMC Oral Health. 2020 Oct 15;20(1):283. doi: 10.1186/s12903-020-01258-5. The authors should be better specified at the end of the introduction section, the rationale of the study.

• Thank you. We cited the references.

In the materials and methods, should better clarify the 2d and 3d morphology, and the LIVE/DEAD assay.

• Thank you. We clarified the figures.

The conclusion should be added with the main findings of the study and reinforce in light of the future directions.

• Thank you. We corrected it.

In conclusion, I am sure that the authors are excellent clinicians who achieve very nice results with their adopted protocol. However, this study, in my view, does not in its current form satisfy a very high scientific requirement for publication in this journal and requests a revision before a further re-evaluation of the manuscript.

• Thank you. As you say, we are clinicians rather than researchers. With your guidance, we can make a better manuscript.

 

 

Minor Comments:

 

 

Introduction:

• Please refer to major comments
  • Thank you. We corrected it.

 

Discussion

• Please add a specific sentence that clarifies the results obtained in the first part of the discussion
  • Thank you. We corrected it.

 

 

Reviewer 3 Report

The authors present an interesting experimental pilot study where they compared the morphology, viability, stemness, and functional gene expression of Periodontal ligament stem cells (PDLSCs) cultured using either 3D or conventional 2D methods. The experiment was adequately performed following acceptable protocols and provide relevant insight on how cells of same origin can have different functions and characteristics depending on the culture method, at the same time the respective limitations are well acknowledged. Some minor concerns are presented following:

Introduction 

Page 2 – Line 54: Scaffolds have limitations that can negatively affect cell stability and cell behavior during incubation.

What are these limitations? Add them for more complete comprehension.

 

Page 2 – Line 64: “Simiarly” is a spelling error? It should be "Similarly".

 

M&M

“2.1.2. D and 3D culture” typo error, should be “2.1. 2D and 3D culture..”

 

Results

In table 1, it is missing a more detailed description of related function for MMP1 and MMP10 like there is in the description for other genes. Example, MMPs are related to degradation of both matrix and non-matrix proteins.Similarly, it is lacking further description for SMOC1.

 

Discussion

Page 10 – Line 294: The sentence: “However, com-pared to the control group, ARS staining showed that the 3D-cultured PDLSCs had significantly higher absorbance than the 2D-cultured PDLSCs (20.8 and 1.6 times higher, respectively). This suggests that more mineralized tissue is produced by the 3D-cultured PDLSCs compared to their 2D counterparts [17]” seems misleading.

Although there was a higher difference in the 3D cultured group (osteo) compared to its respective control (basal media) than in the 2D cultured group (osteo) compared to its respective control (basal media), higher absorbance for alizarin red S staining of on day 14 was presented in the 2D osteo group. Please review this result and discussion.

Author Response

 

Reviewer 3

The authors present an interesting experimental pilot study where they compared the morphology, viability, stemness, and functional gene expression of Periodontal ligament stem cells (PDLSCs) cultured using either 3D or conventional 2D methods. The experiment was adequately performed following acceptable protocols and provide relevant insight on how cells of same origin can have different functions and characteristics depending on the culture method, at the same time the respective limitations are well acknowledged. Some minor concerns are presented following:

Introduction 

Page 2 – Line 54: Scaffolds have limitations that can negatively affect cell stability and cell behavior during incubation.

What are these limitations? Add them for more complete comprehension.

• Thank you. Scaffolds are very good methods. In the case of hydrogel among the scaffolding techniques, there is also a problem that the hydrogel collapses during the media replacement process. We added it in the manuscript.

 

Page 2 – Line 64: “Simiarly” is a spelling error? It should be "Similarly".

• Thank you. We corrected it.

 

 

M&M

“2.1.2. D and 3D culture” typo error, should be “2.1. 2D and 3D culture..”

• Thank you. We corrected it.

 

Results

In table 1, it is missing a more detailed description of related function for MMP1 and MMP10 like there is in the description for other genes. Example, MMPs are related to degradation of both matrix and non-matrix proteins. Similarly, it is lacking further description for SMOC1.

• Yes, you are right. This result was obtained through a gene analysis program, and the program briefly classified gene functions. So, we wrote more about the specific gene, such as MMPS and SMOC1, in the discussion.

 

Discussion

Page 10 – Line 294: The sentence: “However, com-pared to the control group, ARS staining showed that the 3D-cultured PDLSCs had significantly higher absorbance than the 2D-cultured PDLSCs (20.8 and 1.6 times higher, respectively). This suggests that more mineralized tissue is produced by the 3D-cultured PDLSCs compared to their 2D counterparts [17]” seems misleading.

Although there was a higher difference in the 3D cultured group (osteo) compared to its respective control (basal media) than in the 2D cultured group (osteo) compared to its respective control (basal media), higher absorbance for alizarin red S staining of on day 14 was presented in the 2D osteo group. Please review this result and discussion.

• I understand your point, because we had discussed about it. Our conclusion is that it is difficult to compare 3D culture and 2D culture on the same line, and the comparison within the same culture methods is more valid. This is because the number of cells used for 3D and 2D culture is not uniform, and the measurement method is used for 2D until now. Therefore, in the discussion, we stated that the research on 3D measurement method is needed in the future.

 

Reviewer 4 Report

To author:

This manuscript reported the pilot study for the culture methods of periodontal ligament stem cell. Reviewer understand the need for research. Although experiments were logically conducted, the authors should address the following issues:

 

1. [Introduction] Reviewer did not understand why the authors compared 2d and 3d culture of PDLSC instead of MSC, even though there are already many reports on MSC. The author needs to articulate the importance and significance of this study.
2. [Materials and Method] There is no explanation such as the number of cells in 2D culture. Are the number of cells per unit area of 2d culture the same as 3d culture?
3. [line 77] There is a typo.
4. [2.4 Viability analyses…] Can cells in the center of spheroids be measured with CCK-8 or live/dead?
5. [Figure 2] The order of the figure of 2d and figure of 3d should be reversed.
6. [Figure 3] The length of the scale bar should be indicated.
7. [line183-4] The authors state that the cells in the center of the spheroid are dead. However, few cells stained red appear in the center. In addition, the green-stained area and the red-stained area are observed to almost overlap. Please add an explanation about this.
8. [line 197] What is the result of "p <0.05" compared?
9. [Figure 4] The length of the scale bar should be indicated.

Author Response

 

 

Reviewer 4

To author:

This manuscript reported the pilot study for the culture methods of periodontal ligament stem cell. Reviewer understand the need for research. Although experiments were logically conducted, the authors should address the following issues:

 

1. [Introduction] Reviewer did not understand why the authors compared 2d and 3d culture of PDLSC instead of MSC, even though there are already many reports on MSC. The author needs to articulate the importance and significance of this study.
   • Your point is very valid. However, we are clinical dentists rather than researchers. Therefore, we were trying to do research using the periodontal ligament. In some studies, the periodontal ligament stem cell is referred to as MSC, but we specified as a periodontal ligament stem cell in the sense that it was collected from the periodontal ligament.

 

2. [Materials and Method] There is no explanation such as the number of cells in 2D culture. Are the number of cells per unit area of 2d culture the same as 3d culture?
   • Thank you for your comment. We had thought a lot about this. This is because the number of cells can have a great influence on the results of the experiment. Initially, the amount of cells was the same. However, the 3D culture did not work well. The experiment used in this paper used the amount of cells recommended for each cell culture method. During this research, we realized that it was difficult to apply the 2D culture cell method and the measurement method equally to 3D culture, and further research on this was necessary.

 

 

3. [line 77] There is a typo.
   • We are very sorry about that. We did the language revision again.

 

4. [2.4 Viability analyses…] Can cells in the center of spheroids be measured with CCK-8 or live/dead?
   • We thought it was hard to figure out the center of spheroids with CCK-8 or live/dead well, but the results reflected the condition of the spheroids. As mentioned above, proper methods for measuring the 3D culture is needed.

 

5. [Figure 2] The order of the figure of 2d and figure of 3d should be reversed.
   • Thank you. We corrected it.

 

6. [Figure 3] The length of the scale bar should be indicated.
   • Thank you. We corrected it.

 

7. [line183-4] The authors state that the cells in the center of the spheroid are dead. However, few cells stained red appear in the center. In addition, the green-stained area and the red-stained area are observed to almost overlap. Please add an explanation about this.
   • Your point is very sharp. However, if you look closely, the border of green light is bright. The number of cells in this border is less than the number of cells that survived, so if inner cell was alive, it would be brighter than this outside. So we thought more of inner cell was dead. Even in previous studies, there is a result of necrosis from the center due to lack of nutrition and oxygen.

 

8. [line 197] What is the result of "p <0.05" compared?
   • That meant that the absorbance was higher in the osteo group than in the control group in 3D culture (fig. 5). We deleted ‘p <0.05’ in the manuscript for clear understanding.

 

9. [Figure 4] The length of the scale bar should be indicated.
   • Thank you. We added the scale bar.

 

 

 

Round 2

Reviewer 1 Report

Thank you very much for the corrections. Some of my points were not corrected in the new manuscript, please revise:

1.) Line 88 – 90: Missing Space between value and unit (37 °C), missing FBS concentration, manufacturer, gentamicin concentration.

2.) Exact p-values are missing.

3.) Add wavelength in the ordinate of graphs.

The missing proof of stemness is a major problem. Three-linage differentiation is one of the most important steps to proof stemness character of the obtained cells (FACS enhances this, too). This could be done with the cells directly after isolation (buying) and propagation in a monolayer. Maybe the company has done this already and gives a guarantee on this. It was not possible for me to find the company which is selling the cells.

Author Response

Thank you very much for the corrections. Some of my points were not corrected in the new manuscript, please revise:

1.) Line 88 – 90: Missing Space between value and unit (37 °C), missing FBS concentration, manufacturer, gentamicin concentration.

2.) Exact p-values are missing.

3.) Add wavelength in the ordinate of graphs.

=> We were very sorry for this missing and corrected all you pointed.

 

The missing proof of stemness is a major problem. Three-linage differentiation is one of the most important steps to proof stemness character of the obtained cells (FACS enhances this, too). This could be done with the cells directly after isolation (buying) and propagation in a monolayer. Maybe the company has done this already and gives a guarantee on this. It was not possible for me to find the company which is selling the cells.

• Thank you for pointing out the essential part. We requested the manufacturer to receive the product manual. Stemness was confirmed by flow cytometry.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have well addressed to all reviewer's comments. I suggest the acceptance of this interesting manuscript.

Author Response

Reviewer 2

The authors have well addressed to all reviewer's comments. I suggest the acceptance of this interesting manuscript.

• Thank you for your kind comment.

Reviewer 4 Report

The reviewer has a different perception of the authors' comments (No. 7; Figure 3, line203). A small number of cells, both red-stained dead cells and green-stained live cells, are observed in the center compared to the outer edge. Reviewer thought that this mean that there are very few cells in the center, not mean that the cells in the center are dead. As the author commented, there is a lack of nutrition and oxygen in the center. Reviewer thought that the cells in the center move outward, causing cavitation. Reviewer do not think the author can conclude that the cells in the center are dead (line203).

Author Response

The reviewer has a different perception of the authors' comments (No. 7; Figure 3, line203). A small number of cells, both red-stained dead cells and green-stained live cells, are observed in the center compared to the outer edge. Reviewer thought that this mean that there are very few cells in the center, not mean that the cells in the center are dead. As the author commented, there is a lack of nutrition and oxygen in the center. Reviewer thought that the cells in the center move outward, causing cavitation. Reviewer do not think the author can conclude that the cells in the center are dead (line203).

 

Thank you for your comment. We corrected the manuscript reflecting your comment.