Enhanced Osteogenic Differentiation Based on Combining Pulp Stem Cells with Ultralong Hydroxyapatite Nanowires and Cellulose Fibers
, Chenglong Yang 1, Lifeng Yuan 1, Changning Hao 2, Junli Duan 2,* and Yongjian Lu 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsHello, dear authors. Your article is an interesting research. I was glad to read it. However, I have a number of comments.
232-233. Alizarin red staining was used to test mineralized tissue formation of hDPSCs in the control group (F) and experimental group (G) on days 14 post-osteoinduction
Cells in the control group were not stained with alizarin red? A drawing of the control group stained with alizarin red should be provided. Fig.F and G were taken in different modes.
255-257After being cultured for 1, 4, and 24h, compared with the control group, the HAPNW/CF group showed significantly increased cell density, better cell spreading morphology
In 1 hour, DPSC cells do not have time to adhere to the surface, which corresponds to your illustration. Why then did you provide this data? I recommend explain its necessity.
324-326 In vivo studies conducted on rat cranial defects have demonstrated that the utilization of HAPNW/CF paper significantlyenhances new bone formation compared to the control group.
342-345 Clinical, X-ray, and histological tests conducted 3 months later indicated complete re-generation of the bone tissue, and 1 year later, the regenerated bone tissue function reached its best, suggesting that dental pulp stem cells can fully restore human mandibular bone defects.
These are data from in vivo research. Your work did not conduct such research. Why are these data presented in the discussion? 348-349 Additionally, due to the relatively rough surface of the HAPNW/CF biomaterial scaffold, it was beneficial for cell adhesion and proliferation.You have not provided any rough surface data.
352-355 This was because the HAPNW/CF biomaterials provided more attachment sites for hDPSCs due to their higher specific surface area, which further promoted early adhesion of hDPSCs on the composite membrane 354 surface.
The specific surface area data you refer to in your discussion is not provided.
Sincerely
Author Response
Dear reviewers:
We are truly grateful to you for your critical comments and constructive suggestions. Based on these comments and suggestions, we have made careful modifications to the original manuscript and made elaborate responses to all the comments. We hope that the revised manuscript will receive a favorable decision. Below you will find our point-by-point responses to the reviewers' comments and questions.
Comment 1: 232-233. Alizarin red staining was used to test mineralized tissue formation of hDPSCs in the control group (F) and experimental group (G) on days 14 post-osteoinduction.
Cells in the control group were not stained with alizarin red? A drawing of the control group stained with alizarin red should be provided. Fig.F and G were taken in different modes.
Response 1: Thank you very much for your comment. Based on your comment, we have replaced Figure 2F, a drawing of the control group stained with alizarin red. (Page 6, Line 233-241, Figure 2)
Comment 2: 255-257After being cultured for 1, 4, and 24h, compared with the control group, the HAPNW/CF group showed significantly increased cell density, better cell spreading morphology.
In 1 hour, DPSC cells do not have time to adhere to the surface, which corresponds to your illustration. Why then did you provide this data? I recommend explain its necessity.
Response 2: Thank you very much for your comment and suggestions. After reviewing relevant literature, we observed that hDPSCs exhibited a certain adhesion capacity after 1 hour of culture. In accordance with Luo et al.'s study [1], hDPSCs were stained following incubation at 37°C for 1 hour, revealing significant differences in cell adhesion outcomes between the control and treatment groups.
Comment 3: 324-326 In vivo studies conducted on rat cranial defects have demonstrated that the utilization of HAPNW/CF paper significantly enhances new bone formation compared to the control group.
342-345 Clinical, X-ray, and histological tests conducted 3 months later indicated complete re-generation of the bone tissue, and 1 year later, the regenerated bone tissue function reached its best, suggesting that dental pulp stem cells can fully restore human mandibular bone defects.
These are data from in vivo research. Your work did not conduct such research. Why are these data presented in the discussion?
Response 3: Thank you very much for your comment. We are very sorry for the results caused by our mistakes and carelessness. The in vivo experiment is the experiment we need to continue, and the in vivo experiment is not completed yet, so we will delete the part about the in vivo discussion.
Comment 4: 348-349 Additionally, due to the relatively rough surface of the HAPNW/CF biomaterial scaffold, it was beneficial for cell adhesion and proliferation.
You have not provided any rough surface data.
Response 4: Thank you very much for your comment. The materials utilized in this experiment are those previously published by our research group in "Coatings". Please refer to our previous article [2] for the material characterization, which is also cited as a reference in this paper.
Comment 5: 352-355 This was because the HAPNW/CF biomaterials provided more attachment sites for hDPSCs due to their higher specific surface area, which further promoted early adhesion of hDPSCs on the composite membrane 354 surface.
The specific surface area data you refer to in your discussion is not provided.
Response 5: Thank you very much for your comment. The biomaterials utilized in this experiment are those previously published by our research group in "Coatings". Please refer to our previous article [2] for the material characterization, which is also cited as a reference in this paper.
In the previous article[2], as shown in Figure 4A,B, the BET specific surface area of the HAPNW paper is measured to be 47.29 m2·g−1 , and that of the CF paper is very small (0.84 m2·g−1). Although the presence of CFs will decrease the specific surface area of the HAPNW/CF biopaper, the value is maintained at a relatively high level of 36.84 m2·g−1.
Reference
- Luo, Z.; Li, D.; Kohli, M.R.; Yu, Q.; Kim, S.; He, W. Effect of BiodentineTM on the Proliferation, Migration and Adhesion of Human Dental Pulp Stem Cells. Journal of Dentistry 2014, 42, 490–497.
- Gao, J.; Hao, L.-S.; Ning, B.-B.; Zhu, Y.-K.; Guan, J.-B.; Ren, H.-W.; Yu, H.-P.; Zhu, Y.-J.; Duan, J.-L. Biopaper Based on Ultralong Hydroxyapatite Nanowires and Cellulose Fibers Promotes Skin Wound Healing by In-ducing Angiogenesis. Coatings 2022, 12, 479.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsReview: Manuscript ID: coatings-2814726
Title: Enhanced osteogenic differentiation based on combining pulp 2 stem cells with ultralong hydroxyapatite nanowires and celluose fibers
Authors: Kai Liu , Wen Lai , Yuan-kang Zhu , Liang-shi Hao , Jing Gao, Cheng-long Yang, Li-feng Yuan, Jun-li Duan and Yong-jian Lu.
The authors present an intereting topic but the manuscript need major improvements before being considered for publication.
1. Figure 6 is missing
2. Figure 5 A has poor resolution. You cannot draw any conclusion based on the figure.
3. Figure 4, in the figure legend, the scale is declared 100 µm while in the figure, the scale bar is 500 µm.
4. Figure 3, in the figure legend, the scale is declared 400 µm while in the figure, the scale bar is 500 µm.
5. Figure 4 is of poor quality and cannot be used to draw a solid conclusion.
6. Also, some physico-chemical studies on the samples should be added in order to better understand the structure and morphology of the samples. It is well knowns that the structure and morphology of the samples play a crucial role in their biological properties.
Comments on the Quality of English LanguageThe English should be be improved cause there are minor typos through the text.
Author Response
Dear reviewers:
We are truly grateful to you for your critical comments and constructive suggestions. Based on these comments and suggestions, we have made careful modifications to the original manuscript and made elaborate responses to all the comments. We hope that the revised manuscript will receive a favorable decision. Below you will find our point-by-point responses to the reviewers' comments and questions.
Comment 1: Figure 6 is missing
Response 1: Thank you very much for your comment. We are very sorry for the missing of Figure 6 due to technical reasons. Now we add the figure to the article.(Page 9, Line 309-313, Figure 6)
Comment 2: Figure 5 A has poor resolution. You cannot draw any conclusion based on the figure.
Response 2: Thank you very much for your comment and suggestions. Based on your comment, we have substituted the high-resolution Figure 5A. (Page 8, Line 293-297, Figure 5)
Comment 3: Figure 4, in the figure legend, the scale is declared 100 µm while in the figure, the scale bar is 500 µm.
Response 3: Thank you very much for your kind reminding. We are very sorry for the inconsistent expressions caused by our carelessness. The scale bar in the figure should be consistent with the figure legend, both of which are 100 μm. The modified section is highlighted in red font. ( Page 7, Line 270, Figure 4)
Comment 4: Figure 3, in the figure legend, the scale is declared 400 µm while in the figure, the scale bar is 500 µm.
Response 4: Thank you very much for your kind reminding. We are very sorry for the inconsistent expressions caused by our carelessness. The figure legend should be consistent with the scale bar in the figure, both of which are 500 μm. The modified section is highlighted in red font. ( Page 7, Line 255, Figure 3A)
Comment 5: Figure 4 is of poor quality and cannot be used to draw a solid conclusion.
Response 5: Thank you for your comment and suggestions. We have changed the pictures in Fig. 4A to a more pronounced level, kindly verify. ( Page 7, Line 269-271, Figure 4)
Comment 6: Also, some physico-chemical studies on the samples should be added in order to better understand the structure and morphology of the samples. It is well knowns that the structure and morphology of the samples play a crucial role in their biological properties.
Response 6: Thank you very much for your comment. The biomaterials utilized in this experiment are those previously published by our research group in "Coatings".Please refer to our previous article [1] for the material characterization, which is also cited as a reference in this paper.
In the previous article[1], the morphologies of CFs, HAPNWs, and the HAPNW/CF biopaper are shown in Figure 2. In many cases, HAPNWs are self-assembled along their longitudinal direction into highly aligned nanowire bundles with larger diameters, and the diameter of a single nanowire is only about 10 nm (Figure 2A–C). The self-assembled alignment structure of HAPNWs (nanowire bundles) can enhance their mechanical strength and flexibility. It is observed that ultralong HAP nanowire bundles can naturally bend without fracture. The diameters of CFs are tens of micrometers, and a higher-resolution SEM image reveals that the CFs also consists of many approximately parallel fine fibers (Figure 2D,E).
Through a vacuum-assisted filtration process, HAPNWs and CFs were synergistically interwoven with each other to form the HAPNW/CF biopaper. The SEM image of the HAPNW/CF biopaper shows that CFs were embedded in the ultralong HAP nanowire network, which plays an important role as the reinforcement agent, similar to the steel bars in reinforced cement (Figure 2F). In a higher-magnification SEM image of the HAPNW/CF biopaper, a porous network structure constructed with HAPNWs and CFs is clearly observed (Figure 2G). Moreover, the cross-section SEM observation of the HAPNW/CF biopaper exhibits a layered structure, and the thickness of one single layer is about 10 µm( Figure 2H ). Thus, the CF-reinforced ultralong HAP nanowire network as well as the layered structure in the HAPNW/CF biopaper cooperate to facilitate the high mechanical performance. Compared with the conventional brittle HAP ceramics, the as-prepared HAPNW/CF biopaper is highly flexible, and can be bent at a large angle (nearly 180â—¦ ) without obvious cracking (the inset of Figure 2I). In addition, a large-sized HAPNW/CF biopaper with a diameter of 9 cm (Figure 2I) is fabricated rapidly and conveniently through the above-mentioned method for large-area skin wound dressings.
Response to Comments on the Quality of English Language
Thank you very much for your kind reminding. We have made modifications to the English language issues identified in the original manuscript, which are highlighted in red font within the article. Kindly review and verify.
Reference
- Gao, J.; Hao, L.-S.; Ning, B.-B.; Zhu, Y.-K.; Guan, J.-B.; Ren, H.-W.; Yu, H.-P.; Zhu, Y.-J.; Duan, J.-L. Biopaper Based on Ultralong Hydroxyapatite Nanowires and Cellulose Fibers Promotes Skin Wound Healing by In-ducing Angiogenesis. Coatings 2022, 12, 479.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
I read the paper:
Enhanced osteogenic differentiation based on combining pulp stem cells with ultralong hydroxyapatite nanowires and cellulose fibers
The paper is interesting, but I have some flaws.
Line 128: How many teeth were used? What is the gender of the patients?
Line 133: “The endodontium tissues were gently isolated…”
Did the patients give informed consent for the experimental work? How did you remove the pulp tissue?
Line 303: The authors describe the legend in figure 6, but there is no figure 6 in the text:
Line 406: The authors refer “The HAPNW/CF biopaper, owing to itsoinductive properties…”.
What is this property?
Author Response
Dear reviewers:
We are truly grateful to you for your critical comments and constructive suggestions. Based on these comments and suggestions, we have made careful modifications to the original manuscript and made elaborate responses to all the comments. We hope that the revised manuscript will receive a favorable decision. Below you will find our point-by-point responses to the reviewers' comments and questions.
Comment 1: Line 128: How many teeth were used? What is the gender of the patients?
Response 1: Thank you very much for your comment. A total of 15 teeth were utilized throughout the course of the experiment. The teeth were sourced from a cohort of ten individuals, comprising five males and five females.
Comment 2: Line 133: “The endodontium tissues were gently isolated…”
Did the patients give informed consent for the experimental work? How did you remove the pulp tissue?
Response 2: Thank you very much for your comment.
(1) The participants were provided with comprehensive information regarding the experiment's content and the purpose of their involvement, and subsequently gave informed consent by signing a document. All tooth extractions in the research were authorized by the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine.
(2) Firstly, teeth requiring extraction due to impacted dentition in individuals aged 18-25 years were collected with the informed consent of the patients. Subsequently, the extracted teeth were placed in a sterile bottle containing PBS buffer supplemented with penicillin and streptomycin at a temperature of 4°C and transported to the laboratory. A groove approximately 1.5mm deep was carefully created around the cervical region of each tooth using a turbine (taking care not to penetrate the pulp), followed by repeated rinsing with PBS within a super clean bench environment. The teeth were then split along this groove under aseptic conditions, allowing for removal of pulp tissue which was subsequently transferred into an EP tube containing PBS buffer.
Comment 3: Line 303: The authors describe the legend in figure 6, but there is no figure 6 in the text.
Response 3: Thank you very much for your comment. We are very sorry for the missing of Figure 6 due to technical reasons. Now we add the figure to the article.(Page 9, Line 309-313, Figure 6)
Comment 4: Line 406: The authors refer “The HAPNW/CF biopaper, owing to itsoinductive properties…”.
What is this property?
Response 4: Thank you very much for your comment. We sincerely apologize for the spelling error resulting from our oversight. The accurate spelling should be " The HAPNW/CF biopaper, owing to its osteoinductive property…". The modified section is highlighted in red font. ( Page 11, Line 408). The HAPNW/CF biopaper comprises hydroxyapatite, the primary inorganic constituent of the human skeleton, known for its remarkable osteoinductive properties[1–3]. Given its potential as a bone regenerative material, the HAPNW/CF biopaper exhibits exceptional bone induction performance.
In the previous article[4], the morphologies of CFs, HAPNWs, and the HAPNW/CF biopaper are shown in Figure 2. In many cases, HAPNWs are self-assembled along their longitudinal direction into highly aligned nanowire bundles with larger diameters, and the diameter of a single nanowire is only about 10 nm (Figure 2A–C). The self-assembled alignment structure of HAPNWs (nanowire bundles) can enhance their mechanical strength and flexibility. It is observed that ultralong HAP nanowire bundles can naturally bend without fracture. The diameters of CFs are tens of micrometers, and a higher-resolution SEM image reveals that the CFs also consists of many approximately parallel fine fibers (Figure 2D,E).
Through a vacuum-assisted filtration process, HAPNWs and CFs were synergistically interwoven with each other to form the HAPNW/CF biopaper. The SEM image of the HAPNW/CF biopaper shows that CFs were embedded in the ultralong HAP nanowire network, which plays an important role as the reinforcement agent, similar to the steel bars in reinforced cement (Figure 2F). In a higher-magnification SEM image of the HAPNW/CF biopaper, a porous network structure constructed with HAPNWs and CFs is clearly observed (Figure 2G). Moreover, the cross-section SEM observation of the HAPNW/CF biopaper exhibits a layered structure, and the thickness of one single layer is about 10 µm( Figure 2H ). Thus, the CF-reinforced ultralong HAP nanowire network as well as the layered structure in the HAPNW/CF biopaper cooperate to facilitate the high mechanical performance. Compared with the conventional brittle HAP ceramics, the as-prepared HAPNW/CF biopaper is highly flexible, and can be bent at a large angle (nearly 180â—¦ ) without obvious cracking (the inset of Figure 2I). In addition, a large-sized HAPNW/CF biopaper with a diameter of 9 cm (Figure 2I) is fabricated rapidly and conveniently through the above-mentioned method for large-area skin wound dressings.
Reference
- Sun, T.-W.; Yu, W.-L.; Zhu, Y.-J.; Yang, R.-L.; Shen, Y.-Q.; Chen, D.-Y.; He, Y.-H.; Chen, F. Hydroxyapatite Nanowire@Magnesium Silicate Core–Shell Hierarchical Nanocomposite: Synthesis and Application in Bone Regeneration. ACS Appl. Mater. Interfaces 2017, 9, 16435–16447.
- Prakasam, M.; Locs, J.; Salma-Ancane, K.; Loca, D.; Largeteau, A.; Berzina-Cimdina, L. Fabrication, Properties and Applications of Dense Hydroxyapatite: A Review. JFB 2015, 6, 1099–1140.
- Sun, T.; Zhu, Y.; Chen, F. Highly Flexible Multifunctional Biopaper Comprising Chitosan Reinforced by Ultralong Hydroxyapatite Nanowires. Chemistry A European J 2017, 23, 3850–3862.
- Gao, J.; Hao, L.-S.; Ning, B.-B.; Zhu, Y.-K.; Guan, J.-B.; Ren, H.-W.; Yu, H.-P.; Zhu, Y.-J.; Duan, J.-L. Biopaper Based on Ultralong Hydroxyapatite Nanowires and Cellulose Fibers Promotes Skin Wound Healing by In-ducing Angiogenesis. Coatings 2022, 12, 479.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have responded properly to all my comments.
