Fabrication of a Porous Three-Dimensional Scaffold with Interconnected Flow Channels: Co-Cultured Liver Cells and In Vitro Hemocompatibility Assessment

Round 1

Reviewer 1 Report

Dear authors,

I would like to congratulate with you about the work that I think it is very interesting, well organised in terms of experimental activities and fruitfully contextualized with the literature. However, some improvements could be included. For this reason, I recommended “major revision” and I would like to propose some suggestions:

1. In the sentence (Pag 1, lines 31-33), the authors say: “These results shows the potential of fabrication of such per-31 fusable 3D scaffolds consisting of interconnected flow channels, perfusable with whole blood, for 32 engineering transplantable liver tissue”. I would like to invite the authors to change/improve this sentence highlighting the real application potential of these constructs toward engineering transplantable liver tissue. Try to avoid repeated words.
2. Revised the introduction because there are some sentences too long. Use the same way of writing, for example “inter-connected or interconnected”, (three dimensional or 3-D” choose one and use in all the paper. Check the pdf attached file to see some of my revisions
   • In the sentence (Pag 1, lines 38-40) the authors open the Introduction of the paper as reported below “Fabrication of 3D construct or macro scaffold have been reported by using stereolithography [1], deposition and layering [2] , silicon micromachining [3], 3D printing [4] and selective laser sintering (SLS) [5]”. I would like to suggest the authors to improve a little bit the sentence such as “Fabrication of 3D construct or macro scaffold by additive manufacturing technologies like stereolithography [1], deposition and layering [2] , silicon micromachining [3], 3D printing [4] and selective laser sintering (SLS) [5] has gained great interest in recent years” for example.
   • The sentence (Pag 2, lines 53-54) “Nevertheless, it is difficult to obtain literature on specific details about scaffold strut thickness” could be converted in “To the best of our knowledge, very few studies have reported specific details about scaffold strut thickness”.
   • The sentence (pages 2-3, lines 98-99) “The effect of growth on cellular metabolism of Hep G2 due to lining by endothelial cells was also investigated” is not clear. Do the growth and the cellular metabolism refer to HepG2? Do the authors mean that the aim to investigate how the scaffold lining with endothelial cells affect the growth and the cellular metabolism of HepG2?
3. In Materials and methods:
   • The methodology used for the scaffolds fabrication could be more easily understandable by means of a scheme showing the fundamental steps
   • Equations (1) and (2) use the same nomenclature in the formula and in the text of the manuscript
   • The authors said in the sentence (page 2, line 125-126) “The scaffold was rinsed carefully with 1X phosphate buffered saline three times to remove the remaining particles of PGA”. How sure are the authors that all the porogen was eliminated? In addition, during the SLS process some of the PCL powder may be trapped in the mesh of the sintered construct. How did that unreacted dust remove?
4. In Results:
   • Make uniform all the micrographs (micro-CT, SEM from Fig. 1 to Fig. 6) using the format of the scale bar. In most of them, the scale bars are non-visible
   • About the caption of the figures, please check my revision in Figure 1 (c) (Page 6, lines 249-251) and use the same for all the micrographas, including in the squared brackets the magnification used “[scale bar 10 mm, Magnif. xxx]”.
   • Please, use the same format also for the histogram graphs, in Fig. 5 (a) for example, the categories names (HepG2 and Co-culture) are out of the graph. Include them as shown in Fig. 5 (b) and use the same format also for the bars in all the graphs
   • Please, could the authors add the % of porosity at which they are referring the sentence “The PGA porogen leaching gave rise to highly porous scaffold” (Page 10, line 345)
   • What do the authors mean with the sentence “The process of leaching out PGA microparticles and the could be easily handles without disintegration of the scaffold”
   • Have mechanical and biodegradation tests been performed on PCL scaffolds produced using PGA as progen versus neat PCL scaffolds? It could be interesting improve the data also with these features
5. Finally, I would like to suggest an English check of the manuscript 

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you very much for appreciating our work and your valuable suggestions have helped us to improve our manuscript. We have attempted to modify the former version to meet the requests from you. We hope you are satisfied with the following answer and revisions.

Thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

• References should be updated to more recent work. There is very little work cited in 2019 and 2020! It is crucial to look into the recent work to find out the knowledge gap. 
• 40-42: The preference of SLS over other 3D printing techniques is not clear. Other 3D printing techniques have the same benefits mentioned for SLS. 
• 59-61: "However, 59 NaCl has high thermal conductivity which leads to growth of the struts due to heat con-60 duction into adjacent powder particles." reference?
• 62-63: It is off-topic! MEMS! "Resolution of 3D fabrication 62 is almost 1000 times higher than MEMS based 2D micropatterning method [3]."
• 69-70: Poorly written: "Tissue engineering in liver have revolutionized the construction of three dimensional 69 liver construct in recent years"
• 154: "The scaffolds were rinsed carefully" Rinsed with what?
• 233: What is the slicing software? How did you make the G-code?
• Figure 6: The significance in the difference of data is not clear. It should be clarified that the statistical analysis had been performed for comparing which data.
• 342: Remove the extra period "NaCl salt particles.."
• 342-344: There is no valuable discussion here, and wordiness is evident! No need to mention figures that had been already mentioned. 
• The conclusion doesn't have an appropriate format. It resembles an introduction not a conclusion, in terms of formatting and writing style.
• 4 and 71: "in-vitro" should be italicized: in-vitro.

Author Response

Dear Reviewer,

We would like to extend our gratitude to the reviewer for taking time and effort to review our manuscript. The valuable suggestions and comments have helped us to improve and revise the manuscript. Below we have attempted to reply to the comments considering all of your suggestions.

Thanking you.

Author Response File: Author Response.pdf

Reviewer 3 Report

I have a few questions and comments that need to be addressed.

- p.3 lines 108-110: How does your design mimic the liver structure? Why the varying inner diameters of flow channels in your scaffold?  

- p.3 line 115: the molecular weight of PGA was missing

- p.3 lines 122-126: How did you address the effect of 5% NaOH on PCL during the treatment to elute out PGA particles from the scaffold? Moreover, how did you verify that there was no residual PGA in your scaffold after your treatments?

- p.3 lines 134-135: Operating parameters for μCT scanning, e.g. accelerating voltage, pixel size etc?

- p. 4 lines 155-158: How can you be sure that collagen was adsorbed on to the surface of your scaffold with overnight soaking? What happens if you do not include this step of collagen treatment during your processing?

- p. 4 line 167: Incorrect figure number

- Figure 2c: Your figure caption states “The slices of cross section I (channel diameter 2 mm), cross section II (channel diameter 1.5 mm)”. However, why does the diameter of the channel in cross section I appear smaller than the one in cross section II? The channel seems to be largest in cross section II followed by cross section I and cross section III.  

- Figure 4c: The description of yellow arrows in your figure caption?

- Typos or grammatical errors: Line 71, Line 299, Line 343, Line 347, Line 355-357, Line 365

- Table 1 is shown on p.6 but no mention in the manuscript. Moreover, the unit for spot diameter is missing.

- PGA particles were used instead of NaCl salt particles to limit the strut thickness in this paper. What was the thickness of the struts in your scaffolds? How does the value compare to the one fabricated using NaCl salt particles?  PGA is much more expensive than NaCl so maybe using NaCl salt particles is a more feasible/economical option, if the difference is not significant? Please comment on this.

- Liver is a soft tissue but PCL is semi-crystalline polymer. Please comment on the mechanical mismatch between the two.

- Conclusions?

Author Response

Dear Reviewer,

Thank you very much for taking time and effort for reviewing our manuscript. We have attempted to your valuable comments and suggestions into considerations to improve our manuscript. We hope to meet your comments and be able to answer in the following.

Thank you.

Author Response File: Author Response.pdf

Reviewer 4 Report

Review Comments:

The authors have represented a research article entitled “Fabrication of a porous three-dimensional PCL scaffold with interconnected flow channels using PGA as porogens: liver cell culture, endothelization and in-vitro hemocompatibility assessment”. The aims and objectives of the manuscript are good. The research is in the early stage and needs more confirmation regarding its reproducibility and toxicity. The research study is well represented. There are a few fundamental approaches that need to be resolved before acceptance. 

1. Please try to explain the status of this experiment compared to recently reported literature. Please change the title. Present title contains PCL, PGA abbreviations. Please rewrite or avoid abbreviations in the title and also rewrite the title more precisely. The title must be smart and meaningful which must reflect the entire experimental study.
2. Why the authors choose that particular scaffold design, please explain in more detail.
3. During the cell perfusion study, do the authors observed any leakage or cell loss throughout the scaffold body?
4. During the cell perfusion study, how the authors maintained the CO₂ environment to maintain the pH of the media?
5. What is the targeted in vivo application of such types of scaffolds?
6. Please discuss a little more about hemocompatibility assessment.

Author Response

Dear Reviewer,

We would like to extend our gratitude for appreciating our work. Thank you very much for taking time and effort to review the manuscript. We anticipate answering to the reviewer’s valuable suggestions and comments.

Thanking you.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear authors,

I really appreciated your effort in considering my suggestions. I found the manuscript improved with respect to the previous versions and I have read very carefully your answers. Therefore, I recommended “accepting your manuscript after very few modifications”.

1. Most of my spelling corrections are included in red in the pdf attached file.
2. Would the authors be so kind to include or remove the scale bar dimensions (i.e. 200 microns) in all the pictures, for a more homogeneous style? If you decide to include the scale bar dimensions (i.e. 200 microns), please would you be so kind to enlarge the scale bar characters dimensions (i.e. 200 microns) in order to make them easier to read?
3. Page 11, lines 351-352: What the authors means with the sentence “The observation that the coverage of endothelial cells on Hep G2 were emphasized in live imaging of cell localized on the scaffold”? The meaning is not clear. Please, would be so kind to revised the sentence?

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

On behalf of all the authors, I would like to express our thanks for sharing valuable insight for further improvement in the manuscript. This has helped us a in many ways to improve on our mistake and revise. The editions are highlighted in green color in the manuscript.

Thanking you.

Author Response File: Author Response.pdf