Green Bioprinting with Layer-by-Layer Photo-Crosslinking: A Designed Experimental Investigation on Shape Fidelity and Cell Viability of Printed Constructs
Round 1
Reviewer 1 Report
In General:
- The paper is well structured and the presented results show improvements for green bioprinting
- Language and linguistic style are excellent
The reviewer has the following comments:
Line 45: “However, photo-crosslinking, due to its use of ultraviolet (UV) radiation, may decrease cell viability of printed constructs”. The harmful influence of UV on cells and algae is well researched. Please elaborate on this section, you will easily find more literature about this. Note that often UV-B is more harmful.
Line 81: “UV lightbulbs contained in the print head of the bioprinter were used to photo-crosslink the constructs.” What wavelength spectrum is emitted by the UV lightbulb? Please specify.
Line 185: “ImageJ software (ImageJ, USA) was used to measure the perimeter (total length of the four edges of the construct on the top surface) for each construct image.” The perimeter was manually measured with the help of ImageJ software? Please specify.
Line 198: “The initial cell concentration of each printed construct was 106 cells/milliliter.” I guess that initial cell concentration was also measured with the Auto T4 cell counter. Please detail this.
What is the possible application for your printed construct? Please detail this.
For each experiment condition, three replicate constructs were used for shape fidelity measurements, and also three replicate constructs were used for cell viability measurements. What is the standard deviation? Why is no standard deviation shown in Fig. 6, 7, 8, 9, 10, 11, 12, and 13? Furthermore, you are connecting two points in these figures and therefore implying a linear correlation that cannot be derived from the presented results directly. I suggest adding the standard deviations and removing the lines or presenting the data in bar charts.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In “Green Bioprinting With Layer-by-Layer Photo-Crosslinking: A Designed Experimental Investigation on Shape Fidelity and Cell Viability of Printed Constructs”, the authors investigate the shape fidelity and cell viability in extrusion bioprinted constructs using a bioink based on alginate-methylcellulose-GelMA.
Although the paper is of interest in the field, there are several major concerns for its publication:
- The paper has many typos throughout the text (e.g., in the results and discussion section there is some text left out from the MDPI template). Moreover, the style of citations is not consistent in all sections of the paper.
- The introduction does not cover the relevant literature regarding the assessment of printability and shape fidelity in extrusion bioprinting. For example, there is no clear description of state-of-art methods to assess this property.
- In the materials and methods section, the logic of the work is not very clear. I’d suggest reordering the different sections (e.g., by first describing the bioink preparation, then the printing process). Furthermore, since it is relevant to the work, the design of experiments method should be described in more detail.
- Regarding the experiments design, it could be interesting to add also another variable to the design of experiments, i.e., the presence or not of layer-by-layer crosslinking, which may or may not affect the shape fidelity of the printed shape.
- It is not clear why: i) you chose that specific shape for the printed construct, and ii) why you chose those specific printing parameters. More details should be added (did you do some preliminary optimization?).
- The shape fidelity measurement method is by no means complete. For example, since you have a porous structure, you could evaluate the pore morphology (e.g., are the pores well defined or rounded) and dimensions. I’d suggest a more through analysis of the relevant literature, including the following papers:
Ouyang, Liliang, et al. "Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells." Biofabrication 8.3 (2016): 035020.
Bonatti, A. F., Chiesa, I., Vozzi, G., & De Maria, C. (2021). Open-source CAD-CAM simulator of the extrusion-based bioprinting process. Bioprinting, 24, e00172.
Ribeiro, A., Blokzijl, M. M., Levato, R., Visser, C. W., Castilho, M., Hennink, W. E., ... & Malda, J. (2017). Assessing bioink shape fidelity to aid material development in 3D bioprinting. Biofabrication, 10(1), 014102.
Schwab, Andrea, et al. "Printability and shape fidelity of bioinks in 3D bioprinting." Chemical reviews 120.19 (2020): 11028-11055.
- A separate section should be included in the materials and methods section to report precisely the statistical methods used in the paper. Furthermore, a statistical significance level at 0.1 is quite high, have you tried to lower it?
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
I appreciate the authors for the manuscript on green bioprinting, which will be an important step for the future. Here are my comments:
- Please add more texts explaining the 3D bioprinting technique and different methods. These two articles could be cited for mentioning further details: https://doi.org/10.1002/adfm.202104149 , https://doi.org/10.1089/ten.teb.2021.0012
- Please add a section on the aims and objectives of the study.
- Please add the strengths and limitations of the study at the end of the discussion section.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 4 Report
This manuscript asses the parameters affecting layer-by-layer photo-crosslinking for bioprinting applications. The parameters investigated include extrusion pressure, nozzle size, and ink composition. The article in general is interesting and well organised and presented but may need to rearranged figures and tables. Figures 6-13 can ideally be combined in one figure and captions should include more information to properly describe what is being presented in each figure. Additionally, all figures do not have error bars and experimental trials has to be increased to truly represent a mean of several attempts rather than a single outcome.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Compared to the first version of “Green Bioprinting With Layer-by-Layer Photo-Crosslinking: A Designed Experimental Investigation on Shape Fidelity and Cell Viability of Printed Constructs”, the authors have improved the overall quality of the paper which can be accepted for publication.
Some suggestions to further improve it:
· Pay attention to formatting errors, some sections of the paper are justified left instead of to the center.
· Please add units of measurements to the tables (i.e., Table 4, Table 6).
Author Response
Compared to the first version of “Green Bioprinting With Layer-by-Layer Photo-Crosslinking: A Designed Experimental Investigation on Shape Fidelity and Cell Viability of Printed Constructs”, the authors have improved the overall quality of the paper which can be accepted for publication.
The authors thank the reviewer for the comments.
Some suggestions to further improve it:
Pay attention to formatting errors, some sections of the paper are justified left instead of to the center.
Formatting errors have been rectified.
Please add units of measurements to the tables (i.e., Table 4, Table 6).
The units of measurement have been added to the tables.
Reviewer 4 Report
Authors have addressed comments and updated the manuscript accordingly
Author Response
Authors have addressed comments and updated the manuscript accordingly
The authors are thankful to the reviewer for their comments.
