Research Progress in the Construction and Application of In Vitro Vascular Models

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The scope of the article based on the title is a review of the progress in construction and application of in vitro vascular models. The authors have presented the reasons and challenges associated with engineered vascular models and have done a good job reviewing current state of the art in biofabrication methods and their advantages and limitations. However, the article provides very little emphasis on connecting these advantages and limitations to specific biological challenges. For instance,

1. How does the diameter/curvature of the micro channel engendered by these fabrication methods influence endothelial monolayer formation, barrier function , etc.

2. While the authors have touched upon this, they could go into greater detail on differences in bioprinting as opposed to fabrication of micro channels which are then seeded. How does this change for single cell types and multiple cell types such as pericytes and astrocytes in addition to endothelial cells? Challenges with achieving the spatial arrangements in multiple cell types as well as in uniform distibution.

3. Along the same lines, while the authors have touched upon the subject of organs on a chip, very little time has been spent on describing the challenges in adapting the biofabrication methods into on chip systems. Effects of flow and shear stress on both fabrication as well as biology and the biological relevance of currently achievable flow rates.

 

 

Author Response

Comments 1：How does the diameter/curvature of the micro channel engendered by these fabrication methods influence endothelial monolayer formation, barrier function , etc.

Response 1：Thank you for pointing this out. I agree with this comment. Therefore, I have revised the manuscript to address this issue. Specifically, in the revised manuscript, we discuss how the diameter and curvature of microchannels affect the formation and barrier function of endothelial monolayers. This discussion can be found in lines 921-941 of paragraph 2 on page 23. This section explores how smaller diameters and curvatures promote tighter junctions but may affect cell viability, whereas larger diameters and curvatures facilitate cell migration but may compromise barrier integrity. These additions strengthen the manuscript by elucidating the critical role of microchannel dimensions in vascular model systems.

Comments 2：While the authors have touched upon this, they could go into greater detail on differences in bioprinting as opposed to fabrication of micro channels which are then seeded. How does this change for single cell types and multiple cell types such as pericytes and astrocytes in addition to endothelial cells? Challenges with achieving the spatial arrangements in multiple cell types as well as in uniform distibution.

Response 2: Thank you for pointing this out. I agree with this comment. Therefore, we further elaborated on the differences between bioprinting and pre-manufacturing microchannels followed by cell seeding, with particular emphasis on differences between cell types, including endothelial cells and astrocytes, among other peripheral cells. These revisions can be found in the revised manuscript, lines 470-510 of paragraph 3-4 on page 12. Additionally, we extensively discussed the challenges of achieving spatial arrangement and uniform distribution of multiple cell types in vascular models. These modifications can be found in the revised manuscript, lines 989-1004 of paragraph 3 on page 24.

Comments 3: Along the same lines, while the authors have touched upon the subject of organs on a chip, very little time has been spent on describing the challenges in adapting the biofabrication methods into on chip systems. Effects of flow and shear stress on both fabrication as well as biology and the biological relevance of currently achievable flow rates.

Response 3: Thank you for pointing that out. I completely agree with your view. Therefore, we discussed the challenges of using biomanufacturing methods to construct organ chips. These updates can be found in the revised manuscript, in paragraph 5 on page 21, lines 854-874. Additionally, we elaborated extensively on how factors such as fluid flow, shear stress, and flow rate influence the manufacturing process and their significance in biological research. These updates can be found in the revised manuscript, in paragraph 3 on page 23, lines 942-961.

Author Response File: Author Response.doc

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript aims to provide an overview of the current state of 3D in vitro models for vasculature. The authors successfully outline many important approaches used to build such engineered tissue models and their potential applications. However, the manuscript will benefit from some restructuring to better emphasize the specific strengths and weaknesses of the different methods (in contrast to the general statements currently provided). This will allow the readers to compare and contrast which specific method might be most applicable for their research question, making this article a useful resource. Some examples of the details would include cell types used or tissue sources, size ranges, heterogeneity, complexity, ease of manipulation etc. of the vessels generated by these methods or how the engineering challenges might be overcome. It could also be interesting to discuss how some methods like microfabrication might work better to model large blood vessels but how understanding self-assembly is critical to generate models for capillaries.

Another key advantage of the engineered tissue models is better recapitulating the tissue microenvironment. Some discussion of efforts to engineer the microenvironment by tuning the ECM, growth factors etc could also be useful - especially as this is what sets the 3D models apart from the 2D models. 

While the manuscript focusses a lot on the vascular biology and the details of tissue fabrication techniques, I would strongly encourage to emphasize the vascular-specific applications of these methods. Some of these technical details can be consolidated to leave more space for the specific methods and applications relevant to vascular biology.

The manuscript will also benefit from additional schematics/example images for the types of vascular networks generate by these techniques. For e.g. the current figures showcase the general methods for photolithography and tissue synthesis instead of the specific use case for vascular models or the final outcomes. It would be really helpful to see and compare the engineered tissues generated by the different methods.

Comments on the Quality of English Language

The language used is appropriate. The manuscript will benefit from tightening of the language for clarity in some places. But I have no major comments on the writing.

Author Response

Comments 1: However, the manuscript will benefit from some restructuring to better emphasize the specific strengths and weaknesses of the different methods (in contrast to the general statements currently provided). 

Response 1: Thank you very much for carefully reviewing our submitted manuscript and providing valuable suggestions. Your comments are crucial for highlighting the specific strengths and weaknesses of different methods. We carefully evaluated the time required to complete the restructuring of the manuscript and feel that we cannot currently handle the time pressure it would bring. At the same time, we believe that the current structure and content of the paper still effectively demonstrate the specific advantages and disadvantages of various methods for constructing vascular models. In this manuscript, we categorize extracorporeal vascular model construction methods into two main groups: microfluidic-based and non-microfluidic-based. We provide detailed reviews of photolithography, soft lithography, self-assembly, templating, bioprinting, and laser ablation/hollowing methods. Each method's strengths and weaknesses are specifically discussed, and we use Table 2 to compare their advantages and limitations. These comparisons help readers understand the differences between methods and select the most appropriate approach for their research questions, making this paper a valuable reference resource.

Comments 2: It could also be interesting to discuss how some methods like microfabrication might work better to model large blood vessels but how understanding self-assembly is critical to generate models for capillaries.

Response 2: Thank you for pointing that out. This paper discusses methods for constructing microvascular models and their applications, thus it does not elaborate on the construction methods of large-diameter vessels. Additionally, microfabrication techniques are primarily used to construct microvascular models (soft lithography, 50–200 µm). Synthetic scaffold, natural scaffold, cellular sheets, and decellularization can be used to construct large-diameter vessel models (single tube, mm-scale). Furthermore, 3D printing can be used to construct branched large-diameter vessel models. More information on understanding the importance of self-assembly in generating capillary models can be found in the revised manuscript, lines 595-609, of paragraph 5 on page 14.

Comments 3: Another key advantage of the engineered tissue models is better recapitulating the tissue microenvironment. Some discussion of efforts to engineer the microenvironment by tuning the ECM, growth factors etc could also be useful - especially as this is what sets the 3D models apart from the 2D models. 

Response 3: Thank you for pointing that out. We completely agree with this viewpoint. Therefore, we briefly discussed the roles of ECM and growth factors in the design of vascular models. This addition emphasizes the advantages of 3D engineered tissue models in simulating tissue microenvironments. Specifically, these discussions can be found in the revised manuscript, lines 962-976 of paragraph 4 on page 23. Additionally, we have added figures (Figures 4 and 5) to illustrate the importance of fluid dynamics and growth factors in constructing vascular models. Figure 4 demonstrates how shear stress influences the behavior of endothelial cells through multiple signaling pathways. The study underscores the significance of complex blood flow environments in recruiting leukocytes to endothelial cells and adjacent smooth muscle cells (SMCs), offering new insights into the mechanisms of leukocyte interaction with vascular walls in atherosclerosis patients. These modifications can be found in the revised manuscript, lines 703-709 on page 18. Figure 5 replicates the sprouting process of endothelial cells in tumor environments by adjusting the concentration of growth factors. These changes can be found in the revised manuscript, lines 761-764 on page 19. Compared to traditional 2D models, this 3D model better simulates the process of vascular generation in colorectal cancer environments.

Comments 4: While the manuscript focusses a lot on the vascular biology and the details of tissue fabrication techniques, I would strongly encourage to emphasize the vascular-specific applications of these methods. Some of these technical details can be consolidated to leave more space for the specific methods and applications relevant to vascular biology.

Response 4: Thank you for pointing that out. I agree with this comment. Therefore, I have integrated the technical details of these vascular model fabrication methods and emphasized their significance in specific vascular applications. Specifically, this discussion can be found in the revised manuscript from pages 14 to 17, lines 580 to 673.

Comments 5: The manuscript will also benefit from additional schematics/example images for the types of vascular networks generate by these techniques. For e.g. the current figures showcase the general methods for photolithography and tissue synthesis instead of the specific use case for vascular models or the final outcomes. It would be really helpful to see and compare the engineered tissues generated by the different methods.

Response 5: Thank you for pointing that out. I agree with this comment. Therefore, I have added explanatory figures to illustrate the types of vascular networks produced by these techniques and specific application examples. These additional figures can be found in the revised manuscript at lines 703-709 on page 18, lines 761-764 on page 19, lines 807-811 on page 20, and lines 843-844 on page 21. The purpose of these images is to provide clearer analysis comparing different methods of vascular model construction. We believe these additions better demonstrate the practical applications of these techniques in generating vascular networks, significantly enhancing the manuscript's quality.

Author Response File: Author Response.doc

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made the necessary changes to the manuscript.

Author Response

Comments 1:  I noticed that the newly added text is missing any citations. Please add the missing citations before publication.

Response 1: Thank you for pointing this out. I agree with this comment. Therefore, I have reviewed the revised manuscript and added the necessary references to support the newly added text. Specifically, these additional references can be found in the revised manuscript at:

Lines 470-510 in Paragraphs 3-4 on Page 12

Lines 580-673 on Page 14

Lines 854-874 in Paragraph 5 on Page 21

Lines 921-977 on Page 23

Lines 990-1005 in Paragraph 3 on Page 24

These additions are intended to provide proper attribution and enhance the credibility of the information presented. Thank you for your valuable feedback.

Author Response File: Author Response.doc

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing my comments. I noticed that the newly added text is missing any citations. Please add the missing citations before publication.

Author Response

Comments 1:  I noticed that the newly added text is missing any citations. Please add the missing citations before publication.

Response 1: Thank you for pointing this out. I agree with this comment. Therefore, I have reviewed the revised manuscript and added the necessary references to support the newly added text. Specifically, these additional references can be found in the revised manuscript at:

Lines 470-510 in Paragraphs 3-4 on Page 12

Lines 580-673 on Page 14

Lines 854-874 in Paragraph 5 on Page 21

Lines 921-977 on Page 23

Lines 990-1005 in Paragraph 3 on Page 24

These additions are intended to provide proper attribution and enhance the credibility of the information presented. Thank you for your valuable feedback.

Author Response File: Author Response.doc