Physicochemical and Mechanical Properties of Blow Spun Nanofibrous Prostheses Modified with Acrylic Acid and REDV Peptide

Round 1

Reviewer 1 Report

Dear Authors,

A well-described article with an interesting theme of vascular graffits. In principle, I have no major comments except for the two cosmetic ones, which after taking into account will certainly improve the content of the article:

(1) please correct the descriptions of chemical formulas - you do not use lower indexes but most of the formulas are described by large numbers

(2) refer in the introduction to the current knowledge on the modification of implant surfaces in the cardiovascular system, in the introduction describe something about stents especially DES and modification of the surface of metallic scaffolds, you can also refer to the following publications by authors familiar with cardiovascular system:

• Evaluation of electrochemical properties of antibacterial ZnO layers deposited to 316LVM steel using atomic layer deposition | [Bewertung der elektrochemischen Eigenschaften von antibakteriellen ZnO-Schichten, auf 316LVM-Stahl aufgebracht mittels Atomlagenabscheidung]
• Effect of thin SiO2 layers deposited by means of atomic layer deposition method on the mechanical and physical properties of stainless steel | [Einfluss dünner SiO2-Schichten, die mittels Atomlagenabscheidung aufgebracht wurden, auf die mechanischen und physikalischen Eigenschaften von Edelstahl] 

Best wishes

Reviewer

Author Response

Response to Reviewer 1 comments

Point 1: please correct the descriptions of chemical formulas - you do not use lower indexes but most of the formulas are described by large numbers

Response 1: HNO3has been changed to HNO_3,please see page  3 line 147. (NH4)4Ce(SO4)4has been changed to (NH₄)₄Ce(SO₄)₄, please see page 3 line 149, page 3 table 1, page 4 line 189.

Point 2:refer in the introduction to the current knowledge on the modification of implant surfaces in the cardiovascular system, in the introduction describe something about stents especially DES and modification of the surface of metallic scaffolds, you can also refer to the following publications by authors familiar with cardiovascular system:

• Evaluation of electrochemical properties of antibacterial ZnO layers deposited to 316LVM steel using atomic layer deposition | [Bewertung der elektrochemischen Eigenschaften von antibakteriellen ZnO-Schichten, auf 316LVM-Stahl aufgebracht mittels Atomlagenabscheidung] 
• Effect of thin SiO2 layers deposited by means of atomic layer deposition method on the mechanical and physical properties of stainless steel | [Einfluss dünner SiO2-Schichten, die mittels Atomlagenabscheidung aufgebracht wurden, auf die mechanischen und physikalischen Eigenschaften von Edelstahl] 

Response 2: In the Introduction section, we've added information on the most common methods for modifying cardiovascular implants together with appropriate references. Please see page 2, line 69-89. The information presented concerns vascular prostheses. DES are a separate group of implants with slightly different requirements, which is why we have given up referring to them in introduction.

Reviewer 2 Report

The authors have reported physical and mechanical properties of the blow spun nanofibrous prostheses which modified with AA and REDV. I suggest the authors do some few more experiments to ensure that the AA and REDV are really deposited on the PU structure chemically (via covalent bonds), not physically. Otherwise, I feel it is not convinced enough for readers and is not ready to be published. In addition, I suggest the authors answer and address some questions and requests below:

1. Please add a short sentence/phase of future perspective in the abstract.

2. Please inform the mole ratio of each chemical that employed for the PU modification, and explain carefully why it needs to be applied at that amount based on the chemical interaction ration.

3. On the PU-COOH structure, it is obvious that there are at least 3 positions of carboxylic acid, why the EDC/NHS and the REDV can react only one position?

4. Please remain the polymer parenthesis on the PU-REDV in figure 2 as well as PU-COOH, and PU.

5.  Please inform the concentration of the initiator carefully, "mg" is not a concentration unit. It should be, for example, 10 mg/mL, 10 mg/kg.

6. It was not clear why the S_0.5_25_10 was chosen instead of the S_1_35_10 or S_0.5_25_100?

7. Please note that "significant" term is used in scientific report once it is supported by a statistic study,e.g. t-test or F-test, and so on. Kindly address those terms in the manuscript. 

8. I would suggest to either redo the XPS and FTIR experiments for the AA modified PU and replace in the manuscript. Otherwise, please delete the section and find new methods that support the studies.

9. For all spectroscopic results, please add references for all peak that indicate something important.

I'm happy to help review this manuscript again once it has amended as suggested.

Author Response

Response to Reviewer 2 Comments

The authors have reported physical and mechanical properties of the blow spun nanofibrous prostheses which modified with AA and REDV. I suggest the authors do some few more experiments to ensure that the AA and REDV are really deposited on the PU structure chemically (via covalent bonds), not physically. Otherwise, I feel it is not convinced enough for readers and is not ready to be published. In addition, I suggest the authors answer and address some questions and requests below:

Response: Thank you very much for your review, we are grateful for the valuable comments. As suggested, we have done additional experiments.  We compared surfaces modified with acrylic acid and REDV in both conditions: with and without addition of the cerium salt. Cerium ions are responsible for the activation of carbon atoms in polyurethane structure and formation of new bond between PU and acrylic acid. After modification, the surfaces were rinsed vigorously with buffer to rinse off unbound/loosely bound acrylic acid. Then surface wettability was analyzed. For details please refer to the manuscript: Results section (page 11, line 348-366)

Point 1: Please add a short sentence/phase of future perspective in the abstract.

Response 1: We have added sentences in the abstract, page 1, line 21-24.

Point 2. Please inform the mole ratio of each chemical that employed for the PU modification, and explain carefully why it needs to be applied at that amount based on the chemical interaction ration.

Response 2: The concentration of two major modifying agent, acrylic acid and REDV, was selected based on our previous studies. When choosing reagent concentrations, we took into account the effect on the morphology of the material and the growth of endothelial cells. In the case of acrylic acid, we tested various concentrations >3%, which caused visible micro- and macroscopic changes in the structure of fibrous material. At the same time, concentrations <1% did not give a positive effect on endothelial cell growth. As far as REDV is concerned, the effect of the concentration on endothelial cell growth has been studied. Cell adhesion has not been enhanced for higher REDV concentrations (>5mM). Lower REDV concentrations (<5mM) were not tested. The concentration of other components e.g.: EDC, NHS were selected based on the concentrations used in the literature and our previous studies. 

Point 3. On the PU-COOH structure, it is obvious that there are at least 3 positions of carboxylic acid, why the EDC/NHS and the REDV can react only one position?

Response 3: We have no knowledge of how many carboxyl groups react with REDV. However, we assume that peptide does not react with each carboxyl group due to steric hindrance.That is why in Figure 2 we present REDV chain attached to one selected COOH group.

Point 4. Please remain the polymer parenthesis on the PU-REDV in figure 2 as well as PU-COOH, and PU.

Response 4: The missing parentheses have been added in Figure 1.

Point 5:  Please inform the concentration of the initiator carefully, "mg" is not a concentration unit. It should be, for example, 10 mg/mL, 10 mg/kg.

Response 5: We have corrected misspelled units. Page 3, table 1; page 7, line 283; page 8, line 301; page 10, line 341 and Table 6; page 18, line 543.

Point 6:It was not clear why the S_0.5_25_10 was chosen instead of the S_1_35_10 or S_0.5_25_100?

Response 6: When choosing the final material variant, we took into account fibers morphology. In particular, we paid attention to any damage to the fibers. Various fiber damage has occurred at higher concentrations of the initiator (Figure 6). Therefore, variants with concentrations of Ce(SO4)4 = 0.05% and 0.1% were rejected. As far as the S_1_35_10 variant is concerned, for most materials modified at 35, fibre damage was also occurred in particular in combination with a longer response time (Figure 5). That is why the modification was ultimately carried out at 25°C.

Point 7. Please note that "significant" term is used in scientific report once it is supported by a statistic study,e.g. t-test or F-test, and so on. Kindly address those terms in the manuscript. 

Response 7: We have added statistical analysis (ANOVA with post hoc Tukey test) for wettability results (please see Table 2 and Table 3). In case of results for which statistical analysis has not been carried out, the expression 'significant' has been deleted and replaced.

Point 8: I would suggest to either redo the XPS and FTIR experiments for the AA modified PU and replace in the manuscript. Otherwise, please delete the section and find new methods that support the studies.

Response 8: We have redone XPS analysis, new results were added to the manuscript. A new description has also been included (page 15, line 598-613). We decided to delete FTIR analysis.

Point 9:For all spectroscopic results, please add references for all peak that indicate something important.

Response 9: We have added literature references to all mentioned peaks (page 15, line 598-613).

Reviewer 3 Report

The manuscript by Kuźmińska et al. on "Physicochemical and mechanical properties of blow spun nanofibrous prostheses modified with acrylic acid and REDV peptide" described a two step modification process involving a hydrophilic linker and an REDV peptide. The authors previously described this approach in great details in a recent publication by Butruk-Raszeja BA, et al. on Endothelial cell growth on polyurethane modified with acrylic acid and REDV peptide in Surface Innovations Journal in 2019 (reference 17). 

Data presented in this paper appeared to be part of that study given previously described study design and tested conditions. In fact, authors provided a more sounded study in that work and included biological evaluation of this coating. 

Having said that, I have several concerns regarding this manuscript including:

1. Introduction was heavily focused on vascular prostheses haemocompatibility and why REDV sequence incorporation is important, however no where within the introduction section, they cover importance of physicochemical and mechanical properties described prostheses.
2. Figure 1 is poorly prepared. The idea of prosthesis’ internal surface modification and schematic of the flow system used for modification needs to be re-designed in more scientific fashion.
3. Mechanism of a two-step reaction for the surface modification process was described in better details in Butruk-Raszeja, Beata A., et al. "Endothelial cell growth on polyurethane modified with acrylic acid and REDV peptide." Surface Innovations 8.1–2 (2019): 89-104. Please check the attachment. 
4. I found several mis-spelling and grammatical errors through out this manuscript. For instance: Table 2. Contact angle values for each surface condition. For acronym definitions, see Tab.1.
5. Pore size evaluation of surface before and after modification using SEM is not the most reliable technique given this imaging takes place at high vacuum which could compromise the microstructural integrity of those samples. 
6. Provided spectroscopic analysis of surface modification reactions did not match up with previously published data (Table 1 in Butruk-Raszeja, Beata A., et al. Surface Innovations 8.1–2 (2019): 89-104.). 
7. Peak maxima for water is around at 3450 cm⁻¹, however authors described this peak as the characteristic signature peak for PU_REDV which is not correct. 

Overall, I am not confident given current status of this manuscript with several serious flaws and duplication/similarity of findings with Authors' recent publication in Surface Innovations, makes this work so original and novel with significant content as a new publication. 

Comments for author File: Comments.pdf

Author Response

 Response to Reviewer 3 Comments

The manuscript by Kuźmińska et al. on "Physicochemical and mechanical properties of blow spun nanofibrous prostheses modified with acrylic acid and REDV peptide" described a two step modification process involving a hydrophilic linker and an REDV peptide. The authors previously described this approach in great details in a recent publication by Butruk-Raszeja BA, et al. on Endothelial cell growth on polyurethane modified with acrylic acid and REDV peptide in Surface Innovations Journal in 2019 (reference 17). 

Data presented in this paper appeared to be part of that study given previously described study design and tested conditions. In fact, authors provided a more sounded study in that work and included biological evaluation of this coating. 

Response: Thank you very much for your review, we are grateful for the valuable comments. The research presented in our previous publication (Butruk-Raszeja et al, Surface Innovations, 2020) concerns the modification of flat polyurethane films. When we have tried to modify fibrous materials under the same process conditions, we encountered problems related to morphology change and fiber degradation. Therefore, a number of experiments were carried out to adapt the conditions of the modification process. Discussion of the effect of these variables on the properties of nanofibrous prosthesis is one of the goals of this article. In addition, in the article we analyze the possibility of carrying out modifications under flow conditions, so that only the inner surface of the cylindrical structure is modified. In previous studies, the modification was carried out in a classical way, by immersing the material in modifying solutions. In conclusion, the studies are different both in the type of material modified (flat films vs cylindrical nanofibrous prosthesis) and how the modification reaction is conducted (classical immersion vs. modification in flow).

Point 1: Introduction was heavily focused on vascular prostheses haemocompatibility and why REDV sequence incorporation is important, however no where within the introduction section, they cover importance of physicochemical and mechanical properties described prostheses.

Response 1: We have added the missing information in the Introduction section, please see page 1, line 25-44 and page 2, line 58-68.

Point 2: Figure 1 is poorly prepared. The idea of prosthesis’ internal surface modification and schematic of the flow system used for modification needs to be re-designed in more scientific fashion.

Response 2: Figure 1 has been redesigned.

Point 3: Mechanism of a two-step reaction for the surface modification process was described in better details in Butruk-Raszeja, Beata A., et al. "Endothelial cell growth on polyurethane modified with acrylic acid and REDV peptide." Surface Innovations 8.1–2 (2019): 89-104. Please check the attachment. 

Response 3: Yes, that is correct and that's why in the current article we only mention in a few sentences what is the mechanism of the modification. In our opinion, such short information will help readers to understand the changes that occur in the prosthesis at the various steps of modification. For more details, we refer to our previous articles.

Point 4: I found several mis-spelling and grammatical errors through out this manuscript. For instance: Table 2. Contact angle values for each surface condition. For acronym definitions, see Tab.1.

Response 4: We have re-checked the text and made language corrections.

Point 5: Pore size evaluation of surface before and after modification using SEM is not the most reliable technique given this imaging takes place at high vacuum which could compromise the microstructural integrity of those samples. 

Response 5: Available techniques for measuring pore size include scanning electron microscopy (followed with image analysis), BET, mercury intrusion porosimetry, and liquid extrusion porosimetry. The BET method requires a sample preparation procedure, which could damage the peptide structure on nanofibrous materials' surface (even more than SEM vacuum). Intrusion and extrusion methods use pressurized liquid to flow through the samples, which could flush any modification from the nanofibrous system. With initial sputtering, only SEM allows us to investigate our sample's morphology and measure its physical characteristics without damaging the coating. The applied procedure is one of the most common ways to investigate pore size and pore size distribution within polymeric nanofibrous systems [1].

[1] Eichhorn, S., Sampson, W. (2005). Statistical geometry of pores and statistics of porous nanofibrous assemblies Journal of The Royal Society Interface 2(4), 309-318. https://dx.doi.org/10.1098/rsif.2005.0039

Point 6: Provided spectroscopic analysis of surface modification reactions did not match up with previously published data (Table 1 in Butruk-Raszeja, Beata A., et al. Surface Innovations8.1–2 (2019): 89-104.). 

Response 6: The materials described in the previous publication were flat PU films. In the manuscript we present and fibrous materials. In addition, the process conditions were different (reagent concentration). After all, both analyses were performed in other laboratories, using different equipment.

Point 7: Peak maxima for water is around at 3450 cm⁻¹, however authors described this peak as the characteristic signature peak for PU_REDV which is not correct. 

Response 7: We decided to remove FTIR analysis, as suggested by the other reviewer.

Round 2

Reviewer 2 Report

The MS has been improved as suggested, therefore, it is acceptable to be published in this journal.

Author Response

The MS has been improved as suggested, therefore, it is acceptable to be published in this journal.

Response: Thank you for re-reviewing the MS.We re-checked MS for language and made the necessary corrections.We also added information on the thickness of the prosthesis' wall (table 3).

Reviewer 3 Report

The authors successfully addressed all concerns raised and revised the manuscript accordingly. The edited text and modified figures brought the manuscript to a much better place quality-wise. I appreciate their efforts and I am pleased with the current version of the manuscript and recommend it to the editor for publication. 

Author Response

The authors successfully addressed all concerns raised and revised the manuscript accordingly. The edited text and modified figures brought the manuscript to a much better place quality-wise. I appreciate their efforts and I am pleased with the current version of the manuscript and recommend it to the editor for publication. 

Response: Thank you for re-reviewing the MS.We re-checked MS for language and made the necessary corrections.We also added information on the thickness of the prosthesis' wall (table 3).