Molecular Interaction, Chain Conformation, and Rheological Modification during Electrospinning of Hyaluronic Acid Aqueous Solution

Round 1

Reviewer 1 Report

This article provided research concerning how PEO affects suitability for an HA/PEO blend in producing fibers in ES. The paper is well written, and the figures and plots are high quality. The following recommendations will improve the quality of the article:

(1) In Table 1 please ensure significant figures are reviewed for the "Surface tension" column. I would suspect three or four significant figures would be suitable.

(2) In Fig. 2 (b) the x-axis label is cutoff.

(3) I recommend using a non-breaking half-space between the number and the degree sign when temperatures are reported throughout the article.

(4) In Fig. 4 (b) the font is not consistent with the other plots in the article. Please modify the font for consistency and clarity.

(5) In Fig. 5 further description in the caption should be added. Please add wording that highlights the major results shown in the figure.

(6) In Fig. 6 the scale bar size needs to be defined.

Author Response

Response to Reviewer 1 Comments

This article provided research concerning how PEO affects suitability for an HA/PEO blend in producing fibers in ES. The paper is well written, and the figures and plots are high quality. The following recommendations will improve the quality of the article:

Point 1: In Table 1 please ensure significant figures are reviewed for the "Surface tension" column. I would suspect three or four significant figures would be suitable.

Response 1: We ensure that there are three significant figures for the “Surface tension” column in Table 1.

Point 2: In Fig. 2 (b) the x-axis label is cutoff.

Response 2: the x-axis label in Fig. 2(b) has been fixed.

Point 3: I recommend using a non-breaking half-space between the number and the degree sign when temperatures are reported throughout the article.

Response 3: We agreed with your recommendation and have revised them in the paper.

Point 4: In Fig. 4 (b) the font is not consistent with the other plots in the article. Please modify the font for consistency and clarity.

Response 4: The font of Fig. 4(b) has modified and been consistent with other plots in paper.

Point 5: In Fig. 5 further description in the caption should be added. Please add wording that highlights the major results shown in the figure.

Response 5:

The chain entanglement model: (a) Pure HA, stiff polymer chains, only form few flections, and polymer chains will slip away when elongated by electric field; (b) HA/PEO-100 blends. Flexible PEO-100 chains pass through HA and entangle with other PEO molecules, and stabilize electric spinning process; (c) HA/PEO-2 blends. PEO-2, much smaller molecules, reduce the electrostatic repulsive forces and hydrogen bonding interaction, to make HA chains more flexible and form efficient entanglements.

Point 6: In Fig. 6 the scale bar size needs to be defined.

Response 6: In Fig. 6, the scale bar is 5 μm

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript provides good perspective to understand the effects of PEO on the electrospinning pf HA in HA/PEO solution and usage in tissue engineering of several tissues such as bones, skin and soft tissues.

Author Response

Response to Reviewer 2 Comments

The manuscript provides good perspective to understand the effects of PEO on the electrospinning pf HA in HA/PEO solution and usage in tissue engineering of several tissues such as bones, skin and soft tissues.

Response 1: We would like to express our sincere thanks to you for your high recognition and support for our article. It will make us more motivated and persistent in our future research work.

Author Response File: Author Response.docx

Reviewer 3 Report

The paper presented by Chen et al is interesting. The authors have discussed about electrospinning of PEO and HA using water. The topic is interesting and emergent for tissue engineering applications. However, the authors must improve the quality of the manuscript to be published in Membranes. 

Abstract: The authors should include more experimental results and brief discussion about that.

Introduction:

1 - The authors have included wrong phrases related to electrospinning. As example: "...Electrospinning has attracted considerable attention in the last few years...." this sentence is wrong, double check about the time to elaborate a more concise contextualization. 

2 - ".....Natural polymers with good biocompatibility usually possess poor electrospinning processability compared to synthetic polymers [4-7], especially
from their aqueous solutions. The authors should discuss a litle bit about salt to improve the dielectric constant to improve the electrospin process.

3 - Double check in all sentence to improve the state of art.

Materials and methods: 

1. Include references to support the chosen parameters.

Results and discussion:

1 - Include distribution of diameters collected from SEM analysis (including statistical analysis)

2 - Include a biological analyses to improve the quality of the paper ( cell adhesion, proliferation,...)

Author Response

Response to Reviewer 3 Comments

The paper presented by Chen et al is interesting. The authors have discussed about electrospinning of PEO and HA using water. The topic is interesting and emergent for tissue engineering applications. However, the authors must improve the quality of the manuscript to be published in Membranes

Abstract:

Point 1: The authors should include more experimental results and brief discussion about that.

Response 1: We have carefully revised the abstract and highlighted the important results.  

“Most of natural water-soluble polymers are hard to be electrospun due to their specific chain conformation in aqueous solution, which limits their applications. This study investigated the effects of polyethylene oxide (PEO) on the electrospinning of hyaluronic acid (HA) in HA/PEO aqueous solutions. The rheological properties of HA/PEO aqueous solutions showed polymer chain entanglement in HA was the essential factor affecting its electrospinnability. Wide-angle X-ray scattering and differential scanning calorimetry analyses of PEO crystal showed different crystallization behaviour of PEO chain with different molecular weight, which indicates different interaction with HA. A schematic molecular model has been proposed to explain the effect of PEO on the chain conformation of HA along with the relationship between electrospinnability and chain entanglement. PEO with relatively high molecular weight with limited crystal formation formed extensive chain entanglements with HA, while PEO with relatively low molecular weight weakened the interactions among HA chains. The findings of this study provide a wide perspective to better understand the electrospinning mechanisms of natural polyelectrolytes and usage in tissue engineering.”

Introduction:

Point 2: The authors have included wrong phrases related to electrospinning. As example: "...Electrospinning has attracted considerable attention in the last few years...." this sentence is wrong, double check about the time to elaborate a more concise contextualization.

Response 2: We checked about the time related to electrospinning in biomedical field. And we have revised the correct time in a more concise contextualization as below.

“….. Electrospinning has attracted considerable attention from the beginning of the 21st century (in the last few years) as a simple and versatile method for manufacturing nanoscale structures in tissue engineering and biomedical field, for example, in neuronal and bone and tissue engineering [1-4]……”

Point 3: ".....Natural polymers with good biocompatibility usually possess poor electrospinning processability compared to synthetic polymers [4-7], especially from their aqueous solutions.” The authors should discuss a litle bit about salt to improve the dielectric constant to improve the electrospin process.

Response 3: The brief discussion of adding salt into polymer has been added in the article.

“The addition of salt solution can also increase the conductivity of the polymer solution, increases the surface charge of spinning jet, and makes spinning process more stable.”

Point 4: Double check in all sentence to improve the state of art.

Response 4: We have checked all the sentence and record it in the submitted document.

Materials and methods:

Point 5: Include references to support the chosen parameters.

Response 5:

In Junxinh Li’s research [17], both 1.3 and 1.5 w/v% HA solution can undergo a stable electrospinning process. But as the distance between the electrode and the collector was decreased to 15 cm, the membrane morphology became more uniform when compared with that electrospun at the 25 cm distance undergo a electric field (22kV). The bead-string structure was formed at both 1.3 and 1.5 w/v% HA aqueous solution and the beads were more like spindles. Therefore, an increase in the electric field strength could benefit the fiber formation. Based on references to previous work, in this study, we chosen the HA concentration as 1 w/v%. The electric field was fixed at 20 kV and the tip-to-collector distance was fixed at 15 cm.

According to Shanshan Xu [19] and Doan Van Hong Thien’s work [20], HA was blended with gelatin or chitosan. They proved that HA could be electrospun by blending with other substances. And the concentration of HA in those solution was 1.5 or 2 w/v%.

In Huarong Nie’ study [21], the influence in electrospinning of poly(ethylene oxide) (PEO) and sodium alginate (SA) blend solution was investigated. A small number of beads and bead-on-string structure were obtained when PEO-100 aqueous solution was electrospun at a very low concentration (0.175 w/v%). The PEO-100 concentration increasing could benefit the fiber formation. For all PEO-2 solutions, electrospinning was impossible, even when the concentration approached 35 w/v%.

As for SA/PEO-100 blended solution, spindle-on-string morphologies with a few droplets were collected although the mass ratio of SA to PEO100 was 95/5, that is, the concentration of PEO100 was only 0.175 w/v%. Then more fibers were collected when the mass ratio of SA to PEO100 reached 90/10. Finally, as the mass ratio of SA to PEO100 was 80/20 (PEO100 0.7 w/v%), a continuous stable jet was observed and smooth uniform fibers were also obtained. It can be concluded that PEO100 played a positive role on improving the electrospinnability of SA, and the SA/PEO100 bi-component nanofibers could be fabricated through a stable electrospinning process. However, in SA/PEO2 blended solution, none of the blended solutions can be electrospun, even when the concentration of PEO2 reached a very high concentration of 60 w/v%. It is obvious that PEO-2 cannot improve the electrospinnability of SA. Based on the research result above, We selected the corresponding parameter settings in the article.

All the new references above have been added to article.

Results and discussion:

Point 6: Include distribution of diameters collected from SEM analysis (including statistical analysis)

Response 6: The distribution of diameters collected from SEM analysis was added in paper by using image J (software name).

Point 7: Include a biological analyses to improve the quality of the paper ( cell adhesion, proliferation,...)

Response 7:

We highly agree that the biological analyses can improve the quality of the paper. However, HA is a water-soluble material, in the process of cell adhesion experiment, the HA fibrous membranes will dissolve when it come into water. It brings a great challenge to biological analyses on HA fibrous membranes. But we can change this property by cross-linking HA and gelatin.

According to Shanshan Xu’s research, the HA based fibrous membranes present good cell viability but bad cell proliferation. This phenomenon is possibly related to the intrinsic nature of HA reported in previous literature. Although HA possesses good biological compatibility and nontoxicity, HA is not suitable for cell attachment and proliferation. However, this feature of HA materials can be changed by increasing the gelatin content in HA/gelatin blend. Our further study will be published soon and provide an interesting result of the HA/gelatin bi-component membrane with enriched gelatin content.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The authors have improved the quality of the paper and reply to all comments. However, biological tests should be performed in others experiments.