Early Recognition of the PCL/Fibrous Carbon Nanocomposites Interaction with Osteoblast-like Cells by Raman Spectroscopy

Round 1

Reviewer 1 Report

In the article entitled: “Early recognition of the of PCL/fibrous carbon nanocomposites interaction with osteoblast-like cells by Raman spectroscopy”, the authors describe the production of nanomaterials in the form of membranes composed of a poly(ε-caprolactone) matrix and the introduced: multi-walled carbon nanotubes (MWCNTs); or functionalized multi-walled carbon nanotubes (MWCNT-f); or electro-spun carbon nanofibers (CNFs); or functionalized carbon nanofibers (CNFs-f). The authors seeded the U-2 OS cell on top of the membranes and evaluated cell response and the biomaterial properties up to eight days after cell seeding, and characterized by fluorescence microscopy and confocal Raman microspectroscopy. They concluded that the addition of the carbon additives stimulated the growth of the U-2 OS cells and additionally that the degradation of the material occurred mainly at the amorphous domains of the PCL.

Although the article is interesting, it seems not to be completely novel, and the authors need to highlight throughout the paper the novelty of the study and the materials. There are also some major points that need to be addressed, also statistical analysis are missing throughout the results section, and some figures need to be modified.

Points to considered are:

• Although the paper is mostly well written, it needs some English grammar revision.
• Abstract section:

In this section, the electro-spun carbon nanofibers non-functionalized and functionalized are abbreviated as ESCNFs and ESCNFs-f respectively, while through the other sections of the paper are just abbreviated as CNFs and CNFs-f. Please unified the abbreviations throughout the whole paper

In the abstract section some information is missing that would help the reader to better get the essence of the paper quickly and know the key points from the paper. Some information missing is the main findings of the paper, and at the beginning of the abstract a bit of context or background information would be desirable also.

• Materials and Methods section:

Throughout this section there are some numeric values in which their corresponding units are written without any space (one example is when the authors rite “… 1 to 20KV”). Please correct were needed.

Subsection: 2.1. Fabrication of nanocomposite membranes

It seems that the fabrication of the nanocomposite membranes from PCL and MWCNTs and MWCNTs-f have been already described by the authors in a previous paper. This information is missing. Please add this information together with the reference of the corresponding paper (Anna Kołodziej, Aleksandra Wesełucha-Birczyńska, Małgorzata Świętek, Łukasz Skalniak, Marta Błażewicz. A 2D-Raman correlation spectroscopy study of the interaction of the polymer nanocomposites with carbon nanotubes and human osteoblast-like cells interface. Journal of Molecular Structure. Volume 1212. 2020, 128135, ISSN 0022-2860, https://doi.org/10.1016/j.molstruc.2020.128135). In case that the preparation of other materials have also been published before please add the information in the corresponding section.

Some information is missing in the figure legend from Figure 1, since only the parts (a) and (b) are written in the legend and the bottom part of the figure is not mentioned in the figure legend. Also in this figure, separate the time units (min) from the numeric values. Similar with the hours (h) and its values.

Also in this subsection the authors state that the diameter range of the carbon nanofibers range between 0.6 to 1.2 mm while in the case of the MWCNTs the range is 10-30 nm. The units of the carbon nanofibers are correct?

I assumed from the pictures on Figure 1 that the final composite membranes are circles of 55 mm in diameter. Is that correct? However, the membranes are transferred to 12-well plates (size around 22 mm in diameter) after sterilization for cell seeding. Please clarify the size of the final membranes produced in this section.

Subsection: 2.2. Contact angle measurements and surface free energy evaluation

Did the authors applied statistical analysis to the calculations of the average values obtained by contact angle measurements?

If so, please specify which test was performed and the values obtained to be able to understand the reproducibility of this values in the different batches obtained from the fabrication of these composites and if there are significant differences between the different type of membranes produced.

Please indicate how many independent replicates were performed.

Subsection: 2.3. Cell culture

Similar to the comment on subsection 2.1., the same procedure was already described by the authors in a previous paper. This information is missing. Please add this information together with the reference of the corresponding paper (Anna Kołodziej, Aleksandra Wesełucha-Birczyńska, Małgorzata Świętek, Łukasz Skalniak, Marta Błażewicz. A 2D-Raman correlation spectroscopy study of the interaction of the polymer nanocomposites with carbon nanotubes and human osteoblast-like cells interface. Journal of Molecular Structure. Volume 1212. 2020, 128135, ISSN 0022-2860, https://doi.org/10.1016/j.molstruc.2020.128135).

Subsection: 2.4. Fluorescence microscopy

In this section, please specify the information on the objective used (type, magnification and numerical aperture).

Subsection: 2.5. Raman microspectroscopy

In the introduction section, the authors stated in the last paragraph the following: “Raman microspectroscopy was also applied to simultaneously verify interactions between the nanomaterial' phases, i.e., at the interface of the fibrous carbon-based nanoparticles and polymer, and also at the nanomaterial / cell interface”. However, in the materials and methods section the authors do not mention how they acquired the Raman data in order to differentiate the Raman information obtained at the interface of the fibrous carbon-based nanoparticles and polymer, from the Raman information at the nanomaterial / cell interface, taking into account the resolution limit imposed by the confocal microscope setup.

Some information is missing in this subsection. Please include the following information: objective type and corresponding numerical aperture, pinhole aperture, software used for the acquisition of the Raman spectra (name, version, vendor), integration time of the acquire spectra and number of accumulations, spectral resolution (i.e. in cm-1/point units), spectra processing and software used for the processing. Statistical analysis applied to the spectral comparisons and the software used for that.

• Results and Discussion section:

Subsection: 3.1. Morphology of membranes of PCL/ with fibrous carbon nanoparticles

In this subsection the authors describe the appearance of the material resorting to the brightfield images obtained with a widefield microscope. Since this is images do not possess 3D information, to characterize the topography of the materials the authors need to use a technique that allows the acquisition of 3D information, for example laser scanning confocal microscopy or if they are interested to resolve the topography at the nanoscale level using an atomic force microscope. Additionally by using atomic force microscopy information on the stiffness of the materials can be obtained, which might have an effect on cell growth and response.

In figure 1 the quality of the images does not allow to understand if in the materials there are cells growing, since it seems to be a dim green color in the images corresponding to the different materials. If this is the case, please indicate in the figure legend.

In the image b of the figure 1 it is difficult to perceive the U-2 OS cell, please substitute this image for a better define one. Also clarify in the figure legend if in this image the cell is growing in some material.

Subsection: 3.2. Contact angle measurements and Surface free energy (SFE)

In this section, the information on the statistical analysis performed are missing, please introduce this information since the authors state that: “The results do not indicate the specific diversity between the nanocomposite polymer membranes. Although, the introduction of fibrous carbon nanoparticles into the polymer matrix resulted in a slight decrease in nanomaterial membranes hydrophobicity, the differences in the value of the wetting angle are in the error limit and equal to 94.7 ± 1.2; 88.8 ± 1.3; 90.6 ± 3.7, 89.5 ± 1.5, 208 89.4 ± 1.2 for PCL, PCL/MWCNTs, PCL/MWCNTs-f, PCL/CNFs and PCL/CNFs-f, respectively”.

On table 1 the PLC/CNFs are referred as PCL/CNT, please correct and unify the abbreviations throughout the manuscript.

Subsection: 3.3. The growth of U-2 OS cells on the membranes of PCL/ with fibrous carbon nanoparticles

From the data presented, the attachment and/or growth of the U-2 OS cells is compromised when they are seeded in the PCL membranes without functionalization. Please discussed this data since this material appears in the literature for applications such as regeneration, and others have shown that cells are able to grow normally.

Interestingly the data on SFE from the previous section shows quite similar values from the PCL alone and the PCL functionalized with the CNF, while they show a huge difference in the cell growth capacity. Can the authors comment on that?

The authors referred that they have estimated cell growth “by employing modern, quantitative methods in fluorescence microscopy”. However, this methodology and the software used is a simple and commonly used methodology for quantification of fluorescence intensity in fluorescence microscopy. However, the quantifications of cell growth using this methodology might be underestimating the amount of cells per micrograph, since further in the paper the authors referred to have used a fluorescent nuclear dye (Figure 9) and from the images in this figure it is possible to see some cells with their nuclei stained with DAPI while they appear not positive for GFP expression.

The authors referred “outstanding proliferation of cells on the tested materials”, however the control of the quantification of the number of cells using the same time points and quantification methodology but growing in control surfaces (i.e. the plastic surface of the 12-well plate) are missing.

In this subsection, the information and results on the statistical analysis applied is missing, please add. Statistical significance information needs to be added in Figure 4 also.

Some of the images in Figure 3 seem to be already published in a previous paper published by some of the authors of the current manuscript (images from PCL, PCL/MWCNTs and PCL/MWCNTs-f).

Subsection: 3.4. Raman microspectroscopic analysis of the membranes of PCL/ with fibrous carbon nanoparticles/cells interactions

Please indicate if the Raman spectra on the Figure 5 are representative spectra of the different conditions, or are average spectra of the different conditions. Information on how many spectra per sample was acquired and how many independent replicates were performed is missing.

There are some titles missing on some of the spectra in figure 5. The x-axis title only appears in c and e, and in a additionally the x-values of the axis are also missing, please correct this figure for uniformity. Add also the units of Raman intensity in the y-axis of the different graphs.

Subsection: PCL matrix crystallinity

In table 2 the title state “Observed significant Raman bands…”. Please explain why are they significant. Was some statistical analysis performed? In this table why the authors are showing the peaks appearing in the first day of culture with the cells? The list would be different at day 2, 4, 6, and/or 8?

The authors state that: “The intensity of some marker bands characterizing the polymer crystallinity, i.e., stretching vibrations at 1723 cm-1 due to ν(C=O), 1108 cm-1 band assigned to ν(COC), 913 cm-1 to ν(C-COO), and also the deformation vibrations at δ(CH2) at 1440 and 1417 cm-1, marked with arrows in Figure 5b-e, changed significantly in the first days of culture”. Which statistical analysis was performed. Please indicate the analysis performed and its resulting values. This information is also missing in the corresponding graphs from figure 6. Also indicate the total data contributed to this graphs (number of Raman spectra and number of independent experiments). Moreover in the figure legend of figure 6 remove the cm-1 units after each ratio.

The calculated ratios presented in figure 6, are they already discussed in previous literature as ratios indicated for the assessment of the polymer crystallinity? If so, please add this information in this results and discussion subsection. If not, please explain better the rationale behind the calculations of these ratios, since some of the wavenumbers are not indicated in the first paragraph of this subsection. Also further discussion is missing in this subsection together with references to the literature throughout this subsection.

The authors state: “The intensity ratio of 1108 (cryst)/1097 (amorph) cm-1 ν(COC) vibrations in the PCL chain decreases in the first days of culture for all types of membranes, what indicates a decrease in the crystallinity of the polymer matrix, and then its increase on day 8 (Figure 6a and b)”. Are the observed changes statistically significant? Moreover, if the crystallinity is decreasing over time why it increases at day 8? Besides, how can it be rule out by the intensity of these peaks the crystallinity of the matrices when there are cells growing on the materials that present Raman peaks assigned to cellular content in the range of the peaks used for the calculation of the ratio cryst/amorph? Amongst these peaks 1097 cm-1 for phosphodioxy groups (PO2-), 1095 cm-1 for lipids and DNA: O-P-O backbone stretching. The increase in cellular content can contribute to the decrease observed in this ratio. While in the range of 1100 cm-1 there are assignments for lipids and fatty acids. If the U2-OS cells are increasing their content on fatty acids over time this might reflect also in the corresponding ratio.

In the graph appearing in figure 6b, there is a point missing at 8 days for the PCL/CNFs-f condition.

The authors state: “The characteristics of the adjacent spectral regions give the intensity ratio of 913 (cryst)/864 (amorph) cm-1 due to ν(C-COO) vibrations, which for PCL/MWCNTs-f is similar to the previous ones, but for PCL/MWCNTs it decreases in consecutive days (Figure 6c). However, for PCL/CNF and PCL/CNF-f crystallinity of the polymer matrix does not change significantly (Figure 6d). Variability in the C-C region appears to indicate that cell adhesion is taking place”. Again, statistical information is missing as well as literature references for the statements. Similar considerations on the Raman peaks from cellular content applies in this wavelength ranges. For example, 918 cm-a for proline and hydroxyproline, 915 cm-1 for ribose vibration, 913 cm-1 for glucose, and 864 cm-1 for structural proteins like collagen.

The authors mention: “The band at 1305 cm-1 due to ω(CH2) came from the crystalline and amorphous PCL domains in, while 1285 cm-1 originated only from ω (CH2) in crystalline areas. The intensity ratio of 1285/1305 cm-1 decreases for PCL/MWCNTs, while increases for PCL/CNF & PCL/CNF-f (Figure 6e and f). Interaction with cells seems to influence this process”. Please discuss further this result, and add the statistical information.

In this result: “Another important parameter of crystallinity of the polymer matrix is the intensity ratio of 1723 (cryst)/1732 (amorph) cm-1 reflecting involvement of the C=O group in interactions with cells, which grows for PCL/MWCNTs and PCL/MWCNT-f ,but at 8th day significantly decreases (Figure 6g). it is different for for PCL/CNF for which the intensity ratio decreases and then increases, while for PCL/CNF-f the behavior is directly opposite (Figure 6h)”. Please add the statistical information and references.

The authors wrote: “The relative increase of the intensities of the above mentioned bands indicates the rise of amorphicity in the studied nanomaterials, in comparison to the reference PCL membrane for which changes almost do not happen (Figure 7). The observed trend can be correlated with the increase in the population of the cells, whose extremely strong development in the subsequent days of the culture was monitored in fluorescence microscopy (Figure 3) what modifies the extracellular matrix”. Please discuss further this result, and add the statistical information.

References for the peaks mentioned by the reviewer can be found in the following book: Vibrational Spectroscopy for Tissue Analysis. Series in Medical Physics and Biomedical Engineering. Eds.Ihtesham ur Rehman, Zanyar Movasaghi, and Shazza Rehman 2013. CRC Press, Taylor & Francis Group.

Subsection: 3.4.2. Arrangement of carbon nanostructures

It is not clear to me if the Raman spectra presented in Figure 7 and the results provided in this subsection are performed only in the materials produced or belong to the materials produced with the growing cells. Please clarify in this subsection. I assume that the cells are growing since the measurements are performed using the same time points as in previous results. However, the Raman spectra presented in figure 7a are different from the Raman spectra presented in figure 5 (of the data appearing within the same spectral range), please clarify. Once again the information on the statistical analysis applied is missing within the text and the graphs presented in figure 7 b and c.

If the Raman spectra presented in this subsection come from the different materials with growing cells, how can the authors rule out the contribution of the D and G bands and be sure that there is no information on the cellular content on these wavelengths? Indicate as well how many data points contribute to this data and the number of independent experiments. Also indicate why the ratios calculated are different between the materials (fig 7b and c).

No references are included for the discussion

Subsection: 3.4.3. Raman spectroscopy of U-2 OS cell development on PCL membranes with fibrous carbon nanoparticles

Please provide the information and results on the statistical analysis performed in this subsection and in the corresponding graphs of figure 8.

The authors mentioned: “Actually it is monitored in the Raman spectra as visible bands of 711 cm-1 and 913 cm-1 (Fig. 7A&B; Table 1)”. However, in figure 7a there is no bands appearing at these wavelengths and 7b is a graph for the ratios 1324/1585. Please correct this phrase, since the authors might be referring to figure 5. The table reference might also be not correct since in table 1 are shown the values for the wettability of the materials.

The authors mention: “The cells growth on studied materials is excellent (Figure 3), however, the cells adhesion monitored by Raman spectroscopy proceeds in a different way, possibly due to the presence of the nanoparticle (Figure 6)”. However, no calculations or statistical analysis were shown for the 711 and 913 cm-1 peaks which are just mentioned in the previous paragraph to this assumption.

Figure 8a is not mentioned throughout the text. Please add where needed. In Figure 8 it is shown the values of the ratio (957+970)/913, please add the information behind the information of this calculations, and discuss. If there is any reference, please add it also.

The point of 8 days is missing for the PCL/CNFs-f, please add this information.

Subsection: 3.5. Morphology of U-2 OS cells growing on PCL/MWCNTs materials

Since the authors have images of U-2 OS cells growing on the matrices and stained with GFP and have access to the ImageJ software, please include a quantification of morphological features of the cells for the different conditions and time points. Include number of cells per condition and number of independent experiments together with the statistical analysis.

The authors reported the staining of the cell nuclei at day 2 of cell growth on the materials with the nuclear dye Hoeechst 33342, that stains nuclei from both healthy and compromised cells. In this regard, it is not possible to infer that there are no signs of necrotic disruption of the nuclei with this staining. In order to get this information a different dye can be used, for example propidium iodide. Moreover, quantification of the number of nuclei with and without nuclear fragmentation/condensation is missing (together with the corresponding information on total number of cells counted, number of independent experiments performed and corresponding statistical results).

In figure 9 the blue staining of the nuclei is not perceived, please provide the images with this information, as it is written in the corresponding figure legend. Provide also the information of the scale bar in the images of this figure.

• Conclusions section:

In this section, the authors state the usefulness of these materials to stimulate the growth of bone tissue and cartilage, which was determined. However, the authors did not perform any experiment in which the U-2 OS cells were differentiated. Since there are published works in which these cells have been differentiated it would be of interest to see that this materials are actually stimulating bone tissue formation. An example of a differentiation protocol can be seeing in the following paper: Buchinger B, Spitzer S, Karlic H, Klaushofer K, Varga F. Lysyl oxidase (LOX) mRNA expression and genes of the differentiated osteoblastic phenotype are upregulated in human osteosarcoma cells by suramin. Cancer Lett. 2008 Jun 28;265(1):45-54. doi: 10.1016/j.canlet.2008.02.008. Epub 2008 Apr 18. PMID:18374478.

Author Response

Please, find attached replay to reviewer 1

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors reported sound work regarding the characteristics of  PCL/Fibrous carbon nanocomposite as well as its interaction with  osteoblast-like cells. In particular, the Raman micro-spectroscopy was used to detect or track the dependence of U-2 OS cell development in the nano composite. This is an interesting study, offering basis to further develop effective tissue engineering-based approach for tissue regeneration. However, I have some major comments that need to be addressed before the manuscript is considered for publication.

1. In section of conclusion, the authors demonstrated " the effect of carbon nano additives on the polymer structure and usefulness to stimulate the growth of bone and cartilage tissue were determined.  It is difficult to make such conclusion in terms of fluorescence microscopy and Raman microspectroscopic data. The measurement of osteogenic or chondrogenic markers at gene or protein level should be included. And, the osteoblast-like cell line rather than progenitor stem cell was used in this study. So this cell line may not be suitable for cartilage study.

   2. Figure 9 lacks scale bar/sub-figure number, and looks no blue color on the stain of materials. 

3. Importantly , the entire study lacks the statistic analysis that is a basis for  scientific research and data presentation.  

4. Some "exaggerated" words like "excellent" "extremely" shall be avoided in describing the results.

5. The further improvement on the importance/rationale of this study in the "Introduction" and "Results and Discussion" is needed. 

Author Response

please, find attached replay to reviewer 2

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Although the authors have answered most of the questions that I raised, some important questions remain unsolved. In order to better describe the results there are some statistical tests missing in some of the experimental data.

Also, English grammar needs to be revised throughout the paper.

Additional concerns include:

Section: 3.2. Contact angle measurements and Surface free energy (SFE)

In the contact angle data the authors state that: “The findings do not indicate the 253 specific diversity between the nanocomposite polymer membranes” however they did not perform or show the values of any statistical test. Please include the corresponding statistical test for these comparisons

Include also information on the materials and methods of the statistical test performed

Section: 2.5. Raman microspectroscopy

The authors have improved the information on the Raman spectra acquisition and processing of the spectra, however no information on the statistical test to understand the differences between the peaks in the different materials and composite was provided. Please provide a statistical test for the comparison of these data.

Section: The morphology of membranes of PCL/ with fibrous carbon nanoparticles

Since the authors did not use an image technology that produces 3D information on the samples, it is not accurate to state that there is significant changes in the surface topography. Instead, they can talk about differences in sample morphology.

Section: 3.4. PCL matrix crystallinity

The authors write in the first line of this section: “The observed significant Raman bands and their assignments are collected in Table2.” Since significant might be confusing with statistically significant it is advisable to change to another word such as for example relevant

In this section is still missing the information on the statistical test applied to the Raman data in order to support the statements made by the authors regarding the significant changes that they reported. Please add the information on the statistical test applied and its results to validate your assumptions on the differences reported for different peaks.

Please indicate a reference where the ratio 1723/1732 has been previously used if it exists.

Section: 3.4.2. The arrangement of carbon nanostructures

Please indicate which statistical test and its values was applied for analyzing the results shown in this section. It is important to understand if the fluctuations observed are due to differences or on the contrary there are no differences between the different days.

Sorry but still it is not clear to me if the Raman spectra presented in Figure 7 and the results provided in this subsection are performed only in the materials produced or belong to the materials produced with the growing cells

Subsection: 3.4.3. Raman spectroscopy of U-2 OS cell development on PCL membranes with fibrous carbon nanoparticles

In this subsection the authors have added information on the spectra processing method but they have not included information on the statistical test performed to

The authors did not comment neither correct the following information that I previously reported: The authors mentioned: “Actually it is monitored in the Raman spectra as visible bands of 711 cm-1 and 913 cm-1 (Fig. 7A&B; Table 1)”. However, in figure 7a there is no bands appearing at these wavelengths and 7b is a graph for the ratios 1324/1585. Please correct this phrase, since the authors might be referring to figure 5. The table reference might also be not correct since in table 1 are shown the values for the wettability of the materials.

Author Response

Section: 3.2. Contact angle measurements and Surface free energy (SFE)

In the contact angle data the authors state that: “The findings do not indicate the  specific diversity between the nanocomposite polymer membranes” however they did not perform or show the values of any statistical test. Please include the corresponding statistical test for these comparisons

Include also information on the materials and methods of the statistical test performed

Section 3.2 Contact angle measurements and Surface free energy (SFE) has been revised in accordance with the comments of the reviewer.

Information on the statistical test performed was added.

Section: 2.5. Raman microspectroscopy

The authors have improved the information on the Raman spectra acquisition and processing of the spectra, however no information on the statistical test to understand the differences between the peaks in the different materials and composite was provided. Please provide a statistical test for the comparison of these data.

The results of the Raman data test have been inserted into section 2.5 Raman microspectroscopy - Statistical analysis was performed with PCA with Calibration 99,30505;  Validation 97,97677 (the first measurement day); Calibration 97,08938;  Validation 95,18859 (the third measurement day); Calibration 99,35546;  Validation 98,39545 (the sixth measurement day); Calibration 97,8542;  Validation 94,61504 (the sixth measurement day).

Section: The morphology of membranes of PCL/ with fibrous carbon nanoparticles

Since the authors did not use an image technology that produces 3D information on the samples, it is not accurate to state that there is significant changes in the surface topography. Instead, they can talk about differences in sample morphology.

According to reviewer comment the above expression - there is significant changes in the surface topography – was changed to - there is significant changes in the surface morphology

Section: 3.4. PCL matrix crystallinity

The authors write in the first line of this section: “The observed significant Raman bands and their assignments are collected in Table2.” Since significant might be confusing with statistically significant it is advisable to change to another word such as for example relevant

As suggested by the reviewer Table 2 caption was exchanged at  “Observed characteristic Raman bands [cm^-1] and….”

In this section is still missing the information on the statistical test applied to the Raman data in order to support the statements made by the authors regarding the significant changes that they reported. Please add the information on the statistical test applied and its results to validate your assumptions on the differences reported for different peaks.

Please indicate a reference where the ratio 1723/1732 has been previously used if it exists.

The 1723/1732 intensity ratio was calculated in the publication [20]. Reducing the 1723 band intensity and, at the same time, the 1723/1732  intensity ratio indicates an enlargement of the area of amorphous domains in the polymer matrix. Additional discussion has been attached.

Section: 3.4.2. The arrangement of carbon nanostructures

Please indicate which statistical test and its values was applied for analyzing the results shown in this section. It is important to understand if the fluctuations observed are due to differences or on the contrary there are no differences between the different days.

Sorry but still it is not clear to me if the Raman spectra presented in Figure 7 and the results provided in this subsection are performed only in the materials produced or belong to the materials produced with the growing cells

Figure caption was supplemented with the details suggested by reviewer.

Subsection: 3.4.3. Raman spectroscopy of U-2 OS cell development on PCL membranes with fibrous carbon nanoparticles

In this subsection the authors have added information on the spectra processing method but they have not included information on the statistical test performed to

Information on the statistical test performed was added.

The authors did not comment neither correct the following information that I previously reported: The authors mentioned: “Actually it is monitored in the Raman spectra as visible bands of 711 cm-1 and 913 cm-1 (Fig. 7A&B; Table 1)”. However, in figure 7a there is no bands appearing at these wavelengths and 7b is a graph for the ratios 1324/1585. Please correct this phrase, since the authors might be referring to figure 5. The table reference might also be not correct since in table 1 are shown the values for the wettability of the materials.

We would like to thank the reviewer for this comment and for drawing attention to this sentence.

Reviewer 2 Report

The authors have addressed most comments. The revised manuscript has been improved. However, the writing needs to be further edited. 

Author Response

Additional discussion has been suplemented.