Cu-Doped Mesoporous Bioactive Glass Nanoparticles Loaded in Xanthan Dialdehyde-Alginate Hydrogel for Improved Bioacompatiability, Angiogenesis, and Antibacterial Activity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article's weak points are the poor numbering of the figures and the imprecise referencing of these figures in the text (e.g., lines 117, 279, 282, 291, 304, 314, 345, 350, 387, 405, 433, 462, 468, 481, 503, 512, 457, and 579).

Abbreviation for alginate should be unified ALG line 381, NaALG Line 345, Na-ALG

ICP method should be mentioned in experimental part.

Figure 1, line 304, Figure 3:

Figure 2, line 345, Figure 4).

The abbreviation should be carefully explained in the abstract as well as throughout the article.

The English should be revised, as some of the text looks strange to me, e.g., Chapter 3.5.2, "Cellular Studies."

 Also, the synthesis of Cu-MBGNs should be provided because reference 33 contains several syntheses that lead to materials with different properties.

Figures should be self-explanatory, and abbreviations should be introduced in the description of Figure 1.

Line 201: Why do you use UV-VIS?

On line 222, the equation description should be on one line. Please shorten the text.

Figure 9, "Releasing of Copper from Glass Beads," should be provided because it seems to show the degradation of the hydrogel rather than the release of copper from the glass beads.

Comments on the Quality of English Language

The English should be revised, as some of the text looks strange to me, e.g., Chapter 3.5.2, "Cellular Studies."

Author Response

1. The article's weak points are the poor numbering of the figures and the imprecise referencing of these figures in the text (e.g., lines 117, 279, 282, 291, 304, 314, 345, 350, 387, 405, 433, 462, 468, 481, 503, 512, 457, and 579).

We appreciate reviewers for pointing out the errors in numbering and cross-reference of figures. All figures and their subsequent cross-references are now carefully checked and corrected to ensure consistency. The revised manuscript now reflects correct numbers and accurate cross-referencing at all the suggested line numbers.

2. Abbreviation for alginate should be unified ALG line 381, NaALG Line 345, Na-ALG

Abbreviation for alginate is unified throughout in the revised manuscript particularly in line 381 which is now 388 and 345 which is 353.

3. ICP method should be mentioned in experimental part.

We appreciate the reviewer’s observation, the method of ICP-OES is now added to experimental section from line 229-249 and is pasted below for reviewer’s observation.

“The ion release behavior of doped Cu-MBGNs in XDA/Na-ALG was studied in Phosphate Buffer Saline (PBS) of pH 7.4. Briefly, a 10mm x 10mm x 0.1mm size hydrogel was soaked in 20ml of PBS soln. in a shaking incubator at 37 ^oC at 90 rpm for 14 days. After 3 hrs for an initial 12 hours, 1 day, and after every day for 14 days, 1mL of soln. was taken and replaced with fresh PBS. The ionic concentration was studied by using inductively coupled plasma optical emission spectroscopy (ICP-OES, PerkinElmer Optima 8300) ICP-OES. Briefly, the collected PBS samples were first filtered by employing 0.22 µm filters. Thereafter, they were diluted with 2 v/v % nitric acid in order to stabilize the ions besides matching the matrix of calibration standards. Standard of Cu was used to draw a six-point calibration curve for metallic ion. The ICP-OES was then operated in axial view mode following standard conditions. Minimal spectral interference and accurate results were achieved by measuring Cu ions at wavelengths of 324.754 nm. Moreover, blank and quality control standard (scandium) were also used for accurate results. Herein, the final ionic concentration of Cu ions was calculated by determining the suitable dilution factor and adjusting the collected or added PBS at each time.

In order to avoid any overlap with degradation kinetics of XDA/Na-ALG/Cu-MBGNs hydrogel, the ICP-OES studies were performed separately. As the supernatant was collected primarily for ICP-OES analysis only at each time point, while the hydrogel samples were not taken out or dried for degradation purposes during ion release studies. Herein, ion release studies, subjectively, depict the release of Cu²⁺ from Cu-MBGNs and not the degradation of the hydrogel matrix.”

4. Figure 1, line 304, Figure 3:

In line 304 now line 328, Figure 1 is changed to Figure 3, and whole line is copied below.

“The convex shape curve in the Figure 3 is plotted between the relative pressure (P/P0) at x-axis Vs quantity of gas adsorbed (cm³/g) at y-axis, while there is another inset plot which shows the relation between average pore diameter (nm) and pore volume (ml/g).”

 

5. Figure 2, line 345, Figure 4).

In line 345 now line 252 Figure 2 is changed to Figure 4 and is pasted below for reviewer’s observation.

“Figure 4A and B display ATR-FTIR spectra of the individual components, namely XDA, Na-ALG, and Cu-MBGNs, as well as the ATR-FTIR spectrum of the composite hydrogel formed by combining XDA, Na-ALG, and Cu-MBGNs (referred to as XDA/Na-ALG/Cu-MBGNs composite hydrogel).”

 

6. The abbreviation should be carefully explained in the abstract as well as throughout the article.

Thank you for pointing out the error. The abbreviations have been rechecked and explained both in the abstract and article.

7. The English should be revised, as some of the text looks strange to me, e.g., Chapter 3.5.2, "Cellular Studies."

We appreciate the reviewer’s keen observation. The manuscript has been thoroughly revised by using Grammarly and ensured to keep the flow throughout the manuscript. Moreover, chapter 3.5.2 (cellular studies) is completely revamped to improve the flow and readability. Please consult line 284-300 (copied below for reviewer’s convenience).

“The cellular viability of XDA/Na-ALG and XDA/Na-ALG/Cu-MBGNs against primary dermal fibroblast cells (HDFa) was checked through water soluble tetrazolium salt (WST-8) assay. Briefly, HDFa cells were cultured (at 37 ℃ for 48 hrs) in culture medium (89 % DMEM; 10 % FBS: 1 % Penicillin/Streptomycin) until it reaches at confluency of 80 - 90 %. Afterwards, the culture medium was removed, and cells were washed thrice with sterile PBS, followed by trypsinization for 5-7 mins. The cells were collected via centrifugation and further diluted in freshly prepared culture medium. Then, the cells were counted using hemocytometer and a number of 5 × 10⁴ cells were introduced in each well of 24 well plate except the first well. The first well was loaded with fresh medium only and referred to as blank. While the next three wells (A2-A4) were referred to as tissue culture plate; TCP. And later wells were loaded with XDA/Na-ALG and XDA/Na-ALG/Cu-MBGNs hydrogels. The well plate was then incubated again for 7 days. The 100 µL medium was collected from each well at regular intervals and replaced with fresh medium. The collected medium was transferred to 96 well plate followed by addition of 10 µL of WST-8 dye and its incubation for 3-4 hrs. The incubated plate was then read through ELISA microplate reader (Accuris-9600) at wavelength of 450 nm. The absorbance of TCP on respective time was considered as 100 % and the samples were reported accordingly.”

8. Also, the synthesis of Cu-MBGNs should be provided because reference 33 contains several syntheses that lead to materials with different properties.

We acknowledge the reviewer’s concern to highlight the synthesis of Cu-MBGNs. Therefore, the synthesis of Cu-MBGNs is now added to revised manuscript in line 116-136 and is also copied below for your observation,

“Cu-MBGNs were dispersed into the soln. of XDA/Na-ALG to induce angiogenic and antibacterial effects, with an amount of 2g/L [43]. Cu-MBGNs were prepared using the CuCl₂/Ascorbic Complex precursor, as reported in [33]. The precursor was made by adding 0.2 M CuCl₂ solution in DI water, which was then heated in oil bath at 80 ℃ followed by dropwise addition of 0.4 M L-ascorbic acid solution. The reaction mixture was continuously stirred for 24 hours at similar temperature under dark conditions. Thereafter, the supernatant was collected as CuCl₂/Ascorbic Complex precursor after centrifugation at 7000 RCF for 15 mins. Moreover, MBGNs were prepared via widely accepted sol-gel microemulsion method. Briefly, cetyltrimethylammonium bromide (CTAB) (0.56 g) was dissolved in DI water (26 mL) followed by addition of 8 mL ethyl acetate and 5.6 mL of 1 M ammonia solution. Later, 2.88 mL of tetraethyl orthosilicate and 1.83 g of calcium nitrate tetrahydrate were added. 20-30 mins stirring is mandatory at each step. Finally, 5 mL of CuCl₂/Ascorbic Complex precursor was added, and solution was stirred for another 4 hours. The particles were collected via centrifugation at 7000 RCF for 15 mins followed by drying them in oven at 60 ℃ for 12 hrs. Once dried the particles were calcinated at 700 ℃ for 4 hours with heating and cooling rate of 2 ℃ per minute. Finally, the calcinated particles were ground to fine powder by ball milling the calcinated particles 0.5g of prepared Cu-MBGNs was added to a homogeneous solution of XDA/Na-ALG and ultrasonicated to achieve complete dispersion of particles. The developed XDA/Na-ALG/Cu-MBGNs and XDA/Na-ALG hydrogels were then characterized invitro. The synthesis route adopted for the development of hydrogels is shown in Figure 1 .”

9. Figures should be self-explanatory, and abbreviations should be introduced in the description of Figure 1.

The abbreviations used in Figure 1 are now introduced in caption of Figure in revised manuscript from line 139-141 and is copied below too.

“Figure 1. Schematic representation of Hydrogel synthesis. (XG is xanthan gum, SMP is sodium metaperiodate, XDA is xanthan dialdehyde, CaCl₂ is calcium chloride, Na-ALG is sodium alginate, and Cu-MBGNs) are copper doped mesoporous bioactive glass nanoparticles.”

10. Line 201: Why do you use UV-VIS?

Thank you for pointing out the error as that was written mistakenly and is corrected to weight loss now in line 222.

11. On line 222, the equation description should be on one line. Please shorten the text.

The text is shortened as requested and the equation is in one line now. It is also copied below

……. (4)

 

12. Figure 9, "Releasing of Copper from Glass Beads," should be provided because it seems to show the degradation of the hydrogel rather than the release of copper from the glass beads.

We appreciate the reviewer’s valuable insight. Figure 9 primarily represents the cumulative release of Cu⁺² from Cu-MBGNs embedded in XDA-Na-ALG network. It does not depict the degradation profile of XDA-Na-ALG/Cu-MBGNs hydrogel. The release curve indicates well established sustained Cu release kinetics of Cu-MBGNs in PBS besides diffusion of PBS in XDA/Na-ALG network which exposes Cu-MBGNs with physiological medium with time. It is noteworthy to acknowledge that ion release studies were performed independently from degradation studies. Herein, besides slower initial release with a subsequent burst may visually resemble degradation kinetics but it subjectively reports Cu²⁺ ion dissolution from Cu-MBGNs and does not depict hydrogel degradation. Moreover, the methodology of ion release studies have been improved too for reader’s clarity from line 244-249 and are pasted here too for reviewer’s convenience.

“In order to avoid any overlap with degradation kinetics of XDA/Na-ALG/Cu-MBGNs hydrogel, the ICP-OES studies were performed separately. As the supernatant was collected primarily for ICP-OES analysis only at each time point, while the hydrogel samples were not taken out or dried for degradation purposes during ion release studies. Herein, ion release studies, subjectively, depict the release of Cu²⁺ from Cu-MBGNs and not the degradation of the hydrogel matrix.”

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor,

 

I accurately reviewed the article

 

Manuscript Number: prosthesis-3985779

Title: Cu Doped Mesoporous Bioactive Glass Nanoparticles Loaded in Xanthan Dialdehyde-Alginate Hydrogel for Improved Bioacompatiability, Angiogenesis, and Antibacterial Activity

submitted to Prosthesis.

In the present work a xanthan dialdehyde/sodium alginate/copper doped mesoporous bioactive glass nanoparticles (XDA/Na-ALG/Cu-MBGNs) hydrogel is presented. The hydrogel was fabricated by casting method, followed by its characterization in terms of morphology, surface topography, in-vitro biochemical and physical interactions. Scanning electron microscopy images revealed the rough surface of hydrogel, ideal for cell attachment and proliferation. The nanoporous structure revealed by BET enabled it to hold moisture for an extended span. The nanopores were developed because of the ether linkage developed between XDA and Na-ALG as evident from Fourier Transform Infrared Spectroscopy. The loading of Cu-MBGNs was also confirmed by FTIR. The release of copper ions was sustained throughout the 7 days, it is accounting for about 22 µg/mL in 330 hrs, which follows the degradation kinetics of XDA/Na-ALG/Cu-MBGNs hydrogel. The released copper ions promoted angiogenesis as confirmed by enhanced release of vascular endothelial growth factor (VEGF) for XDA/Na-ALG/Cu-MBGNs hydrogel (275 ng/mL) in comparison to 200 ng/mL of bare TCP. The hydrogel, despite being bactericidal against Escherichia coli and Staphylococcus aureus didn’t show toxicity towards human dermal fibroblasts confirmed via Water-Soluble Tetrazolium 8 assay. Hence, developed XDA/Na-ALG/Cu-MBGNs hydrogel possesses potential to be investigated further in terms of in-vivo interactions

 

 

 

The topic is interesting but the authors have to solve some critical issues.

Introduction

The authors should explain the use of metal nanoparticles as antibacterials, particularly silver and copper, which are widely used. This discussion in the introduction should be continued in the results to compare the literature with antibacterial systems based on silver and copper nanoparticles. Important examples in the literature are:

1. https://doi.org/10.3390/ijms252111563
2. doi:10.3390/ma9121028

 

Experimental part

 

Authors must specify the brand and purity of all chemicals used.

Each measurement must be reported as the average of at least three measurements with the corresponding standard deviation.

 

Results and Discussion

For Cu-MBGNs preparation the experimental parameters are not discussed, but they could be interesting for the readers.

 

Figures

the authors need to improve the resolution of many figures

English requires many corrections. Many sentences are too long and there are several typos.

The conclusions are written roughly, generic: they must be rewritten.

In conclusion, the article is suitable for publication, but only after major revisions.

best reg

Comments on the Quality of English Language

English requires many corrections. Many sentences are too long and there are several typos.

Author Response

Manuscript Number: prosthesis-3985779

 

Title: Cu Doped Mesoporous Bioactive Glass Nanoparticles Loaded in Xanthan Dialdehyde-Alginate Hydrogel for Improved Bioacompatiability, Angiogenesis, and Antibacterial Activity

 

submitted to Prosthesis.

 

In the present work a xanthan dialdehyde/sodium alginate/copper doped mesoporous bioactive glass nanoparticles (XDA/Na-ALG/Cu-MBGNs) hydrogel is presented. The hydrogel was fabricated by casting method, followed by its characterization in terms of morphology, surface topography, in-vitro biochemical and physical interactions. Scanning electron microscopy images revealed the rough surface of hydrogel, ideal for cell attachment and proliferation. The nanoporous structure revealed by BET enabled it to hold moisture for an extended span. The nanopores were developed because of the ether linkage developed between XDA and Na-ALG as evident from Fourier Transform Infrared Spectroscopy. The loading of Cu-MBGNs was also confirmed by FTIR. The release of copper ions was sustained throughout the 7 days, it is accounting for about 22 µg/mL in 330 hrs, which follows the degradation kinetics of XDA/Na-ALG/Cu-MBGNs hydrogel. The released copper ions promoted angiogenesis as confirmed by enhanced release of vascular endothelial growth factor (VEGF) for XDA/Na-ALG/Cu-MBGNs hydrogel (275 ng/mL) in comparison to 200 ng/mL of bare TCP. The hydrogel, despite being bactericidal against Escherichia coli and Staphylococcus aureus didn’t show toxicity towards human dermal fibroblasts confirmed via Water-Soluble Tetrazolium 8 assay. Hence, developed XDA/Na-ALG/Cu-MBGNs hydrogel possesses potential to be investigated further in terms of in-vivo interactions

The topic is interesting but the authors have to solve some critical issues.

 

Introduction

 

The authors should explain the use of metal nanoparticles as antibacterials, particularly silver and copper, which are widely used. This discussion in the introduction should be continued in the results to compare the literature with antibacterial systems based on silver and copper nanoparticles. Important examples in the literature are:

 

https://doi.org/10.3390/ijms252111563

doi:10.3390/ma9121028

We acknowledge reviewer’s suggestion to improve the discussion regarding antibacterial efficacy of Cu ions in introduction section. Therefore, we have included 1^st provided reference in introduction section and is cited as reference 27 now. While, the 2^nd provided reference seems to be focused on silver nanoparticles which seems irrelevant to our work.

 

Experimental part

Authors must specify the brand and purity of all chemicals used.

We really appreciate your deep insight, the source, brand name and purity of chemicals have been added now. The reviewer can refer to line 92-96 of revised manuscript or can consult the copied lines below.

“Xanthan (Xn) from Xanthomonas campestris, Sodium meta periodate (SMP, >99%), Sodium Alginate (Na-ALG) (sodium salt of alginic acid obtained from brown algae), Ethylene glycol (EG, >99% pure). were purchased from Sigma-Aldrich Chemie^® (Sigma Aldrich, Steinheim Germany). Calcium Chloride anhydrous (CaCl₂) (95 % pure) was purchased from Duksan Pure Chemicals (Ansan, South Korea).”

Each measurement must be reported as the average of at least three measurements with the corresponding standard deviation.

The experiments were conducted in triplicate and data is reported as mean of three values in revised manuscript. As an example, you can consult line 580-581, and 614-617 (both are pasted below too).

“The values were taken in mean ± standard deviation of triplicates (n = 3).”

“The values were taken in mean ± standard deviation of triplicates (n = 3). One-way analysis of variance (ANOVA) was applied to the experimental data (n = 3) where “*” represents the significant difference (p < 0.05), and where “#” represents the non-significant difference (p < 0.05).”

 

Results and Discussion

 

For Cu-MBGNs preparation the experimental parameters are not discussed, but they could be interesting for the readers.

We appreciate the reviewer’s effort to make manuscript reader friendly. As suggested the experimental parameters for preparation of Cu-MBGNs have been added from line 111-131 and pasted below too for reviewer’s convenience.

“Cu-MBGNs were dispersed into the soln. of XDA/Na-ALG to induce angiogenic and antibacterial effects, with an amount of 2g/L [43]. Cu-MBGNs were prepared using the CuCl₂/Ascorbic Complex precursor, as reported in [33]. The precursor was made by adding 0.2 M CuCl₂ solution in DI water, which was then heated in oil bath at 80 ℃ followed by dropwise addition of 0.4 M L-ascorbic acid solution. The reaction mixture was continuously stirred for 24 hours at similar temperature under dark conditions. Thereafter, the supernatant was collected as CuCl₂/Ascorbic Complex precursor after centrifugation at 7000 RCF for 15 mins. Moreover, MBGNs were prepared via widely accepted sol-gel microemulsion method. Briefly, cetyltrimethylammonium bromide (CTAB) (0.56 g) was dissolved in DI water (26 mL) followed by addition of 8 mL ethyl acetate and 5.6 mL of 1 M ammonia solution. Later, 2.88 mL of tetraethyl orthosilicate and 1.83 g of calcium nitrate tetrahydrate were added. 20-30 mins stirring is mandatory at each step. Finally, 5 mL of CuCl₂/Ascorbic Complex precursor was added, and solution was stirred for another 4 hours. The particles were collected via centrifugation at 7000 RCF for 15 mins followed by drying them in oven at 60 ℃ for 12 hrs. Once dried the particles were calcinated at 700 ℃ for 4 hours with heating and cooling rate of 2 ℃ per minute. Finally, the calcinated particles were ground to fine powder by ball milling the calcinated particles 0.5g of prepared Cu-MBGNs was added to a homogeneous solution of XDA/Na-ALG and ultrasonicated to achieve complete dispersion of particles. The developed XDA/Na-ALG/Cu-MBGNs and XDA/Na-ALG hydrogels were then characterized invitro. The synthesis route adopted for the development of hydrogels is shown in Figure 1 .”

 

 

Figures

 

the authors need to improve the resolution of many figures

We acknowledge the reviewer’s concern and the resolution of figures have been changed such as Figure 6 and Figure 8 are changed to line graph to depict the data more accurately with clearer signs of error. Both are also pasted below for reviewer’s convenience.

 

English requires many corrections. Many sentences are too long and there are several typos.

We appreciate the reviewer’s keen observation. The manuscript has been thoroughly revised by using Grammarly and ensured to keep the flow throughout the manuscript.

The conclusions are written roughly, generic: they must be rewritten.

The conclusion section is completely revamped as suggested by reviewer. Please consult line 642-656 of revised manuscript, it is also pasted below for your kind observation.

“In this study, a novel XDA/Na-ALG/Cu-MBGNs hydrogel was synthesized and evaluated for its potential application in potential wound healing and burn care treatments. The prepared hydrogel exhibited a porous microstructure conducive for cellular attachment, moisture retention, and sustained release of bioactive metallic ions. In-vitro characterization via FTIR and TGA confirmed effective crosslinking and thermal stability, while BET analysis validated the mesoporous nature of Cu-MBGNs. The hydrogel demonstrated superior swelling capacity and controlled degradation, both essential for dynamic wound environments. Importantly, the sustained release of Cu²⁺ ions not only facilitated angiogenesis as evidenced by enhanced VEGF expression but also provided potent antibacterial activity against E. coli and S. aureus, without compromising cytocompatibility toward human dermal fibroblasts. These findings collectively establish XDA/Na-ALG/Cu-MBGNs hydrogel as a promising multifunctional dressing for burn wound management. Detailed biological characterization and in-vivo studies are recommended to further validate its clinical applicability and optimize its performance in real biological settings.”

In conclusion, the article is suitable for publication, but only after major revisions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The figures must be numbered correctly, such as 1, 2, 3, 4, 5, 6, etc.

It is not possible to have Figure 1 or 2 appear twice in the text. Referencing the figure in yellow is not enough; renumber it!

Equation 4 has bad formatting.

Comments on the Quality of English Language

The English should be revised, as some of the text looks strange to me, e.g., Chapter 3.5.2, "Cellular Studies."

"The cellular viability of XDA/Na-ALG and XDA/Na-ALG/Cu-MBGNs against pri- 285
mary dermal fibroblast cells (HDFa) was checked through water soluble tetrazolium salt 286
(WST-8) assay." 

I don't think that hydrogels are viable.

Author Response

Comment 1

The figures must be numbered correctly, such as 1, 2, 3, 4, 5, 6, etc.

Our Response: The Figure are now numbered correctly. Please see the revised manuscript.

Comment 2

It is not possible to have Figure 1 or 2 appear twice in the text. Referencing the figure in yellow is not enough; renumber it!

Our Response: We have checked the numbering of the figures and cross-referencing as well.

Comment 3

Equation 4 has bad formatting.

Our Response: All the equations has been formatted by using mathematical function.

 

Comments on the Quality of English Language

The English should be revised, as some of the text looks strange to me, e.g., Chapter 3.5.2, "Cellular Studies."

"The cellular viability of XDA/Na-ALG and XDA/Na-ALG/Cu-MBGNs against pri- 285
mary dermal fibroblast cells (HDFa) was checked through water soluble tetrazolium salt 286
(WST-8) assay." 

I don't think that hydrogels are viable.

Our Response: We have checked the English again and tried to remove the errors. I hope that the English is now acceptable.

 

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

References to figures are still missing:

Lines 136, 469, 530, and 554.

Author Response

We have carefully checked the figure numbers and have incorporated the Figure numbers. However, I would like to add this about conversion of word file to the PDF version. We have removed the automatic numbering to address the error. Please check the word file also.