Effect of Low Magnetic Field on Biomimetic Deposition of Hydroxyapatite on Titanium and BIOLINE Stainless Steel

Round 1

Reviewer 1 Report

The manuscript entitled "Effect of Low Magnetic Field on Deposition of Hydroxyapatite on Biocompatibility Materials of Titanium and BIOLINE Stainless Steel" reported about the evaluation of the effect of low magnetic field deposition of hydroxyapatite on different substrates. The work is good but the authors should revised the manuscript based on the following comments.

Line 37 : due to its their mechanical resistance

Line 78 : The full-form of Ta and Ta/N not given; it is given in line 85.

Line 98 : Contact angle measurement – details about the microscope should be given

Line 117 : More details about the simulation will help i.e. software, boundary conditions and more detailed results.

Line 238 . both substrates, being this effect is more pronounced at a higher magnetic

Line 266 : use proper superscript Ca²⁺ and check throughout the manuscript.

Figure 2, 5, 6, 8 text is blur a higher resolution images should be given.

A tabular comparison of results for different materials will be provide good insight about the study.

Author Response

response to Reviewer 1: thanks very much for the comments and corrections. We have try to answered point by point to each comment

Line 37 : due to its their mechanical resistance

Response : this was corrected in the test

Line 78 : The full-form of Ta and Ta/N not given; it is given in line 85.

Response: In this line we describe the general composition of the systems and in line 85 we give the specific details

Line 98 : Contact angle measurement – details about the microscope should be given

Response: we use a CCD camara, modelXXX

Line 117 : More details about the simulation will help i.e. software, boundary conditions and more detailed results.

Response: we use XXXXX

Line 238 . both substrates, being this effect is more pronounced at a higher magnetic

Response: this was corrected (the word being was erase)

Line 266 : use proper superscript Ca²⁺ and check throughout the manuscript.

Response: this was corrected and checked out throughout the manuscript

Figure 2, 5, 6, 8 text is blur a higher resolution images should be given.

Response: Higher resolution images were incorporated

A tabular comparison of results for different materials will be provide good insight about the study.

Response: a comparative table was added

The english has been carefully reviewed

Reviewer 2 Report

This story is based only on speculations. No sufficient characterization data about modified surfaces of the metal substrates and apatite deposition were shown.

In addition to that, the reviewer strongly struggles with what was the novelty of this manuscript.

Additional comments

- Why did the authors use 1.5SBF?

- Fig.1 is not necessary.

- How did the authors weight their samples in Figs. 7 and 9?

- The scale of the SEM photographs in Figs. 3 and 4 should be changed to be the same.

- Why is the contact angle data necessary in this experiment?

Author Response

 RESPONSE TO REVIEWER 2

We thank to the reviewer for the helpful comments and for the careful revision of the manuscript. We have attended all the comments and try to answer them point by point. We also extended the introduction and new references were incorporated

1.This story is based only on speculations. No sufficient characterization data about modified surfaces of the metal substrates and apatite deposition were shown.

RESPONSE: We would like to answer to this argument of the reviewer that the deposition of hydroxyapatite was clearly demonstrated, not only through the electron micrographs, but also by ATR-FTIR . We compared the results against samples that were not subject to a magnetic field and it was observed a higher degree in the mass deposition of Hydroxyapatite in presence of magnetic fields. Although it is true that we did not perform XPS or Low angle X-Ray diffraction (because we do not have these techniques available). However, the deposition of Hydroxyapatite was clearly shown.

2.In addition to that, the reviewer strongly struggles with what was the novelty of this manuscript

RESPONSE: The effect of magnetic fields on the deposition of Hydroxyapatite has been very little studied. We considered that this is important since therapeutic applications in vivo of magnetic field on bone regeneration has been reported as a very successful treatment. But there is a lack of literature on systematic studies in vitro over metallic implants. When an implant is placed in the body for a fracture fixation a natural process of calcium phosphates and hydroxyapatite deposition takes place in order to regenerate the fracture. With this work we have demonstrated that the application of magnetic fields is very effective to accelerate this deposition. We use low magnetic fields (01-03T) since these are the values accepted by the human body

Additional comments

3- Why did the authors use 1.5SBF?

RESPONSE: In general studies on deposition of Hydroxyapatite are performed using 1.5SBF to accelerate the processes of deposition. This concentration has been widely studied and reported and correlate well with the results found with in vivo deposition. This concentration just accelerates the deposition to be able to follow the process in shorter time.

4- Fig.1 is not necessary.

RESPONSE:This figure was remove from the manuscript

5- How did the authors weight their samples in Figs. 7 and 9?

RESPONSE: after the samples were immerse for different periods in SBF in presence or absence of magnetic field. Then they were dried with compressed air at room temperature and weighted using and analytical balance with a precision of 0.0001 g

This was incorporated in Materials & Methods section of the manuscript

6- The scale of the SEM photographs in Figs. 3 and 4 should be changed to be the same.

RESPONSE: the scales were changed in the micrographs to be the same, However, the high-resolution micrographs were left as they were, to be able to appreciate the morphology of the coatings.

7- Why is the contact angle data necessary in this experiment?

RESPONSE: The contact angle is a very important measurement in biomaterials since it gives information on the change of hydrophilicity of the system after the metal is modified with the coating. This has a profound effect on cell attachment; therefore, this measurement is extremely important for biocompatibility of an implant.

Reviewer 3 Report

The subject of the manuscript “Effect of Low Magnetic Field on Deposition of Hydroxyapatite on Biocompatibility Materials of Titanium and BIOLINE Stainless Steel” by Rafael Uribe et al. is focused on the evaluation of the effect of magnetic fields on titanium and BIOLINE SS316LVM substrates with and without Ta and Ta/N coatings, their immersion in SBF and the evaluation of the deposition time and morphology of hydroxyapatite on these types of substrates.   

The manuscript is well-written and the subject proposed by the authors is interesting. It can be accepted for publication after the authors will properly respond to the raised queries (in the order they appear in the manuscript):  

1. Introduction

- the “References” list used in this section should be up-dated with some recent ones.

2. Materials and Methods

- the authors should indicate the source (provider) of the Ti substrates;

- what was the diameter of the water droplet used for CA measurements? This information is important when establishing if the measurements were performed on different areas of the metallic samples;

- if the authors decided to divide Section 2, then the paragraph “The metallic substrates were characterized …. as shown in Figure 1” (page 2, line 94 up to page 3, line 101) should be included in a separate subsection, namely “2.1 Characterization of metallic substrates”. Former subsections “2.1 Immersion…” and “2.2 Characterization….” should be renumbered accordingly;

- the authors should clearly indicate the apparatus used for weighting the samples (producer, model, accuracy of measurement);

- “Scanning electron microscopy (SEM)” (page 4, line 129) – once an acronym was defined, it should be further used in the text. The same applies for “hydroxyapatite” (page 5, line 202).

3. Results and Discussion

- one of the most important demands for a coating to be considered suitable for various medical applications is related to its adherence to the (metallic) substrates. In this respect, there are ISO regulations which clearly indicate values higher than 15 MPa. In this context, did the authors perform such tests? The authors should comment on this important aspect;

- “reported for HAp” (page 6, line 218) – at least one reference should be indicated here;

- how many measurements did the authors perform to obtain the results plotted in Figure 7? What type of statistics did they use for the inferred results? Moreover, are the presented results statistically relevant? The same observation applies also for the results included in Figure 9;

- “the deposition of the HAp layer on the Ta/Ti and Ta/TaN/Ti-covered Ti substrates is uniform” – the authors should reconsider their observation, as Figure 8 (b) seems to present a phenomenon of coalescence, which clearly affects the uniformity of the deposited layers;

- Figure 8 (d) was not introduced in the main text. Moreover, the scale bars in each Figure should be redrawn to be more visible, or at least they should be clearly mentioned in the legend of the figure. The same observation applies also for Figures 5 and 6;

- “PO₄^-3 vibration (1110 - 1020 cm^-1) and OH^- (1630 cm^-1) group” (page 9, line 288), “phosphate at 960 cm^-1” (page 9, line 290) – one has difficulties in easily discerning these values from Figure 10. An inset presenting these regions should be therefore used;

- the authors should comment on the phenomenon leading to the drastic decrease in the CA values after the deposition of HAp (down to 3°).

4. Conclusions

- in this Section, at least one possible application of the obtained results should be clearly indicated.

Some other recommendations follow:

• “due to its mechanical resistance” should read “due to their mechanical resistance” (page 1, line 37);
• “are” should read “area” (page 3, line 122);
• “In the other hand” should read “On the other hand” (page 5, line 198, and page 8, line 272);
• “Ti substrates immerse in SBF” should read “Ti substrates immersed in SBF” (page 5, line 207);
• “Figure 5 (e) near the stoichiometric” should read “Figure 5 (e), which is near the stoichiometric” (page 5, line 210);
• “the elemental analysis of Ca/P is equal to 1.60” (page 6, line 219 to 220) – should be rephrased;
• “Figure 6 (a) shows …. Figure 6 (e)),” (page 6, lines 228 to 231) – the authors are suggested to rephrase this paragraph as the Reviewer had difficulties in understanding its meaning (they should also pay attention to punctuation marks…);
• “the deposition of the HAp layer on the Ta/Ti and Ta/TaN/Ti-covered Ti substrates” (page 7, lines 256 to 257) – the presenting order of the structures should be inversed to match the legend of Figure 8 (a) and (b);
• “for desirable for cell adhesion” (page 9, line 311) – the authors should rephrase;

Author Response

REVIEWER 3

We thank to the reviewer for the helpful comments and for the careful revision of the manuscript. We have attended all the comments and try to answer then point by point. We also extended the introduction and new references were incorporated

The manuscript is well-written and the subject proposed by the authors is interesting. It can be accepted for publication after the authors will properly respond to the raised queries (in the order they appear in the manuscript):  

1. Introduction

- the “References” list used in this section should be up-dated with some recent ones.

RESPONSE

The references were updated, new 12 references were added and the introduction was extended

1. Materials and Methods

- the authors should indicate the source (provider) of the Ti substrates;

RESPONSE

In the materials and methods section all the materials sources are described (Ti, 12 mm of diameter, 99.99% purity, from Sigma-Aldrich)

3- what was the diameter of the water droplet used for CA measurements? This information is important when establishing if the measurements were performed on different areas of the metallic samples;

RESPONSE

The area covered by the droplets on the substrate was calculated and added in Table 1. This was incorporated in the manuscript

4- if the authors decided to divide Section 2, then the paragraph “The metallic substrates were characterized …. as shown in Figure 1” (page 2, line 94 up to page 3, line 101) should be included in a separate subsection, namely “2.1 Characterization of metallic substrates”. Former subsections “2.1 Immersion…” and “2.2 Characterization….” should be renumbered accordingly

RESPONSE:

A separated subsection was incorporated in the manuscript : 2.1 “Characterization of metallic substrates”

5- the authors should clearly indicate the apparatus used for weighting the samples (producer, model, accuracy of measurement);

RESPONSE:

An analytical balance BA-E series with precision of 0.0001 g was used to weight the samples. This was incorporated in Materials and Methods

6- “Scanning electron microscopy (SEM)” (page 4, line 129) – once an acronym was defined, it should be further used in the text. The same applies for “hydroxyapatite” (page 5, line 202).

RESPONSE:

This was corrected over the whole text

3. Results and Discussion

7- one of the most important demands for a coating to be considered suitable for various medical applications is related to its adherence to the (metallic) substrates. In this respect, there are ISO regulations which clearly indicate values higher than 15 MPa. In this context, did the authors perform such tests? The authors should comment on this important aspect;

RESPONSE; we agree with the reviewer that this is a very important point specially for biomedical devices. In our case, there are two stages on coating deposition: In the first stage a very thin layer of a calcium phosphate was deposited on the metallic substrates, This has quite a strong adherence in all the substrates. Higher on Ti than for stainless steel. This stage was tested with a carbon tape compressed over the coating and then peel off. This was a manual method, it was not performed in a commercial equipment, and this is the reason why it was not reported in the paper. But the results found were very promising, this very thin layer did not come off after the carbon tape was removed. The second deposition stage of hydroxyapatite was easy to remove. However, the whole idea of this study is to see if the magnetic field could increase the deposition of hydroxyapatite, since once inside the body this could accelerate bone regeneration. And the effect of the magnetic field on the deposition of hydroxyapatite was demonstrated in vitro

8- “reported for HAp” (page 6, line 218) – at least one reference should be indicated here;

RESPONSE:

A reference was added (Ref 32)

9- how many measurements did the authors perform to obtain the results plotted in Figure 7? What type of statistics did they use for the inferred results? Moreover, are the presented results statistically relevant? The same observation applies also for the results included in Figure 9;

RESPONSE:

Five measurements for each sample were carried out.  Standard deviation was used. We believe the results are relevant since the relative error is very low. This is explained in the manuscript

10- “the deposition of the HAp layer on the Ta/Ti and Ta/TaN/Ti-covered Ti substrates is uniform” – the authors should reconsider their observation, as Figure 8 (b) seems to present a phenomenon of coalescence, which clearly affects the uniformity of the deposited layers;

RESPONSE:

As I said before, the first layer deposited was very uniform, however the following deposition was no  uniform and we can observed some agglomerates on the surface of the substrates. This is typical of this method of deposition with SBF as has been reported by many authors.  The paragraph was modified

11- Figure 8 (d) was not introduced in the main text. Moreover, the scale bars in each Figure should be redrawn to be more visible, or at least they should be clearly mentioned in the legend of the figure. The same observation applies also for Figures 5 and 6;

RESPONSE: The scales bars have been corrected in the figures for a good visibility. And figure 8d has been mentioned in the text, pag 9 line 298 (Fig 7 now)

12- “PO₄^-3 vibration (1110 - 1020 cm^-1) and OH^- (1630 cm^-1) group” (page 9, line 288), “phosphate at 960 cm^-1” (page 9, line 290) – one has difficulties in easily discerning these values from Figure 10. An inset presenting these regions should be therefore used;

RESPONSE:

The FTIR graph (now Fig.9) has been modified in absorbance and the functional groups and frequencies were incorporated in the spectra

13- the authors should comment on the phenomenon leading to the drastic decrease in the CA values after the deposition of HAp (down to 3°)         

RESPONSE: The meaning of this was a diferential decrease of 3 degrees. However, this paragraph was rewritten for better understanding. The coating in all cases induces a higher degree of hydrophilicity

1. Conclusions

14- in this Section, at least one possible application of the obtained results should be clearly indicated.

RESPONSE: These materials are promising for therapeutic applications for in vivo in presence of a magnetic field shortening the period of bone regeneration. This was included in the conclussions

Some other recommendations follow:

• “due to its mechanical resistance” should read “due to their mechanical resistance” (page 1, line 37);
• RESPONSE: Corrected
• “are” should read “area” (page 3, line 122);
• RESPONSE: Corrected
• “In the other hand” should read “On the other hand” (page 5, line 198, and page 8, line 272);
• RESPONSE: Corrected
• “Ti substrates immerse in SBF” should read “Ti substrates immersed in SBF” (page 5, line 207);
• RESPONSE: Corrected

• “Figure 5 (e) near the stoichiometric” should read “Figure 5 (e), which is near the stoichiometric” (page 5, line 210);
• RESPONSE: Corrected

• “the elemental analysis of Ca/P is equal to 1.60” (page 6, line 219 to 220) – should be rephrased;
• RESPONSE: Corrected

• “Figure 6 (a) shows …. Figure 6 (e)),” (page 6, lines 228 to 231) – the authors are suggested to rephrase this paragraph as the Reviewer had difficulties in understanding its meaning (they should also pay attention to punctuation marks…);
• RESPONSE: rephased

• “the deposition of the HAp layer on the Ta/Ti and Ta/TaN/Ti-covered Ti substrates” (page 7, lines 256 to 257) – the presenting order of the structures should be inversed to match the legend of Figure 8 (a) and (b);
• RESPONSE: Corrected
•  
• “for desirable for cell adhesion” (page 9, line 311) – the authors should rephrase;
• RESPONSE: rephased

Reviewer 4 Report

The submitted article entitled “Effect of Low Magnetic Field on Deposition of Hydroxyapatite on Biocompatibility Materials of Titanium and BIOLINE Stain-less Steel ”relates to a very interesting topic and the results obtained can be used in practice. However, it should be corrected before publication.

Figure 1 is unnecessary. It is better to include photos of the tested materials, although it is usually unnecessary.

Figure 2 is illustrative and adds little. Moreover, in item b it is so illegible that it may not exist at all.

In the section on material characteristics, the authors describe that FT-IR spectra were made. Is it correct? The received materials were quite transparent or maybe the Authors forgot to add that it was ATR FT-IR?
The Authors in photos 3 show, among others, materials after treatment with NaOH but in the experimental part (section 2) they do not describe how the reaction was performed. This needs to be completed.

Infrared spectra are better represented in absorbance rather than transmittance, which is a converted quantity. This makes the interpretation easier, especially if the observed changes are small.

The contact angle values ​​alone do not contribute much. Much more information is provided by the values ​​of surface free energy determined on its basis or the values ​​of the components themselves: dispersion and polar. Table 1 should be completed.

Author Response

REVIEWER 4

The submitted article entitled “Effect of Low Magnetic Field on Deposition of Hydroxyapatite on Biocompatibility Materials of Titanium and BIOLINE Stain-less Steel ”relates to a very interesting topic and the results obtained can be used in practice. However, it should be corrected before publication.

We thank to the reviewer for the helpful comments and for the careful revision of the manuscript. We have attended all the comments and try to answer then point by point. We also extended the introduction and new references were incorporated

1.Figure 1 is unnecessary. It is better to include photos of the tested materials, although it is usually unnecessary.

RESPONSE:Fig. 1 was removed from the paper

2.Figure 2 is illustrative and adds little. Moreover, in item b it is so illegible that it may not exist at all.

RESPONSE:

Figure 2a and figure 2b were improved to be legible. We left this figure to illustrate the homogeneity of the magnetic field with distance

3.In the section on material characteristics, the authors describe that FT-IR spectra were made. Is it correct? The received materials were quite transparent or maybe the Authors forgot to add that it was ATR FT-IR?

RESPONSE: The reviewer is right we perform these analyses with an ATR-FTIR equipment, the details of the equipment were added in the materials and methods section.

4.The Authors in photos 3 show, among others, materials after treatment with NaOH but in the experimental part (section 2) they do not describe how the reaction was performed. This needs to be completed.

RESPONSE:

The reaction with NaOH was incorporated in the text

5.Infrared spectra are better represented in absorbance rather than transmittance, which is a converted quantity. This makes the interpretation easier, especially if the observed changes are small.

RESPONSE:

IR spectra were converted to absorbance and the functional groups and their specific vibration were indicated in the graph for better interpretation

6.The contact angle values ​​alone do not contribute much. Much more information is provided by the values ​​of surface free energy determined on its basis or the values ​​of the components themselves: dispersion and polar. Table 1 should be completed.

RESPONSE:

The work of adhesion was calculated and incorporated in table 2

Round 2

Reviewer 1 Report

We have checked the manuscript and the responses given by the authors. We have found that they have somehow revised the paper, but the claim that they mention in the response was not incorporated in the revised manuscript. For example, when you first mention Tantalum they have to mention tantalum (Ta)/ Tantalum Nitride (TaN).

They have mentioned that, they have used CCD camera for contact angle measurement, but it is not included on the revised manuscript. This is not a good practice. 

Each response should be genuine or rebuttal should be given.

Author Response

We thank the reviewer for the 2^nd revision of the manuscript and for the helpful comments. We have attended all the comments and try to answer them point by point

We have checked the manuscript and the responses given by the authors. We have found that they have somehow revised the paper, but the claim that they mention in the response was not incorporated in the revised manuscript.

Reviewer 1. For example, when you first mention Tantalum they have to mention tantalum (Ta)/ Tantalum Nitride (TaN).

Response: This was corrected in the manuscript (line 102 and 112)

Reviewer: 2. They have mentioned that they have used CCD camera for contact angle measurement, but it is not included on the revised manuscript. This is not a good practice. 

Response 2: we use a Thorlabs CCD camera, model DC1240C, TYPE CMOS, 1280X1024 pixels, this was incorporated in the manuscript

Reviewer 3 Report

The authors responded to most of the raised queries but there are still some things that need a close attention:

1. Why did the authors choose to change the title by adding the word “Biocompatible”? I do not consider this appropriate since only the SBF testing was performed, and no other in vitro tests. The authors should therefore adopt the initial title;
2. I had difficulties in understanding the phrase “Nevertheless, titanium has some disadvantages, low osseointegration cause of less bioactive of the material” (page 2, lines 44-45);
3. “sputtering coating” should read simply “sputtering” (page 2, line 51);
4. “pulsed lasers” cannot be considered a method…maybe “Pulsed laser deposition” – the authors should correct this in the main text;
5. The phrase “This study also discusses challenges and areas for future research.” (page 2, lines 54-55) is irrelevant;
6. “The method to obtain HAp as bone substitute should offer specific qualities such as, high purity, performance, low cost and has to be bioactive, biocompatible, and osteoconductive [21-24].” – what method should be bioactive, biocompatible?! The authors should rephrase the whole paragraph;
7. The authors should provide more details on how they calculated the area covered by the droplets on the substrate;
8. If the authors state that “five repetitions of the contact angle were carried out”, I was wondering if there was enough space for all of them to be performed on different areas of the same substrate. If not, is there a possibility that one measurement to influence the next one(s), when speaking of tests performed on the same spot of the surface? The authors should explain;
9. I do not really think that “the work of adhesion” provides the same information as the surface free energy, which is calculated on the basis of the CA values. The authors should reconsider this calculation and introduce the corresponding explanations for the inferred results (dispersion and polar components).

Author Response

We thank the reviewer for the careful 2^nd revision and the helpful comments of the manuscript. We have attended to all the comments and tried to answer them point by point.

The authors responded to most of the raised queries but there are still some things that need close attention:

1. Why did the authors choose to change the title by adding the word “Biocompatible”? I do not consider this appropriate since only the SBF testing was performed, and no other in vitro tests. The authors should therefore adopt the initial title;

Response: The new title id=s: Effect of Low Magnetic Field on Deposition of Hydroxyapatite on Titanium and BIOLINE Stainless Steel

2. I had difficulties in understanding the phrase “Nevertheless, titanium has some disadvantages, low osseointegration cause of less bioactive of the material” (page 2, lines 44-45);

Response: this phrase was changed for: Nevertheless, titanium presents a low bioactive surface with low osseointegration and a reference supporting this statement was added  [REF}

3. “sputtering coating” should read simply “sputtering” (page 2, line 51);

Response: this was corrected

4. “pulsed lasers” cannot be considered a method…maybe “Pulsed laser deposition” – the authors should correct this in the main text;

Response: this was corrected in the test

5. The phrase “This study also discusses challenges and areas for future research.” (page 2, lines 54-55) is irrelevant;

Response: this line was deleted

6. “The method to obtain HAp as a bone substitute should offer specific qualities such as, high purity, performance, low cost and has to be bioactive, biocompatible, and osteoconductive [21-24].” – what method should be bioactive, biocompatible?! The authors should rephrase the whole paragraph;

Response: this was changed in the test for: “The method to obtain HAp as a bone substitute should offer specific characteristics such as high purity, performance, low cost [21-24]”.

7. The authors should provide more details on how they calculated the area covered by the droplets on the substrate;

Response: The area covered by the drop over the different substrates was calculated assuming that the drop covers a circular area and from the diameter of the deposited drop the covered area was calculated.

 This was incorporated in the text

8. If the authors state that “five repetitions of the contact angle were carried out”, I was wondering if there was enough space for all of them to be performed on different areas of the same substrate. If not, is there a possibility that one measurement to influence the next one(s), when speaking of tests performed on the same spot of the surface? The authors should explain;

Response: The five repetitions were performed on the same substrate but at different times. This is a measurement of contact angle on one substrate was made, then this substrate was washed and dried and a repeated measurement was made again on the same conditions and this procedure was repeated five times on each substrate.

9. I do not really think that “the work of adhesion” provides the same information as the surface free energy, which is calculated on the basis of the CA values. The authors should reconsider this calculation and introduce the corresponding explanations for the inferred results (dispersion and polar components).

Response: We know that to measure surface energy from a contact angle two liquids of different polarity  (polar and non-polar such as water and diiodomethane) should have been dropped on the surface of the sample and measured the drop. However, we did not carry out this experiment, we only measured the contact angle with water. Therefore, we calculated the work of adhesion instead. We agree that the work of adhesion does not provide the same information as the surface energy. But it provides information of the work required to separate two surfaces and from this, we can have some information on how the hydroxyapatite coating may adhere to the surface. From  table 1 we can observe that Ti, Ta/Ti, Ta/TaN/Ti and Ta/TaN/SS316SVM have the highest work of adhesion and this was consistent with the results obtained after SBF immersion 

Reviewer 4 Report

Thank the Authors for taking into account the suggestions and improving the manuscript which I believe is ready for publication.

Author Response

Thanks very much to the reviewer

I am sending the final version revision

Round 3

Reviewer 1 Report

The authors have incorporated the corrections in the revised manuscript. Hence I recommend this manuscript for publication.

Author Response

We sincerely thank the reviewers for their suggestions.

Reviewer 3 Report

The authors took into consideration all the remaining issues. The manuscript is now ready for publication.

Author Response

We sincerely thank the reviewers for their suggestions.