Al₂O₃-Phosphate Bioceramic Fabrication via Spark Plasma Sintering-Reactive Synthesis: In Vivo and Microbiological Investigation

Round 1

Reviewer 1 Report

This research introduces a method to enhance biocompatibility of bioinert Al2O3-based ceramics by incorporating calcium phosphates (hydroxyapatite (HAp) and tricalcium phosphate (TCP)) into alumina via spark plasma sintering-reactive sintering (SPS-RS). In addition, the antibacterial properties of the prepared ceramics were evaluated by biological experiments under soft tissue of animals.This study has important implications for regeneration and bone reconstruction surgery. However, there are still many questions before it is accepted for publication.

1.  The content of this article is good, but the layout is not very reasonable, the text and the picture are misaligned, it looks inconsistent, and should be adjusted.

2.  The caption in Figure 8 has a different font size than the rest of the captions, this paper should not contain such elementary errors.

2. The experimental process, like the experimental report, is too lengthy (for example, the relevant animal law and its authorized authority in section 2.4 should be placed at the bottom or end of the article page, not in the text), scanning electron microscopy does not give or does not have specific pictures of the specific status of biofilm morphology, there are too many legends, and some unimportant pictures need to be deleted Leave pictures with substantial use.

3. In addition, the article does not mention what are the advantages of developing AL2O3 ceramics on the basis of ZrO2 ceramics, which lacks the embodiment of certain research value, and the reasons should be given in the introduction or conclusion.

4. During the preparation of section 2.1, a picture of the sintered sample (cylindrical ceramic sample) should be given to increase the authenticity of the article.

5. The specific data of ceramic samples should be given in the form of a chart, but this article only has a text description, which is not intuitive enough

many grammar errors

Author Response

Re: «Al₂O₃-phosphate bioceramic fabrication via spark plasma sintering-reactive synthesis: “in vivo” and microbiological investigation» by Papynov E.K. and et al. (Manuscript Number: jcs-2635558)

Dear dear Editors, dear Reviewers,

We deeply appreciate the time you spent reviewing our paper and the valuable recommendations you made. All the comments are taken into account and corresponding changes are made to the manuscript’s body text. Detailed point-by-point answers are presented below.

On behalf of co-authors,

Oleg Shichalin, Researcher, Ph.D.

Response to Reviewers

Reviewer #1:

This research introduces a method to enhance biocompatibility of bioinert Al2O3-based ceramics by incorporating calcium phosphates (hydroxyapatite (HAp) and tricalcium phosphate (TCP)) into alumina via spark plasma sintering-reactive sintering (SPS-RS). In addition, the antibacterial properties of the prepared ceramics were evaluated by biological experiments under soft tissue of animals.This study has important implications for regeneration and bone reconstruction surgery. However, there are still many questions before it is accepted for publication.

Comment #1.

The content of this article is good, but the layout is not very reasonable, the text and the picture are misaligned, it looks inconsistent, and should be adjusted.

Response to the Comment #1:

Thanks to the reviewer for the comment. The layout of the article, namely the arrangement of text, images, and graphs is the responsibility of the journal editorial board. For the visual component (design of the article), the authors rely on the editorial office of the journal according to the requirements.

We are ready to format the article according to any editorial requirements.

Comment #2.

The caption in Figure 8 has a different font size than the rest of the captions, this paper should not contain such elementary errors.

Response to the Comment #2:

Thanks to the reviewer for the comment. The caption for Figure 8 has been corrected

Comment #3.

The experimental process, like the experimental report, is too lengthy (for example, the relevant animal law and its authorized authority in section 2.4 should be placed at the bottom or end of the article page, not in the text), scanning electron microscopy does not give or does not have specific pictures of the specific status of biofilm morphology, there are too many legends, and some unimportant pictures need to be deleted Leave pictures with substantial use.

Response to the Comment #3:

Thanks to the reviewer for the comment. The section behind the animal law from 2.4 has been moved to the Informed Consent Statement section at the end of the article.

Figures 15 and 16 show SEM images of the biofilm on the surface of the samples. Moreover, Figure 16 shows the dynamics of biofilm growth on samples of different composition. We agree with the reviewer and remove the number of legends and captions to improve the visualization of SEM images. However, we ask the reviewer to agree this number of SEM images without modification, as each image reflects a specific result and allows us to prove changes in dynamics. We thank you in advance for your understanding.

Comment #4.

In addition, the article does not mention what are the advantages of developing AL2O3 ceramics on the basis of ZrO2 ceramics, which lacks the embodiment of certain research value, and the reasons should be given in the introduction or conclusion.

Response to the Comment #4:

The following additions have been made to the text of the article (Introduction):

Aluminum oxide (Al2O3) based bioceramics have several advantages over zirconium oxide (ZrO2) based bioceramics:

Higher mechanical strength and hardness. Aluminum oxide has very high strength characteristics, which is important for implants with high stresses.

Greater stability in aggressive environments. The corrosion resistance of Al2O3 is much higher than that of ZrO2. This is important for long-term functioning in the body.

Better bioinertness. Al2O3 is less susceptible to biodegradation and ion leaching, which reduces toxicity risks.

Higher temperature resistance. Al2O3 can be operated at higher temperatures.

Better compatibility with bone tissue by creating porous hydroxyapatite coatings.

Easier production and processing technology. Production of Al2O3 products is cheaper.

No risk of aging and structure degradation inherent in ZrO2.

1. doi: 10.3390/ma14216246
2. https://doi.org/10.1016/0142-9612(95)98860-G
3. https://doi.org/10.1179/1743676111Y.0000000012
4. DOI 10.1088/1757-899X/620/1/012107
5. https://doi.org/10.1007/s12034-018-1612-4
6. DOI: 10.17222/mit.2016.191

Comment #5.

During the preparation of section 2.1, a picture of the sintered sample (cylindrical ceramic sample) should be given to increase the authenticity of the article.

Response to the Comment #5:

Thanks to the reviewer for the comment.

Figure 1 shows the implantation process of ceramic materials obtained by the above method in paragraph 2.1. These samples are represented as ceramic cylindrical specimens. An inset image of a ceramic specimen has been added.

Comment #6.

The specific data of ceramic samples should be given in the form of a chart, but this article only has a text description, which is not intuitive enough

Response to the Comment #6:

Thanks to the reviewer for his comments.

The available important data on the characteristics of the ceramic samples are presented in the text. In particular, specific surface area values of the samples are presented in Figures 7 and 8, relative density and compressive strength values are presented in Figure 12, and Vickers microhardness values are presented in Figure 13.

We have provided all available data. We ask the reviewer to agree with the data presented as they are an important result of the study.

Reviewer 2 Report

The authors present fabrication of alumina/calcium phosphate composites by Spark Plasma Sintering. Different characterizations were made on the powder mixtures and on the sintered samples. The objectives are well presented. The steps of fabrication and the results of characterization are well detailed. However, this paper cannot be published without addressing some comments/issues. Some results can be better explained. 

1) In the introduction, the authors quote different studies about fabrication of composites based on alumina or zirconia and a calcium phosphate phase. They mention the enhanced properties of ceramic composites containing a resorbable phase. But they did not mention the problems related to the secondary phases which can be formed during sintering between alumina or zirconia and calcium phosphate phase. Different authors observed this problem. Besides, in this study, a calcium aluminate phase is formed during sintering of the samples. Can the authors comment this point ?

2) Page 5: The authors present results of granulometric analyses carried out on the powder mixtures. Why did the authors not measure the particle size distributions of the raw powders (i.e., alumina, calcium oxide and calcium hydrophosphate) alone ? It would have enabled to determine the contribution of each powder to the results presented in Figure 2. Moreover, a granulometric analysis carried out before and after the planetary milling could show a possible refinement of the particle size after milling.

3) Page 8-9: The authors mention the phase transformation of tricalcium phosphate from the β to the α phase at around 1180°C. They indicate that XRD peaks of α-TCP are detected on the XRD pattern. But, these peaks are not clearly indexed on the XRD patterns (Figure 6). The peaks corresponding to β-TCP and α-TCP are not distinguished.

4) Page 9: "In our case, Al³⁺ can act as such a substituent coming from Al₂O₃ as shown in [13], which further confirms the beneficial role of the composition chosen for the bioceramics studied here."

With this sentence, the authors claim that Al³⁺ can be a stabilizing cation for β-TCP. But, previously, they highlighted the presence of peaks of α-TCP in their XRD patterns. Can they clarify this point ?  

5) Page 11: Can the authors explain how they identified HAp, TCP or calcium dialuminate in the SEM images ?

6) Page 13 : The authors claim that the 50 wt%HAp-Al₂O₃ samples have higher final relative densities than 20 wt% HAp-Al₂O₃. But this is wrong for the samples sintered at 1200°C.

Minor corrections of English language are needed :

Page 1: Please correct the following sentence: ‘Bionert ceramics is often used’

Page 2: Please correct the following sentence: 'One to the ways...'

Page 2: Please correct: ‘Such resorbable ceramic composites becomes…’

Page 8 : Please correct : ‘On the contrary, (50 wt.% HAp)-Al2O3 sample still contain HAp’

Author Response

Re: «Al₂O₃-phosphate bioceramic fabrication via spark plasma sintering-reactive synthesis: “in vivo” and microbiological investigation» by Papynov E.K. and et al. (Manuscript Number: jcs-2635558)

Dear dear Editors, dear Reviewers,

We deeply appreciate the time you spent reviewing our paper and the valuable recommendations you made. All the comments are taken into account and corresponding changes are made to the manuscript’s body text. Detailed point-by-point answers are presented below.

On behalf of co-authors,

Oleg Shichalin, Researcher, Ph.D.

Response to Reviewers

Reviewer #2:

The authors present fabrication of alumina/calcium phosphate composites by Spark Plasma Sintering. Different characterizations were made on the powder mixtures and on the sintered samples. The objectives are well presented. The steps of fabrication and the results of characterization are well detailed. However, this paper cannot be published without addressing some comments/issues. Some results can be better explained. 

Comment #1.

• In the introduction, the authors quote different studies about fabrication of composites based on alumina or zirconia and a calcium phosphate phase. They mention the enhanced properties of ceramic composites containing a resorbable phase. But they did not mention the problems related to the secondary phases which can be formed during sintering between alumina or zirconia and calcium phosphate phase. Different authors observed this problem. Besides, in this study, a calcium aluminate phase is formed during sintering of the samples. Can the authors comment this point ?

Response to the Comment #4:

Thanks to the reviewer for the comment. Indeed, secondary phases take place during sintering. As indicated by our diffractograms (Figure 6), the secondary phase of calcium aluminate appears at temperatures above 1000 °C. However, following the research objective, it was necessary to determine the optimum temperature to obtain a ceramic biocomposite with an optimal composition. In our opinion, the optimum sintering temperature is 1000 °C, since there is no complete destruction of the hydroxyapatite phase, secondary phases of calcium aluminate are not formed and there is no complete destruction of the porous structure of ceramics. In this regard, all in vivo studies were carried out with samples obtained at 1000 °C.

Comment #2.

2) Page 5: The authors present results of granulometric analyses carried out on the powder mixtures. Why did the authors not measure the particle size distributions of the raw powders (i.e., alumina, calcium oxide and calcium hydrophosphate) alone ? It would have enabled to determine the contribution of each powder to the results presented in Figure 2. Moreover, a granulometric analysis carried out before and after the planetary milling could show a possible refinement of the particle size after milling.

Response to the Comment #2:

Thanks to the reviewer for his comment. According to the well-known principles of ceramic materials production by sintering methods, a significant contribution to the quality of the formed ceramics is made by the starting mixture, namely particle size. The particle size-structure relationship is well known (the smaller the particle size, the more structured the material). Agglomerates also make a significant contribution to the organization of the ceramic structure. In order to improve the quality of ceramics, the powder mixture is preliminarily subjected to the process of mechanical activation (in this case grinding in a ball mill). It is also known that the ball mill grinding method can achieve particle sizes of 1-10 µm, which was obtained and is presented in Figure 2. Taking this into account, the authors do not see the expediency of determining the particle size distribution of the initial powders that make up the powder mixture for further sintering, because in any case the size distribution of the starting mixture will be reduced to the values that allow to obtain ball milling. The dimensionality of the starting powders in this case has no significant influence on the mixture, given the composition of the powders and the material of the ball bodies.

We respectfully ask the reviewer to accept our answer as reliable, since the authors do not see the point of conducting these analytical experiments, since the important indicator for sintering are the parameters of the already milled mixture.

Comment #3.

3) Page 8-9: The authors mention the phase transformation of tricalcium phosphate from the β to the α phase at around 1180°C. They indicate that XRD peaks of α-TCP are detected on the XRD pattern. But, these peaks are not clearly indexed on the XRD patterns (Figure 6). The peaks corresponding to β-TCP and α-TCP are not distinguished.

Response to the Comment #3:

Figure 6 is complementary. We thank the reviewer for a thorough review.

Comment #4.

4) Page 9: "In our case, Al³⁺ can act as such a substituent coming from Al₂O₃ as shown in [13], which further confirms the beneficial role of the composition chosen for the bioceramics studied here."

With this sentence, the authors claim that Al³⁺ can be a stabilizing cation for β-TCP. But, previously, they highlighted the presence of peaks of α-TCP in their XRD patterns. Can they clarify this point ?  

Response to the Comment #4:

Thanks to the reviewer for the comment.

Indeed the stabilization effect exists and has been described in the literature. In our study, we refer to an open source of information and rely on the validity of the statement of fellow scientists. In our case, we have identified the α-TCP phase, however, judging from the experimental data, its amount is very small. That is, its formation is quite possible even at stabilization in the presence of Al3+. First, this may be due to the lack of Al3+ to stabilize the entire amount of the β-TCP phase. Secondly, non-uniform diffusion of Al3+ into the volume of the β-TCP phase is probable due to the inefficient homogenization of the initial mixture of powders during sintering. Third, insufficient rate and depth of Al3+ diffusion into the β-TCP structure. Fourth, the limit of the temperature value at which stabilization may no longer occur has not been determined. In general, this is a separate fundamental study, which was not our goal in this work. Again, the optimal temperature for these samples was set at 1000 ºC.

Comment #5.

5) Page 11: Can the authors explain how they identified HAp, TCP or calcium dialuminate in the SEM images ?

Response to the Comment #5:

Thanks to the reviewer for his comment. The fact of presence of phases indicated on SEM images is proved by XRD analysis. Differences in surface morphology of different phases on the samples are visually noticeable. Based on the phase composition, as well as taking into account the composition of the samples, we can come to the logical conclusion that the areas with loose structure are represented by unreacted aluminum and calcium oxides. Accordingly, the regions of monolithic structure consist of Hap and TCP. This is also confirmed by the EDX analysis, since phosphorus is present in the monolithic structure regions. An increase in temperature above 1000 °C is accompanied by the formation of calcium aluminate, which also forms a monolithic structure. Indeed all three phases Hap, TCP and calcium aluminate cannot be separated in SEM images as they are represented by monolithic agglomerates, however these agglomerates are clearly visible. Separate identification of each of these phases was not an objective of the study. Samples with the aluminate phase were not optimal for the study and were not considered for further biological investigation. Samples with GAP and TCF phases do not require phase separation in terms of bioceramic properties and utilization, as both phases have similar biocompatible properties.

We ask the reviewer to accept our explanation and agree with it.

Comment #6

6) Page 13 : The authors claim that the 50 wt%HAp-Al₂O₃ samples have higher final relative densities than 20 wt% HAp-Al₂O₃. But this is wrong for the samples sintered at 1200°C.

Response to the Comment #6:

Thanks to the reviewer for the comment. We have rechecked the data and verified that the RD values presented in Figure 12 are correct. Corrections to the text have been made. We thank you for your careful review of the results.

Round 2

Reviewer 1 Report

The authors have revised the paper. It is ready for publication.

Reviewer 2 Report

The authors answered to all the questions. I recommend the acceptation of the article. 

Ok