Plasma-Sprayed Hydroxyapatite Coatings and Their Biological Properties

Round 1

Reviewer 1 Report

This paper reports the characterization of a method to coat titanium with hydroxyapatite via air plasma spraying, with studies of the structural properties of the deposited material and assessment of its bio-compatibility with in vitro tests. The results are interesting and could be useful for the community working on materials for implantology and I think it could be published after some minor revisions.

-          Size of Ti substrate: it is reported as 300x10x3 mm (line 151) without saying which one is the height, width etc, i.e. without specifying on which side deposition is made. Why then this size later on becomes 10x10x3 (line 245, with a typo)? Does other substrate have been used or simply some samples portions have been cut out from the originally deposited one? Why are not specified different substrate sizes for different test in the deposition section 2.1?

-          Please clarify the meaning of the so-called “powder parameters” d10, d50 and d90 at line 275

-          SEM images: I cannot find any experimental detail to explain how does cross section of samples have been obtained. Was it a mechanical cutting, an ion milling process, a combination of the above or other? Was the sample previously embedded in some kind of material, resin or similar (it looks so from fig 2c and 4a,c,e but nothing is specified)? Furthermore, the quality of the reported images is very poor. In particular, I cannot see the “lamellar structures” claimed by the authors in fig 4 (claim at line 286). Higher resolution and/or much higher quality images should be provided to support the claim. About the surface morphology in figure 5, all images show pretty bad horizontal lines likely due to charging of sample under electronic beam. Wasn’t it possible to find current/acceleration voltages combination to suppress this bad effect (by the way, authors should also provide this information for all SEM images shown)? If not, isn’t it possible to coat with some very thin layer of metal the surface to avoid this? Furthermore, the sentence at lines 306-307 does not make any sense.

-          Statements from line 344 to line 348 are not much convincing: actually, all these parameters in table 2 of surface morphology (Sku, Sp and Sv) have values that falls pretty much within each other’s uncertainty so in my opinion are not so different to draw any such conclusion. The authors mention in the methods section a statistical test they used to assess the surfaces have statistically different morphologies are different, but they do not detail if the statistical method was used for all the different parameters. While Sa ad Sq seems to show a clear variation in the values from one sample to the other, the same does not seem to happen so clearly for the other parameters. Furthermore about table 2, why is uncertainty of Sku value of HA1 sample so incredibly small compared to other values? Same anomalous small uncertainty results for Sp of the same sample.

-          In general, I am not convinced the reported results support the statement that “The results obtained from the physicochemical and biological characterizations presented in this study suggested that plasma-spraying distance during the HA coating process had a significant impact on the parameters of the obtained coating. The best results were obtained for a distance of 120 mm” (Line 465 and followings), which is repeated and stressed also in the abstract and introduction. The clear difference between the surfaces is a certain level of surface roughness, but all cell viability and biocompatibility as well as bacterial adhesion tests do not give results that justify this assertion. In all these tests numerical results fall well within each other’s uncertainty, so it is not clear to this reader why the distance plays such an important role for the final results, i.e. the application as a coating for medical implants.

-          Reference 4 seems to be in Polish: doesn’t it exist a translation in English? Please consider removing or replacing it

 Few mistakes along the text:

-          At line 60 APS is not introduced as an acronym (it was in the abstract but it is usually good practice to do it independently in the text)

-          Line 123 change “associates with; … “ with “associated with …“

-          Line 136 replace “novelty” with “new” or any other equivalent adjective you might prefer

-          2 mistakes in figure 1, “gade” instead of “grade” and “Bacterias” instead of “Bacteria”

Author Response

Dear Reviewer 1,

Thank you for your reliable assessment and comments regarding our manuscript entitled "Plasma sprayed hydroxyapatite coatings and their biological properties". The paper has been improved, taking into consideration your suggestions. We have attached the revised paper with corrections introduced in the text. The added and corrected text is red-marked. Below, please, find enclosed answers to your comments:

-          Size of Ti substrate: it is reported as 300x10x3 mm (line 151) without saying which one is the height, width etc, i.e. without specifying on which side deposition is made. Why then this size later on becomes 10x10x3 (line 245, with a typo)? Does other substrate have been used or simply some samples portions have been cut out from the originally deposited one? Why are not specified different substrate sizes for different test in the deposition section 2.1?

Thank you for this remark. Typically, sprayed samples are deposited on the largest surface of the substrate, and then cut into samples of the required shape and quantity needed for testing. In this case, the sprayed Ti substrate was sized; 300 mm long, 10 mm width and 3 mm thick. The HA coating was sprayed over an area of 300 mm x 10 mm. Then, for further research, it was cut into smaller samples 10 mm long. The description was supplemented with these stepson lines 155-160.

„HA coatings were deposited from XPT-D-703 powder (Sulzer Metco, Salzgitter, Germany) by a plasma-spraying process on 300×10×3 mm bars of grade 2 titanium on the one 300x10 mm side. The plasma-spraying process was performed by axial powder injection on an Axial III system (Northwest Mettech Corp., Surrey, Canada) with a Thermal Miller 1264 powder feeder. After deposition bars were divided into smaller square samples with dimensions 10 x 10 mm with a diamond saw.”

-          Please clarify the meaning of the so-called “powder parameters” d10, d50 and d90 at line 275

Thank you for this comment. Powders of the same grain size range may have different grain size distribution. That is, for example, it may contain a lot of fine grains or, on the other hand, it may contain a lot of coarse grains. The given parameters of the powder are ; d10 – the portion of particles with diameter smaller than this value is 10%, d50 - the portion of particles with diameter smaller and larger than this value are 50% and d90 - the portion of particles with diameters below this value is 90%.

-          SEM images: I cannot find any experimental detail to explain how does cross section of samples have been obtained. Was it a mechanical cutting, an ion milling process, a combination of the above or other? Was the sample previously embedded in some kind of material, resin or similar (it looks so from fig 2c and 4a,c,e but nothing is specified)? Furthermore, the quality of the reported images is very poor. In particular, I cannot see the “lamellar structures” claimed by the authors in fig 4 (claim at line 286). Higher resolution and/or much higher quality images should be provided to support the claim. About the surface morphology in figure 5, all images show pretty bad horizontal lines likely due to charging of sample under electronic beam. Wasn’t it possible to find current/acceleration voltages combination to suppress this bad effect (by the way, authors should also provide this information for all SEM images shown)? If not, isn’t it possible to coat with some very thin layer of metal the surface to avoid this? Furthermore, the sentence at lines 306-307 does not make any sense.

Thank you for this comment. The specimens for microstructure analysis were cut into a size of 10 mm x 10 mm x 3 mm (perpendicular to the deposited coating). Then samples were embedded in resin and polished using silicon carbide (SiC) paper, with ethanol as a lubricant, starting from 600 to 1200 grit and finishing with diamond suspension. Next gold sputter coating was deposited onto HA coating cross-section. Appropriate descriptions have been introduces in Fig. 4. Moreover, additional Figures have been added to show the lamellar structure of HA coatings. As above described, the surface of HA coatings (Fig. 5) were also covered with a layer of gold. However, the surface of HA coatings after spraying is not equal as in the case of the polished cross-sections. Therefore there are slight shadows that are difficult to avoid. The sentence at lines 306-307 has been deleted.

-     Statements from line 344 to line 348 are not much convincing: actually, all these parameters in table 2 of surface morphology (Sku, Sp and Sv) have values that falls pretty much within each other’s uncertainty so in my opinion are not so different to draw any such conclusion. The authors mention in the methods section a statistical test they used to assess the surfaces have statistically different morphologies are different, but they do not detail if the statistical method was used for all the different parameters. While Sa ad Sq seems to show a clear variation in the values from one sample to the other, the same does not seem to happen so clearly for the other parameters. Furthermore about table 2, why is uncertainty of Sku value of HA1 sample so incredibly small compared to other values? Same anomalous small uncertainty results for Sp of the same sample.

Thank you for thorough analysis of the results. Sa is the extension of Ra (arithmetical mean height of a line) to a surface. It express, as an absolute value, the difference in height of each point compared to the arithmetical mean of the surface. This parameter is used generally to evaluate surface roughness. However, it is only a mathematical mean, therefore all surface parameters were measured in accordance with the ISO 25178 standard. The results obtained are the average of three measurements. In the case of parameters Sa, Sq, Sv and Sz, there is almost a 20% difference between the HA2 coating and the HA1and HA2 coatings. Ssk represents the degree bias of the roughness shape. Values for all HA coatings are Ssk>0, which means, that in all cases height distribution is skewed blow the mean plane. In the case of Sku all values hover aroud 3 which means even distribution both hills and valleys on the surface of the HA coatings. Sp is the height of the highest peak with the defined area. The difference between HA2 and HA1 coating is 6% and between HA2 and HA3 is 1%. No statistical tests have been conducted. Statements from line 344 to line 348 have been modified; “In the case of the HA3 coating sprayed from a distance of 140 mm, the 20% lower value of this parameter was a result of the much longer residence time of the HA powder grains in the plasma stream, which resulted in their greater melting and deformation. The highest value of the Sa, Sq, Sp and Sz parameters in the case of the HA2 were the result of a lower melting of the powder grains (Table 2).”

   -    In general, I am not convinced the reported results support the statement that “The results obtained from the physicochemical and biological characterizations presented in this study suggested that plasma-spraying distance during the HA coating process had a significant impact on the parameters of the obtained coating. The best results were obtained for a distance of 120 mm” (Line 465 and followings), which is repeated and stressed also in the abstract and introduction. The clear difference between the surfaces is a certain level of surface roughness, but all cell viability and biocompatibility as well as bacterial adhesion tests do not give results that justify this assertion. In all these tests numerical results fall well within each other’s uncertainty, so it is not clear to this reader why the distance plays such an important role for the final results, i.e. the application as a coating for medical implants.

Thank you for insightful analysis of the results. The conclusions have been revised based on your suggestions: “The results obtained from the physicochemical and biological characterizations presented in this study suggested that plasma-spraying distance during the HA coating process had a little effect on their biocompatibility. The results obtained for a distance of 120 mm showed a slight increase in the biological properties tested.”

-          Reference 4 seems to be in Polish: doesn’t it exist a translation in English? Please consider removing or replacing it

Thank you for this remark. This book, has not been translated into English. For the convenience of readers, we have replaced this item with one of his team's numerous articles that cover the topics discussed. Due to the length of the scientific article, they are described in a much more simplified form.

Few mistakes along the text:

-          At line 60 APS is not introduced as an acronym (it was in the abstract but it is usually good practice to do it independently in the text)

-          Line 123 change “associates with; … “ with “associated with …“

-          Line 136 replace “novelty” with “new” or any other equivalent adjective you might prefer

-          2 mistakes in figure 1, “gade” instead of “grade” and “Bacterias” instead of “Bacteria”

Thank you very much for pointing it. Errors have been corrected.

Reviewer 2 Report

This manuscript presents the hydroxyapatite coating with different plasma-spray distances. Valuable results were obtained. The following concerns, however, must be addressed:

1. A careful reading of the text should be done to suppress language errors.

2. In "Abstract", don’t need to detail the material specifications (e.g., Commercial Sulzer Metco HA powder) and the specific name of the equipment (e.g., Scanning Electron Microscopy (SEM), optical profilometry, MTT (3-(4,5-Dimethylthiazol-2-yl) reduction assay). Please pay more attention to new findings.

3. In Section 3, it is mentioned that “Cross-sections of coatings HA1, HA2, and HA3 are shown in Figure 4 a–f, … also resulted in part from the significant differentiation of the remelting of HA powder grains, which led to a wide grain size distribution.” Is it possible to analyze the grain size distribution from Figure 4? Please explain.

4. Please rewrite “4. Conclusions”.

Author Response

Dear Reviewer 2,

We want to thank you for spending your time and effort to improve the quality of our manuscript. We highly appreciate it. The paper has been improved, taking into consideration your suggestions. We have attached the revised paper with corrections introduced in the text. The added and corrected text is red-marked. Below, please, find enclosed answers to your comments:

1. A careful reading of the text should be done to suppress language errors.

Thank you for this remark. At present, it is really difficult to find native speaker with technical background in surface engineering and biological science.  Moreover, this manuscript has been translated by professional company. We have tried out best to check and polish the English. If accepted the manuscript will be improved by COATINGS editorial office.

2. In "Abstract", don’t need to detail the material specifications (e.g., Commercial Sulzer Metco HA powder) and the specific name of the equipment (e.g., Scanning Electron Microscopy (SEM), optical profilometry, MTT (3-(4,5-Dimethylthiazol-2-yl) reduction assay). Please pay more attention to new findings.

Thank you for this comment. The abstract section has been corrected.

3. In Section 3, it is mentioned that “Cross-sections of coatings HA1, HA2, and HA3 are shown in Figure 4 a–f, … also resulted in part from the significant differentiation of the remelting of HA powder grains, which led to a wide grain size distribution.” Is it possible to analyze the grain size distribution from Figure 4? Please explain.

Thank you for insightful analysis of the manuscript. It is very difficult to analyse the grain size distribution from cross-section of plasma sprayed coating. This statement has been deleted. 

4. Please rewrite “4. Conclusions”.

Thank you for this remark. Conclusions has been rewritten; “The results obtained from the physicochemical and biological characterizations presented in this study suggested that plasma-spraying distance during the HA coating process had a little effect on their biocompatibility. The results obtained for a distance of 120 mm showed a slight increase in the biological properties tested.”

Round 2

Reviewer 2 Report

The authors stated that modifications were made according to Comments 2 and 3. In fact, they did not do so.

Author Response

Dear Reviewer 2,

The authors stated that modifications were made according to Comments 2 and 3. In fact, they did not do so.

Thank you for your comments regarding our manuscript entitled "Plasma Sprayed Hydroxyapatite Coatings and Their Biological Properties". The paper has been improved, taking into consideration your suggestions. We have attached the revised paper with corrections introduced in the text. The added and corrected text is red-marked. Below, please, find enclosed answers to your comments:

2. In "Abstract", don’t need to detail the material specifications (e.g., Commercial Sulzer Metco HA powder) and the specific name of the equipment (e.g., Scanning Electron Microscopy (SEM), optical profilometry, MTT (3-(4,5-Dimethylthiazol-2-yl) reduction assay). Please pay more attention to new findings.

Thank you for this comment. The abstract section has been corrected and all trade and apparatus  markings have been removed. Below, please, find enclosed Abstract after correction.

Air plasma spraying (APS) is a common method of producing hydroxyapatite (HA) coatings for alloprosthetic implants. Modification of HA spraying potentially may diminish the risk of inflammation and local infection during bone implantation. Titanium implants were HA coated with different deposition process parameters. HA powder was deposited using APS with axial powder injection at three different distances (100, 120, and 140 mm). Surface morphology were examined and direct contact cytotoxicity of HA coatings was evaluated according to norm ISO 10993 5:2009. The response of monocytes to HA was assessed via the activation of transcription nuclear factor. All coatings had a lamellar structure. HA sprayed at a distance of 120 mm showed the highest roughness and little phase change. The analysis of the results of the conducted research showed that plasma-spraying distance during the HA coating process had a negligible impact on their biocompatibility. The results obtained for a distance of 120 mm showed a slight increase in the biological properties tested. Moreover, HA coatings sprayed with different distances were not cytotoxic and did not stimulate the NF-kB. Bare titanium was less susceptible to colonization by Staphylococcus aureus than HA coated surfaces. HA constitutes a potentially good, low-cost, non-cytotoxic material for joint prostheses.

3. In Section 3, it is mentioned that “Cross-sections of coatings HA1, HA2, and HA3 are shown in Figure 4 a–f, … also resulted in part from the significant differentiation of the remelting of HA powder grains, which led to a wide grain size distribution.” Is it possible to analyze the grain size distribution from Figure 4? Please explain.

Once more thank you for careful analysis of ours paper. The sprayed hydroxyapatite powder consists of grains of very different sizes. There are 10% grains with diameter smaller than 3.80 µm, 50% grains with diameter smaller and larger than 69.82  µm and 90% of particles with diameters below 146.30 µm. During the plasma spraying process the melt to varying degrees, depending on size and position in the plasma stream and form lamellas of varying thickness. Moreover, not all the powder grains form the coating, some of the small grains evaporate and some of large unmelted grains are reflected from the surface. Therefore it is very difficult to analyse the grain size distribution from cross-section of plasma sprayed coating.

Cross-sections of coatings HA1, HA2, and HA3 are shown in Figure 4 a–i, respectively, which clearly revealed their lamellar structures. The highly deformed lamellas were characterized by significant internal porosity, which stemmed from the porosity of the HA powder grains. It also resulted in part from the significant differentiation of the remelting of HA powder grains depending of their size and position in the plasma stream.