The Study of Nanosized Silicate-Substituted Hydroxyapatites Co-Doped with Sr²⁺ and Zn²⁺ Ions Related to Their Influence on Biological Activities

Round 1

Reviewer 1 Report

I would like to thank for the opportunity to review this paper.

This paper focused on the nanosized silicate-substituted hydroxyapatites co-doped with Sr2+ and Zn2+ ions. The authors characterized it by XRD (X-Ray Powder Diffraction), the Fourier-Transformed Infrared spectroscopy (FT-IR), and Scanning Electron Microscopy techniques (SEM). The antimicrobial activity, antibiofilm activity, hemolytic activity, and MTT assay were performed by the authors to evaluate the impact of this product on biological activities. The Ames test was also performed to investigate mutagenic potential of this product towards bacterial. The authors concluded that this product exhibit dose-dependent antibacterial activity against Klebsiella pneumoniae and Escherichia coli, but however, no inhibition of biofilm formation. Also, no hemolytic and mutagenic properties were found for this material. The authors also claimed that the biological activity of Sr2+/Zn2+-doped Si-HAp is increased with the increase of Zn2+ ions content as well as the number of silica groups.

 

 

Here are some comments for this paper:

This paper investigates the nanosized silicate-substituted hydroxyapatites co-doped with Sr2+ and Zn2+ ions, however, what are the advantages to use both Sr2+ and Zn2+? I did not find any comparison between dual ions and singular ion materials (such as Zn only or Sr only).

 

Line 253: in the part of 3.2. SEM-EDS analysis

The unit of mol% means the amount of the specific component to the total using the same units (As an example: 10 mol% means 10 moles of that components mixed with 90 mole of the rest of components). Please describe the equation in more detail. Such as in equation 1, mol% presents in both numerator and denominator, so the unit should be 1, not mol%. And on table 1. If Sr2+, Zn2+ and (SiO4)4 (should this be SiO4?) added up to less than 5 mol%, what are the rest 95 mol?

Figure 3a and 3b. From the SEM images, I can not tell that obtained powders are of round shaped.

Figure 4. Where is the peak for Sr2+ in the orange (bottom) curve? And this figure did not include the transmission FT-IR spectra of Ca9.8-xSr0.2Znx(PO4)6-y(SiO4)y(OH)2 (where: x = 0.5 – 3.5 [mol%] and y = 4 – 5 [mol%]) which has both Sr2+ and Zn2+. For the red curve in the middle (5mol%Zn2+:4Si-HAp), why in this curve the OH and H2O close to 3500 cm-1 are significantly different from the other curves?

Figure 5. What is the control group? As Ca9.8-xSr0.2Znx(PO4)6-y(SiO4)y(OH)2 is a solid material, the control should be similar particle but with no activity.

Figure 6. the images are too dim, and a bright field image should be included. A positive control will better support the results.

 

Line 87-88: “Surprisingly, even the prolonged exposure on silica nanoparticles both by skin exposure, oral exposure and even respiratory system exposure is confirmed to be safe.” Any evidence for this? As we know, Pneumoconiosis may be caused by inhaling silica dust.

 

Line 139-141: “Bacterial suspensions were incubated for 18 h at 37°C with shaking (120 rpm) in the presence of 10, 25, 50 and 100 μg/ml of the tested silicate-substituted strontium-hydroxyapatite (colloidal solutions prepared in the saline).” What is the media used for the bacterial culture, Lysogeny broth (LB)? or just the saline (0.9% NaCl)? Why incubate for 18 h at 37°C? The incubation time may be too long. Growth curves with gradient material concentrations may be better to indicate the antimicrobial activity of this material.

Author Response

Dear Editor,

 

We would like to express our sincerest gratitude to the Reviewer for the enormous efforts in criticizing the manuscript. We have taken into account all raised question here follows the detailed answers to the Reviewer.

 

 

Reviewer 1:

 

Question 1: This paper investigates the nanosized silicate-substituted hydroxyapatites co-doped with Sr²⁺ and Zn²⁺ ions, however, what are the advantages to use both Sr²⁺ and Zn²⁺? I did not find any comparison between dual ions and singular ion materials (such as Zn only or Sr only).

 

Answer: There has been shown only the sample doped with Sr²⁺ ions in this study. The sample is marked as 0.0 mol% Zn²⁺/Sr²⁺:Si₄-HAp. Moreover, in our previous research it was evaluated antibacterial activity of Sr²⁺ ion-doped Hap (https://doi.org/10.1016/j.jinorgbio.2019.110884) and Zn²⁺ ion-doped HAp (https://doi.org/10.1016/j.msec.2021.112547), separately. Our research revealed that strontium did not possess antibacterial properties but as has been mentioned, in the literature, the doping of the Sr²⁺ ions can help to dissolve the apatite compounds (“Moreover, the Sr²⁺ ion was added to enhance water-solubility”). Therefore. It was decided to co-dope the HAp with Sr²⁺ and Zn²⁺ ions showing an antibacterial effect as in our previous research. Hence, it was chosen to use strontium-doped hydroxyapatite as a matrix in this research. In conclusion, the aim of the research was to improve both physical and biological properties of hydroxyapatite to enhance its applicability. 

 

Question 2: Line 253: in the part of 3.2. SEM-EDS analysis

The unit of mol% means the amount of the specific component to the total using the same units (As an example: 10 mol% means 10 moles of that components mixed with 90 mole of the rest of components). Please describe the equation in more detail. Such as in equation 1, mol% presents in both numerator and denominator, so the unit should be 1, not mol%. And on table 1. If Sr2+, Zn2+ and (SiO4)4 (should this be SiO4?) added up to less than 5 mol%, what are the rest 95 mol?

Answer: It is described in the line 117 in the main text. The molar content od dopant ions (x mol%) means the fraction of calcium ions concentration. The HAp molecule is constructed with the 10 calcium ions. In case of presented study the molar concentration of dopant ions is associated with this value.

Question 3: Figure 3a and 3b. From the SEM images, I cannot tell that obtained powders are of round shaped.

Answer: We would like to thank you for pointing it out. We agree, and it was a mistake. The HAp crystal are not exactly round. The shape of the crystals is more irregular, and not perfectly rounded. It is clarified in the main text.

 

Question 4: Figure 4. Where is the peak for Sr²⁺ in the orange (bottom) curve? And this figure did not include the transmission FT-IR spectra of Ca_9.8-xSr_0.2Zn_x(PO₄)₆-y(SiO₄)y(OH)₂ (where: x = 0.5 – 3.5 [mol%] and y = 4 – 5 [mol%]) which has both Sr²⁺ and Zn²⁺. For the red curve in the middle (5mol%Zn²⁺:4Si-HAp), why in this curve the OH and H₂O close to 3500 cm-1 are significantly different from the other curves?

Answer: We are grateful for your astute review. The FT-IR spectra have been changed. The inscription of the sample has been corrected. Currently, all samples are presented on the spectra. There are presented the spectra of the investigated samples in the range of 4000 – 3000 cm^-1. In this range bands associated with OH^- groups and water molecules are located. It is marked with dotted line for OH^- groups. In the case of all samples this band is located at 3569 cm^-1. The wide band associated with the water molecules is detected in the range of 3780 – 3250 cm^-1. The water contained in the crystals and also water absorbed on the surface can influence on the bands intensity. Therefore, the intensity of this band is not equal to the water concentration in the hydroxyapatite powder.

Question 5: Figure 5. What is the control group? As Ca_9.8-xSr_0.2Zn_x(PO₄)₆-y(SiO₄)y(OH)₂ is a solid material, the control should be similar particle but with no activity.

Answer: In our opinion, it would be really hard to find “similar particle but with no activity” because all nanoparticles have activity on bacterial cell which is by no means dependable on their physicochemical properties. Therefore, it was decided to use standard control (bacterial suspension in the saline) as well as hydroxyapatite without zinc ions. It turned out that the bacterial survivability is quite similar in both cases. We used that protocol in the previously published research (https://doi.org/10.3390/biom11091388, https://doi.org/10.1016/j.jinorgbio.2019.110884).

 

Question 6: Figure 6. the images are too dim, and a bright field image should be included. A positive control will better support the results.

 Answer: Thank you for the suggestion. We brightened all images by 20%. Taking microscopy images on pellets with bright field is rather hard to be done because of pellet’s thickness that prevent light transmission. In case of the positive control, it is always advisable to use one in the research, however, in our experiment it would be difficult to find the appropriate one (similarly as in the case of non-active nanoparticles).

Question 7: Line 87-88: “Surprisingly, even the prolonged exposure on silica nanoparticles both by skin exposure, oral exposure and even respiratory system exposure is confirmed to be safe.” Any evidence for this? As we know, Pneumoconiosis may be caused by inhaling silica dust.

Answer: Thank you for pointing this out. The evidence has been provided in the main text. However, it has also been proven that exposure to silica nanoparticles can lead to respiratory diseases but it is caused by occupational silica dust exposure. Moreover, to develop chronic radiographic silicosis person should be exposed at least for 40 to 45 years at level of silica particles of 0.05 mg/m³ in the air [https://doi.org/10.1111/all.14202]. The obtained nanomaterials are substituted with silica and phosphate groups in hydroxyapatite crystal lattice. Therefore, there are not silica particles and the risk for exposure is none.

 Question 8: Line 139-141: “Bacterial suspensions were incubated for 18 h at 37°C with shaking (120 rpm) in the presence of 10, 25, 50 and 100 μg/ml of the tested silicate-substituted strontium-hydroxyapatite (colloidal solutions prepared in the saline).” What is the media used for the bacterial culture, Lysogeny broth (LB)? or just the saline (0.9% NaCl)? Why incubate for 18 h at 37°C? The incubation time may be too long. Growth curves with gradient material concentrations may be better to indicate the antimicrobial activity of this material.

Answer: We used that protocol in the previously published research (https://doi.org/10.3390/biom11091388, https://doi.org/10.1016/j.jinorgbio.2019.110884). We did not use bacterial culture media because they influence particles “behavior” which we discussed previously. Moreover, the inorganic and organic components of culture media may inhibit the activity of divalent ions, like Zn²⁺ ions, which we described here: https://doi.org/10.3390/biom12070963. Hence, we decided to use the most “neutral” solution (0.9% NaCl). The incubation time was set based on previous research and its results. The standard incubation time for bacteria is usually 24 h, however, we found out that it was too long and the results were not replicable because a lot of bacterial cells were dying. On the other hand, when we tested more active Ag⁺ we exposed bacteria for only 2 hours on reduced concentrations of hydroxyapatites (described here: https://doi.org/10.1021/acsomega.0c03044). Zinc is, by no means, less active than silver, so we decide to expose bacteria for a longer time which was experimentally set at 18 h. We agree that growth curves could be better, however, as we mentioned above we decided against using culture media in the research, hence, only survival rate in different incubation times could be applied in that case. In our opinion such modification was not necessary here.

Author Response File: Author Response.pdf

Reviewer 2 Report

Figure 1. Y axis - remove arbitary units, label the miller indices represented by the peaks

Figure 3. Nanoparticles are aggregated. please explian why

Figure 4 . Please improve the quality of the figure

Explain the reasons behind choosing hydroxyappatite and silica

Author Response

Dear Editor,

 

We would like to express our sincerest gratitude to the Reviewer for the enormous efforts in criticizing the manuscript. We have taken into account all raised question here follows the detailed answers to the Reviewer.

 

 

Reviewer 2:

 

Question 1: Figure 1. Y axis - remove arbitary units, label the miller indices represented by the peaks

Answer: It is changed.

Question 2: Figure 3. Nanoparticles are aggregated. please explain why.

Answer:  The self-aggregation process is common in case of nanocrystals. The forces that are responsible for this feature are associated with intramolecular or intraparticle interactions. The presence of der Waals forces, dipole−dipole force, electrostatic interaction, and hydrogen bonds results in the self-aggregation of the particles [https://doi.org/10.1021/jp802361r; https://doi.org/10.1038/35008037]. Moreover, preparation for SEM imaging does not counteract the aggregation process. The explanation has been added to the main text.

 

Question 3:Figure 4 . Please improve the quality of the figure

Answer: We can submit the figures as high quality attachments directly to the MDPI system if there is a need for that. We are open for the Editor’s suggestions in that case. 

Question 4: Explain the reasons behind choosing hydroxyapatite and silica.

Answer: The main reason for choosing hydroxyapatite is the fact that HAp-based nanomaterials are known for their biocompatibility and non-toxicity towards eukaryotic cells. Moreover, hydroxyapatite, as well as silica, are known for their regenerative properties which is widely studied on osteoblast’s model. Hydroxyapatite is also an “attractive” matrix due to the fact that calcium cations are easily substituted with other cations, for example Zn²⁺, Cu²⁺, Ag⁺ which are known for their antibacterial properties.

To sum up, we decided to use hydroxyapatite matrix modified with silicate groups and partly substituted with zinc ions to combine regenerative properties with antibacterial activity.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors addressed most of my concerns. 

The only question is the usage of the saline (0.9% NaCl) for bacteria culture. And 18 h at 37°C is too long incubation time. In this condition, bacteria may not be health.

Author Response

Dear Editor,

 

We would like to express our sincerest gratitude once again to the Reviewer for the enormous efforts in criticizing the manuscript. We have taken into account all raised question here follows the detailed answer to the Reviewer.

 

 

Reviewer 1:

 

Question 1: The only question is the usage of the saline (0.9% NaCl) for bacteria culture. And 18 h at 37°C is too long incubation time. In this condition, bacteria may not be health.

Answer: As has been mentioned in the previous round of reviews we set that incubation time based on the preliminary studies. The Figure 5 shows the CFU/ml values (Log) of the control sample spread on the agar plate directly after preparing bacterial suspension in the saline. Moreover, the same sample (control) but after 18 h of incubation and bacterial suspension with 1% of hydroxyapatite doped with Eu³⁺ ion (the results for Eu³⁺ are published here: https://doi.org/10.1016/j.jinorgbio.2019.110884, https://doi.org/10.3390/biom11091388).

It turned out that CFU/ml value is almost the same after P. aeruginosa incubation in the presence of hydroxyapatite as it was in the 0h control sample. The value for the control after 18h of incubation was slightly lower. It has caught our attention and we decided to check it for other bacteria and surprisingly the values in the controls (0h and 18h) for E. coli were exactly the same. But there is an explanation of this phenomenon and, in short, P. aeruginosa cell tend to agglomerate in the pure saline in time that is prevented in the nanoparticle-supplemented saline. E. coli is less prone to this process. Therefore, we started to treat P. aeruginosa samples with “special care” which consisted on vortexing them longer and more vigorously and it helped. The literature indicates that this problem could be also solved by adding chemicals (for example Tween) to the saline but we decided against, because it could also influence the obtained results. In this case, we are convinced that our incubation time is not too long and unhealthy for bacteria.

Author Response File: Author Response.pdf