Evaluation of Mesenchymal Stem Cells and Osteoblasts’ Adhesion and Proliferation in the Presence of HA-AL Biomaterials

Round 1

Reviewer 1 Report

The manuscript is quite well written. It deals with evaluation of the biocompatibility of physically deposited composite of hydroxyapatite and alendronate (HA-AL). The authors used the Matrix Assisted Pulsed Laser Evaporation technique to develop HA-LA film onto titanium surface, which afterwards can be characterized by improved bone cells and human mesenchymal stem cells adhesion and increased number of cell-biomaterial focal points. The presented research can be considered as an interesting development of the research done with HA_AL composite material and neat hydroxyapatite deposited by MAPLE  technique. Therefore, this manuscript is recommended for publication after addressing the following items.

1. Line 31; the acronym MAPLE is repeated (line 27)

2. Lines 95-105 and 106-114; wet method used to obtain HA and the HA-AL synthesis have been reported previously what should be mention somewhere in the text (as they are not new methods).

3. Line 111; figure 1 is not very aesthetic - different letter sizes are used

4. Lines 117-120; there is a lack of the proper description of the infrared spectroscopy and X-Ray diffraction measurements. It is not clear if the presented results are done and analyzed for the purpose of this paper or they are cited form the previous work.

5. Line 121; fluence and the target-substrate distance are missing.

6. Line 148 the explanation of the abbreviations are missing

7. Line 177; Figure 2 – wavenumbers indication to two decimal places has no physical sense, they are beyond the capabilities of the spectrometer measurement. Does the spectrum 'a' belong to the deposited composite film?

8. Lines 179-184; Authors mention that “Both spectra show the 1644 cm ‐1 peak that corresponds to the N-H scissoring vibration.” I see it only on the spectrum 'b'. The sentence seems to be wrong or it is very unclear. Similarly (line 182-3) “The HA‐AL composite spectrum also shows the 1565 cm ‐1 peak that characterizes the –OH stretching vibration of hydroxyapatite molecule.” – there is no such a peak (absorption band) on the spectra (Fig 2 a or b) and what is more, typical -OH stretching vibration is around 3500cm-1 not 1565 cm-1 so the authors statement has to be more explained.

9. There is missing an important information – the conformation if the structure of the HA-AL composite is preserved after the MAPLE process. If the material is considered as a biomaterial with potential use in medicine, the chemical structure is very important otherwise it is unknown what kind of material shows a good biocompatibility and the research cannot be treated as repeatable.

10. Line 186; Figure 3 should be enlarged and the scale is missing.

11. Line 242; Figure 7 is not very aesthetic

12. Lines 261-263; Sentence “Furthermore, the wavelength that appears between 3000 ‐ 3600 cm‐1 for all composites suggests that there are certain interactions between alendronate and hydroxyapatite” is very unclear, the explanation why this region indicates the authors statement is needed.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Authors showed that human sarcoma originated cells were attached to HA+AL combination coating. However, it is not strong evidence for a viable solution when including a bisphosphonate in the coating materials of prosthetic metal components. The drug in bisphosphonate has been shown strong anti-angiogenic effect. As angiogenesis is a vital component for bone regeneration, current experiment was insufficient. According to previous report (Choi JY, et al., Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2007), bisphosphonate combination graft showed no bone formation. In addition, locally high concentration of bisphosphonate may induce osteonecrosis.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Authors in the present manuscript demonstrate the preparation of hydroxyapatite–alendronate composites for the coating of Ti surface for potential orthopedic applications. While the manuscript has severe issues with technical English, it also suffers to deliver the impact of the work. I strongly recommend authors to get their manuscript checked from native English speaker.

1. Line 53, Line 57: There is no connectivity between the paragraphs. The introduction lacks justification for why this work has been carried out. They should highlight the lacuna or the knowledge gap in the existing literature. Similarly in Line 78-84; the Authors mentioned different methods of synthesis of HA but again no further discussion about these methods, their advantage/disadvantage with other methods of HA synthesis. No proper continuity and connectivity between the paragraphs. 

2. Line 96-99: Please cite the proper reference from where they adapted the HA synthesis procedure.

3. FTIR characterization of the surface is not properly explained neither the difference between HA-AL-5mM and 1:1 hydroxyapatite–alendronate is clear. It is difficult to understand what authors are trying to explain in the FTIR. It should be re-written for the understanding of the readers. 

4. I suggest that authors characterize the modified Ti surfaces using XPS (X-ray photoelectron spectroscopy) and water contact angle for confirmation of successful modification.

5. Line 190: Testing the biocompatibility of HA-AL biomaterials is poorly written and requires extensive editing.

6. Figure 7: Authors should proper label the y-axis.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Regarding to the IR spectra analysis:

10 cm-1 shift of absorption band (line 191) cannot be considered as "slight" shift.

Figure 2 a and b: last part of the spectra are cut - missing 600-400 cm-1 range (in the previous version it was ok).

Regarding to the question about the structure of the HA-AL composite after the process:

I agree that SEM-EDX proved the presence of the carbon, sodium and nitrogen suggesting the success of the process, however this technique cannot confirmed if the chemical structure is unchanged/preserved, especially as the results are presented in qualitative and not quantitative way. 

Author Response

We thank the reviewer for the positive feedback. We tried to address the points as follows 

We fixed the figures and modified the text. We added the characterization of the MAPLE deposited HA-AL composite using FTIR

Reviewer 3 Report

Surface characterization post-modification (i.e post MAPLE deposition of HA-AL) is still not convincing. EDS analysis is normally used as a supplementary or supporting data and not as primary evidence of surface modification. Successful surface coupling of HA-AL on Ti implants should be properly confirmed/characterized before claiming the surfaces to be modified. As suggested previously, it is crucial for authors to confirm HA-AL coating by FTIR-ATR if XPS is not available. The water contact angle is a simple experiment wherein authors can check the change in the hydrophobicity of the surfaces with respect to unmodified Ti surfaces using water droplet (Langmuir, 2018, 34 (28), 8178-8194).

Author Response

We thank the reviewer for the feedback. We tried to address the point by  characterizing the MAPLE deposited HA-AL composite using FTIR

Round 3

Reviewer 3 Report

The authors have improved the manuscript as suggested and now can be accepted for publication.