The authors have performed a thorough in vitro investigation of the behavior of a substrate of porous titania as a surface for incubation of living cells or bone and cartilage. The results are very interesting, of potentially high impact. The technical and scientific work is sound. The quality of the presentation should be definitely improved: make figure text readable, and rearrange the figures to select only the important ones and pack together into fewer illustrations. Also, next time authors please have an english mother tongue colleague go through the paper and fix the language issues. After major revisions and a second review round to verify them, the work should be acceptable.

Detailed comments:

Mandatory corrections of technical/scientific contents:

1. In the title, ‘allow’ is a bit weak: many surfaces ‘allow’, mean they are not cytotoxic or too smooth. Rather make it stronger by replacing wil ‘allow enhanced’, or more simply, ‘enhance’ or ‘promote’

2. In the abstract line 2, I wouldn’t write that Ti-alloy is a low-modulus material. I can imagine that the authors mean, low with respect to unalloyed Ti (say, perhaps, 105 GPa instead of 110), anyway this decrease is not that much, and still classifies the material as a high modulus one. One wouldn’t use it for dental or orthopedical applications if it was a low modulus material! Anyway, low or hi are always qualitative statements, depends on application, term of comparison etc, what is low in one case may be hi in another case. So, simply: ‘properties, tensile strength, biocompatibility, low modulus and corrosion resistance’ change to ‘properties, such as high tensile strength, good biocompatibility, appropriate modulus and low corrosion resistance’

3. Line 3: ‘Earlier studies…metals’: this is of no interest, remove it. You should focus only on your workin the abstract, the Introduction will come later.

4. ‘Microscopic examination’ change to ‘We carried out microscopic examination’

5. ‘, for evaluate’ change to ‘ to evaluate’

6. ‘reveals increased cell adhesion, and cellular biocompatibility’ change to ‘to investigate cell adhesion and cellular biocompatibility’

7. ‘for growing of’ change to ‘to grow’

8. Intro, paragraph 2: ‘Pathologies like’ change to ‘These tissues are affected by pathologies like’

9. ‘; osteoporosis is defined as’ change to ‘and osteoporosis,’ Plus: ‘It is considered as the second’ change to ‘These pathologies are considered second’

10. In the intro, among the cited literature of TiO2 applications, a recent advanced characterization of nanotubuklar TiO2 would better be added, which is ‘Anodic oxidation of titanium in sulphuric acid and phosphoric acid electrolytes’, by Sureeporn Uttiya, Daniele Contarino, Sonja Prandi, Maria Maddalena Carmasciali, Gianluca Gemme, Lorenzo Mattera, Ranieri Rolandi, Maurizio Canepa, Ornella Cavalleri, Journal of Materials Science & Nanotechnology, in press. In this paper, by SEM, AFM and XPS, it is discussed that anodization in phosphoric acid instead of sulphuric acid would be better for bioapplications, thanks to the inclusion of likely bioactive phosphates.

11. ‘This work strategy’ change to ‘Our work strategy’

12. ‘Chondrocyte growth, adhesion and biocompatibility on 80 nm TiO2 nanotubes had not been reported.’: remove! redundant

13. Subsequent, sentence: ‘In vitro … response.’ Is already reporting results, which is not appropriate for the Intro. Either move this to the Conclusion, integrating it with already existing summary there, or simply remove!

1. Section ‘Synthesis…’: ‘Electrolytically anodized’: seems the Ti-alloy come from the company already anodized, is this the case? Hope not. If not, remove! (it’s you who anodize, right?)

15. After writing the complex solution used for anodization, you should write if this is overall acid or not, e.g. write its typical pH. Please also specify that the anodization was carried out at RT, if this is the case, as T is important to control during anodization. What is the reason to add salts to the solution, in addition to the acid? Anodization is usually carried out only in simple acidic solutions.

16. ‘using an acoustic chamber to prevent electromagnetic noises’: funny! Anti-acoustic chamber is used against mechanical noise vibration transmitted through the air. Do you mean that the antiacoustic box is also metal coated to shield em noise? Specify it, this is not its default application (very often the box is just made of soft coating inside like PS foam, and of wood outside as a supporting material structure).

17. ‘A 20 um scanner’ change to ‘A 20 um X-Y scanner’, the Z scope of scanner is also there, have to avoid confusion. And, how much was the scope in Z? 2 um? 5 um?

18. ‘cells; isolated’ change to ‘cells were isolated’

19. ‘experiments were conducted for PCC cultures at passage 1 - 3 to avoid any lose of phenotype. For PPO cultures passages 1-3 were used.’ So, both 1-3 passages? If so, what is the reason to write this complicate sentence structure? Whay not to write simply: ‘experiments were conducted for both PCC and PPO cell cultures at passage 1 - 3 to avoid possible loss of phenotype.’

20. ‘cell viability experiment was evaluated’: remove experiment!

21. I suggest to change the times from h to days, because it’s simpler to understand; so use 1 day in vitro (DIV) instead of 24 h, and 3 DIV instead of 72 h.

22. Sputter coated with gold: what approximate thickness? 10 nm? 50 nm?

23. In the Experimental section, I recommend to change the title of subsection ‘Surface characterization’ to ‘Substrate surface characterization’, Stressing that this is done on substrates without cells. Also, remove the sub-subsections SEM, EDS and AFM, and put all in the same subsection level.

24. When later on the subsections come titled ‘Cell morphology analyzed by scanning electron microscopy’ and ‘Cell topography analyzed by atomic force microscopy’, use technique acronyms already in the title, and avoid duplication of intormation: remove the initial ones on SEM and AFM, and include the correspoding info into the subsequent ones. For example:’ The morphology of PCC and PPO cells were observed under a SEM (JSM-6360, JEOL) and the images were taken at a 5kV accelerating voltage.’ Change to only: The morphology of PCC and PPO cells were observed under SEM. (typical conditions already specified previously). And subsequently, delete ‘(Quesant Q-Scope 350, AMBIOS, Agura Hills, Ca, USA)’, and ‘were examined using a 20-μm scanner equipped with silicon tips with 10 nm tip curvature. In addition, the cells were scanned at 1 Hz over a 10 x 10μm region at scale angle of 0°.’ Change to ‘were examined by scanning at 1 Hz over a 10 x 10 μm^2 region’’

25. Previously, you wrote ’25 um^2’ for a scan area, now ’10 um x 10 um’ (BTW, it was ‘10x10 um’, wrong’. Please use a uniform style! Either 5 um x 5 um and 10 um x 10 um, or 5x5 um^2 and 10x10 um^2.

26. I actuanlly don’t know the style of the journal, but, generally, whereas the Experimental section is conveniently separated into different subsections corresponding to the different techniques, this should not occur for the results section: there, the results from different techniques should be merged into a single body. Thus, I suggest to remove the subsections in the Results section. Going to new paragraph (new line) should suffice, when changing the technique.

27. Fig.1: the size (ie magnification) of the image in Fig.1a must be same as the size (ie magnification) of Fig.1b and c: otherwise, it is not possible to say that there are no pores in 1a! Even if there, they would not be visible, due to the pixel size (100 times larger)

28. Still in Fig.1: magnification of 1b and 1c is the same! The bar is half length on the image, in 1c, and is also half length in the real space of the sample (100 nm instead of 200). So, remove either 1b or 1c, as these are duplicated. Since the pores in 1b look strangely stretched, I would remove 1b, and rename current 1c as new 1b.

29. Fig.2: clamp the y-axis to 2.5 (or even less: 2.3, 2.4) values.

30. Legends are not visible! Diminish the number of tick labels, but increase the font such that one can read them!

31. Merge current Fig.2 with Fig.1: So new Fig.1 will be: Panel 1a: current 1a Panel 1b: current 1c Panel 1c: current 2a Panel 1d: current 2b. This will help limit the number of figures (15 is crazy!) and compact the information so as to make it more readable.

32. Current Fig.3: One doesn’t need both top-view and 3D of same image! So, choose one or another. Since 3D is more for posters and presentation slides, while top-view is more scientific, I would leave top-view only. Also, put Ti first, vs anodized-Ti, same as previous figs (no reason to change). Also, another chance to compact: merge into Fig.1. So, in summary: Current panel 3a, 3b: removed; Current 3c becomes 1e Current 3d becomes 1f (Make characters readable!)

33. Current fig.4 becomes fig.2, good as is.

34. ‘Also anodized titanium illustrates a tubular, uniform structure along the surface’ It is false the tubules are visible in the AFM image. However, it is quite acceptable, due to small pore size. So, replace above text with the following: ‘The tubule mouths could unfortunately not be resolved, due to convolution with the used probe tips, which were not ultrasharp. However, anodized titanium showed much increased roughness, which is clearly associated with the tubular porous surface.’

35. Fig.5: again legends are terrible in font size and distorsion (compressed? Why?) Also, the white bars are not clearly visible: fill also themn, just use a different shade of gray.

36. I would rather make current fig.6 come earlier, soon after current fig.4, and then the extracted quantitative information of current fig. 5 and 7 come together in a single fig. So I recommend to take current Fig.6 and rename it 3, and merge current 5 and 7 into panels 4a and 4b of 4 (again after fixing characters!)

37. Moreover was observed Moreover it was observed

38. At each interval time At each time interval

39. Current figs.8,9: That the corresponding position image show more blue spots for 9, is true for panels a and b, but not for c and d. Additionally, current 9c is same as 8c, just blurred and decreased in intensity: please fix. Select appropriate representative images for panels c and d. (Anyway, these figs. Should be now no. 5 and 6).

40. Current figs. 9 and 11 should be merged into panels a and b of new fig. 7.

41. Fig.12 rename to fig.8 And 13 rename to 9.

42. Fig.14 and 15 merge into a and b of fig. 10. Again, only keep the more professional and serious style of top-view figures only (remove the childy 3D, it was used to present them some 20 yrs ago, at the birth of AFM!)

43. ‘showed a greater flatter’? maybe ‘appeared flatter’?

44. ‘enhance a better osteoblast morphology’ change to ‘enhance osteoblast-like morphology’

45. ‘better number’ change to ‘higher number’ or ‘increased number’

46. ‘showing a high increased area of 508.4 nm’: what does it mean?!? First area would be nm^2, not nm. You mean height? Ok, write that. However, no need to put it 3D to display height: rather, use a color bar by the side! Or simply write the min-max range for the colors (eg 0-1300 nm, in the figure legend text).

47. Beginning of Discussion: ‘Improve tissue development around grafts is a process, which’ change to ‘Enhancing tissue development around grafts is a process that’

48. ‘, on Ti and its alloys’: remove, trivial: it is required on whatever the substrate.

49. ‘. Although’ change to ‘, while’

50. ‘25.51% of oxygen, attributed to the induced oxidation and 3.28% of flour associated to the electrolyte used for anodization’: Change flour to fluor…

51. ‘AFM micrographs demonstrate’ change to ‘AFM micrographs suggest’

52. ‘But we detected’, change to newline + ‘Unexpectedly, we detected’

53. Which is agree’ change to ‘which is in agreement’

54. Final lines of discussion: ‘Inthis study… interface’ sound more like conclusion. However, the same info is already presented in the actual Conclusion. So, simply remo ve these duplicated lines.

55. Do you really need to repeat the abbreviations for each legend? They are defined once forever in the manuscript body text.