Vitamin D3-Coated Surfaces and Their Role in Bone Repair and Peri-Implant Biomechanics

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.  The authors may be confused about what vitamin D3 is.  Vitamin D3 is not 1,25-dihydroxyvitamin D.  It is true that 1,25-dihydroxyvitamin D3 is the biologically active form.  It is not cholecalciferol.  Vitamin D3 is cholecalciferol.
2. . Therefore vitamin D3  needs to be metabolized in the liver to 25-hydroxyvitamin D and then in the kidneys to 1,25-dihydroxyvitamin D before he can be active on osteoclast and osteoblasts.
3.  The underlying hypothesis therefore is flawed since the authors used a commercial form of vitamin D3.  Vitamin D3 does not directly have any effect on  bone cells.  It is possible that these observations are related to simply having a sterol on the surface of the implant that may be helping cell viability.  
4.  This study would have been greatly strengthened if they had a negative control i.e. some other sterol like cholesterol to demonstrate that vitamin D3 itself is having some direct effect i.e. promoting an increase in biomechanical capacity and peri-implant bone repair micro architecture.
5.  It would have been ideal if they had to used 1,25-dihydroxyvitamin D3.  
6.  The authors imply in the conclusion that others studies have shown vitamin D3 effects on implants.  Reference 47 for example does not show vitamin D3 having an effect on implant directly but rather animals given vitamin D3 which likely was converted to 1,25-dihydroxyvitamin D3 showed some effect.

Author Response

Initially, we appreciate the comments and points that the reviewer has made to our manuscript. The alterations in the text are highlighted in YELLOW.

1. The authors may be confused about what vitamin D3 is.  Vitamin D3 is not 1,25-dihydroxyvitamin D.  It is true that 1,25-dihydroxyvitamin D3 is the biologically active form.  It is not cholecalciferol.  Vitamin D3 is cholecalciferol.

RESPONSE: We appreciate the information from the reviewer, although we understand that there was some confusion with the text. As informed, vitamin D is 1,25-dihydroxyvitamin D, but there was some confusing interpretation of vitamin D3 and cholecalciferol. Cholecalciferol, also known as vitamin D3, is a prohormone indicated for use as a dietary supplement. Therefore, the text has been reorganized for a better understanding.

2. Therefore vitamin D3  needs to be metabolized in the liver to 25-hydroxyvitamin D and then in the kidneys to 1,25-dihydroxyvitamin D before he can be active on osteoclast and osteoblasts.

RESPONSE: As mentioned, our study used vitamin D3 (1,25-dihydroxyvitamin D3) to coat the implants.

3. The underlying hypothesis therefore is flawed since the authors used a commercial form of vitamin D3.  Vitamin D3 does not directly have any effect on  bone cells.  It is possible that these observations are related to simply having a sterol on the surface of the implant that may be helping cell viability.  

RESPONSE: As mentioned in several topics of the manuscript, the vitamin D3 used in this study is commercially available (Addera D3) and its formula contains only vitamin D3 and the dilution vehicle, without any other steroid as suggested by the reviewer. Composition of Addera D3: medium-chain triglycerides, vitamin D3 (cholecalciferol) and antioxidant: DL-alpha-tocopherol. In addition, osteoblasts, the bone-forming cells, have vitamin D receptors. These receptors are activated by the active form of vitamin D (VDR), 1,25-dihydroxyvitamin D3, which promotes the renewal and maintenance of bone mass. Also, the results show that the medication has an effect on bone metabolism, increasing the microarchitectural parameters and biomechanics of the peri-implant bone.

Ref: Rillaerts K, Verlinden L, Doms S, Carmeliet G, Verstuyf A. A comprehensive perspective on the role of vitamin D signaling in maintaining bone homeostasis: Lessons from animal models. J Steroid Biochem Mol Biol. 2025 Mar 22;250:106732. doi: 10.1016/j.jsbmb.2025.106732. Epub ahead of print. PMID: 40122304.

4. This study would have been greatly strengthened if they had a negative control i.e. some other sterol like cholesterol to demonstrate that vitamin D3 itself is having some direct effect i.e. promoting an increase in biomechanical capacity and peri-implant bone repair micro architecture.

RESPONSE: In our research group, we developed implant surfaces using different substances, and it would be very interesting to compare vitamin D3 with other steroids in the family. However, the action of the compound is visible when compared to the group with no medication used to create the films (Ti group). Thus, our initial question and the aim of this study was the action of vitamin D3, which was observed in all the analyses of this research, where all the results obtained were superior to conventional titanium implants. Therefore, this comparison could be carried out in the future, as suggested by the reviewer.

5. It would have been ideal if they had to used 1,25-dihydroxyvitamin D3.  

RESPONSE: As mentioned, this study used commercially available vitamin D3 (Addera D3 - Mantecorp Farmasa. São Paulo, São Paulo, Brazil), which is widely used by the population and in several studies. Thus, we are convinced that if the medication were ineffective or even inappropriate, as suggested by the reviewer, the results of the study would not show improvements in peri-implant repair or cellular characteristics.

6. The authors imply in the conclusion that others studies have shown vitamin D3 effects on implants.  Reference 47 for example does not show vitamin D3 having an effect on implant directly but rather animals given vitamin D3 which likely was converted to 1,25-dihydroxyvitamin D3 showed some effect.

RESPONSE: The 47th study in question was carried out by our research group. In theory, the medication was metabolized by the kidneys, but we did not carry out any kind of analysis to confirm the reviewer's assertion. Furthermore, in this same study, the results obtained were aimed at assessing peri-implant repair, as in the manuscript being reviewed. Comparing the two studies, we found that the local use of vitamin D3 had a more positive and beneficial effect on peri-implant repair than the systemic use of the medication. Thus, even if it is local, the medication needs to be absorbed by the body and, even in cell culture studies, we can observe its action on the viability, organization and development of bone tissue.

Reviewer 2 Report

Comments and Suggestions for Authors

This is a nicely written and impactful paper showing new technology to coat bone with vitamin D a) works successfully and b) has significant anabolic effects when implanted into bone. The data are plentiful and results considered from both mlorphological and biochemical perspectives. The results are consistent and appear properly analyzed statistically. 

Author Response

We appreciate the comments and points that the reviewer has made to our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript reports the in vitro and in vivo evaluations of titanium (Ti) implants coated with vitamin D3. The research plan carried out in the study was constructed using methods in accordance with the literature, and the results associated with the research plan were presented in this manuscript. However, the novelty of the research was found to be low. Some specific points to be addressed by the authors during the revision process are as follows:

- The abstract does not provide any insight into the coating technique.

- The introduction starts with the term “nanotechnology,” but there is no indication that the research focuses on nanotechnology for implant coating. Only dip-coating was applied, but there is no characterization nor evaluation of coating properties such as thickness, density, stability etc.

-  Figure 1 is given to express dip coating schematically but the information given in the method nor the figure does not provide satisfactory information (if the blue spheres are vD3, then what biomolecules are the other spheres? There are four layers depicted, which are composed of three sub-layers; therefore, if the Ti implants are coated by dipping with vD3 solution, then how these different layers can be formed? According to the method described, this depicted structure cannot be formed.)

-  There are two concentrations studied, 40uL and 400uL. How were these concentrations determined for coating dose selection?

- ISO-10993-1 is the guide for using the standard. The tests to be conducted are selected in accordance with the medical device and target application.  

- SEM micrographs with different magnifications were presented in Figure 3. Non-coated Ti implant surfaces should be given in the same magnifications.

- It has been mentioned that vD400μl coating showed 10x more vitamin D3 molecules than the vD40μl group. The SEM images do not provide a comparable view to evaluate 10x times more molecules were sitting on the surface. Also, how the stability of the layers was concluded should be clarified.

- Level of significance (p) should be written in italics.

- “At 7 days of exposure to MTT” statement should be corrected since the cells were stained after 7 days of culture.

- The materials compared do not seem to be the same; the surface topography of Ti and vD surfaces are not comparable according to the SEM images provided. This point needs to be clarified (It cannot be explained by coating).

- The coating process remains in the air since insufficient description is provided in the methodology. Grafe IV Ti was double acid etched and then used in the coating experiments. If it is acid-etched, then how is it possible to mention TiO2 for chemical bonding? DMSO is a solvent; how is it used as a substrate?

- In the discussion section, it was mentioned that UV-C was used during coating; at what conditions is it used? And any effect on stability (of vD3) evaluated as it is known that lower energy UV-B transforms vD2 to vD3?

Comments on the Quality of English Language

The language needs minor improvements.

Author Response

Initially, we appreciate the comments and points that the reviewer has made to our manuscript. The alterations in the text are highlighted in BLUE.

1. The abstract does not provide any insight into the coating technique.

RESPONSE: We appreciate the mention of this fact by the reviewer. The topic has been inserted in the abstract of the manuscript.

2. The introduction starts with the term “nanotechnology,” but there is no indication that the research focuses on nanotechnology for implant coating. Only dip-coating was applied, but there is no characterization nor evaluation of coating properties such as thickness, density, stability etc.

RESPONSE: The term “nanotechnologies” was used to introduce all the perspectives involving the modification of implant surfaces. Regarding the characteristics mentioned about the dip-coated surface, Professor Paulo Noronha has been working with the development of materials for years, and in his studies, he made a comparison between some incorporation techniques, where it was possible to observe a slightly thicker surface than some other techniques. One point to note is that the choice of 5 immersions in solution is precisely to create a homogeneous, uniform and suitable film for the peri-implant repair process.

3. Figure 1 is given to express dip coating schematically but the information given in the method nor the figure does not provide satisfactory information (if the blue spheres are vD3, then what biomolecules are the other spheres? There are four layers depicted, which are composed of three sub-layers; therefore, if the Ti implants are coated by dipping with vD3 solution, then how these different layers can be formed? According to the method described, this depicted structure cannot be formed.)

RESPONSE: The diagram was created just to represent the technique, however, in order to avoid this kind of confusion, we opted to remove it from the text. We appreciate the reviewer for pointing this problem out in the image.

4. There are two concentrations studied, 40uL and 400uL. How were these concentrations determined for coating dose selection?

RESPONSE: Initially, we conducted a bibliographic review, taking into account other studies that had used vitamin D3. Therefore, we found the most commonly used dose (40 ul) and adapted it for use in a liquid solution. The selection of the highest concentration was made in accordance with other studies by our research group, where we compared it with a 10x higher concentration. This fact was included in the manuscript on “Vitamin D3 Surface - Coating Technique”

5. ISO-10993-1 is the guide for using the standard. The tests to be conducted are selected in accordance with the medical device and target application. 

RESPONSE: There was a mistake about the ISO mentioned in the study, in fact ISO 10993-5 “Tests for Cytotoxicity - In Vitro Methods” was used. We appreciate the reviewer for indicating this error in the manuscript, the information is now corrected.

6. SEM micrographs with different magnifications were presented in Figure 3. Non-coated Ti implant surfaces should be given in the same magnifications.

RESPONSE: There was some confusion about the image reported by the reviewer. Figure 3 is the SEM comparing vD40 and vD400 surfaces, where all the magnifications are the same between the groups. Could the reviewer confirm if anything needs to be changed in this image?

7. It has been mentioned that vD400μl coating showed 10x more vitamin D3 molecules than the vD40μl group. The SEM images do not provide a comparable view to evaluate 10x times more molecules were sitting on the surface. Also, how the stability of the layers was concluded should be clarified.

RESPONSE: The information has been reorganized in the text.

8. Level of significance (p) should be written in italics.

RESPONSE: The information has been reorganized in the text.

9. “At 7 days of exposure to MTT” statement should be corrected since the cells were stained after 7 days of culture.

RESPONSE: The error was corrected in the text.

10. The materials compared do not seem to be the same; the surface topography of Ti and vD surfaces are not comparable according to the SEM images provided. This point needs to be clarified (It cannot be explained by coating).

RESPONSE: In fact, as the dip-coating technique promotes an increase in surface thickness, there is this difference when comparing it to the titanium surface. This viscous and dense film is responsible for aiding the mechanical adhesion process between the layers, as well as keeping the vitamin d3 molecules in contact with the titanium surface.

11. The coating process remains in the air since insufficient description is provided in the methodology. Grafe IV Ti was double acid etched and then used in the coating experiments. If it is acid-etched, then how is it possible to mention TiO2 for chemical bonding? DMSO is a solvent; how is it used as a substrate?

RESPONSE: The double acid etching treatment is carried out by the company providing the implants, and this type of treatment only increases the roughness of the titanium surface. Thus, for functionalization with vitamin d3, UV-C light promotes destabilization between the Ti of the implant and atmospheric oxygen (O2) and hydrogen. At this stage the hydroxylation process takes place, leaving an -OH group which becomes available and binds to the substrate of the solution through a covalent (chemical) bond. These points are set out in the discussion. As for the choice of DMSO, it is a solvent widely used in the pharmaceutical industry to carry and diffuse different compounds. In addition, in our research group we have carried out tests in other studies, where DMSO proved to be safe for this purpose. In addition, as mentioned, DMSO has a very important characteristic for the dip-coating process: viscosity. As DMSO has a higher density than water, it prevents the compound from flowing off the surface of the implant, as well as allowing adhesion between the layers of vitamin d3 film.

12. In the discussion section, it was mentioned that UV-C was used during coating; at what conditions is it used? And any effect on stability (of vD3) evaluated as it is known that lower energy UV-B transforms vD2 to vD3?

RESPONSE: UVC light was used in a camera customized for this function. The implants are first exposed for 15 minutes to allow the aforementioned hydroxylations process. Then, at the end of the immersion in the vitamin D3 solution, the implants are once exposed to UVC light for 15 minutes, this time for a bactericidal action, since UVC light has high energy. Tests were not carried out for the transformation of vitamin D2 into D3, since our study already uses vitamin D3, and there is a specific energy range for the transformation of D2 into D3 (UVB light - 290 and 315 nanometers). The exposure to UVC light was included in the text.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

As mentioned, our study used vitamin D3 (1,25-dihydroxyvitamin D3) to coat the implants

Unfortunately it remains unclear what vitamin D compound they used.  Vitamin D3 is cholecalciferol  and 1,25-dihydroxyvitamin D3 is calcitriol.  They are not the same.  This needs to be corrected throughout the manuscript.  Otherwise the manuscript is uninterpretable.

Vitamin D is a Seco steroid hopefully that makes sense to the authors.  Therefore it would seem  that to know whether they are simply observing an artifact due to the coating with a sterol i.e. vitamin D3 versus the effect of vitamin D3 on osteoblasts will be important.  The only way to determine this is to use a negative control sterol-like 7 dehydrocholesterol.

Author Response

REVIEWER 1

1- As mentioned, our study used vitamin D3 (1,25-dihydroxyvitamin D3) to coat the implants. Unfortunately it remains unclear what vitamin D compound they used.  Vitamin D3 is cholecalciferol  and 1,25-dihydroxyvitamin D3 is calcitriol.  They are not the same.  This needs to be corrected throughout the manuscript.  Otherwise the manuscript is uninterpretable. Vitamin D is a Seco steroid hopefully that makes sense to the authors.  Therefore it would seem  that to know whether they are simply observing an artifact due to the coating with a sterol i.e. vitamin D3 versus the effect of vitamin D3 on osteoblasts will be important.  The only way to determine this is to use a negative control sterol-like 7 dehydrocholesterol.

RESPONSE: We appreciate the reviewer's comments on the manuscript, the alterations in the text are highlighted in pink. As mentioned in the text, we used vitamin D3 to functionalize the implants. We believe that there is a misinterpretation in the introduction because it mentions that the active form of vitamin D3 is 1,25-dihydroxyvitamin D3, as mentioned by the reviewer. However, in order to avoid any further misunderstandings, we have removed the sentence from the text, so we hope it is clear to the reviewer that we use vitamin D3 to functionalize the implants. The medication used throughout the study is clear, as explained in several sentences during the manuscript. As we have mentioned many times, we use a formula that is widely used and available on the Brazilian commercial sector, and which has been safely manufactured and registered with the national regulatory agencies. Addera D3 has several formulas, including compressed formulas. However, to facilitate dilution, we opted for its drop formulation, which is composed only of vitamin D3 (cholecalciferol) diluted in vehicle. The product information is available online. We understand the reviewer's concern about the action of the solution, however, when looking at the results, it is clear that it acts on bone tissue. Furthermore, these results are part of a study in which several other analyses were carried out, including RT-qPCR, immunohistochemistry and histological analysis. We believe that the reviewer should understand that we are using a safe product, whose formulation contains only vitamin D3 (cholecalciferol), and this medication on the surface of the implants had an effect on bone metabolism, as shown in the results provided in this manuscript. Our initial purpose is to characterize this surface in comparison to a conventional titanium surface, thus, with our results, we believe that our surface has been shown superior to titanium implants, in addition to the fact that we are also working with a commercially available and safe medication for the population.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors revised the manuscript according to the reviewers’ reports and responded to the comments. The authors’ responses provide satisfactory answers in general but still there are some points to be addressed.  
-    It was meant about the SEM images that SEM images of the non treated Ti surfaces shall be provided in Figure 2 to express the comparison between non treated and vD3 coated Ti surfaces (there should be 12 images in Figure 2). And the SEM images Does not show a significant change with the coating concentration 1x and 10x.  
-    The information asked about UV-C treatment parameters was the UV-C energy acting on unit area (watts/cm2), distance from UV source together with the treatment period where only treatment period (15 minutes) was given.
-    The comment on UV-C stability was to clarify if the vD3 remains stable during coating and sterilisation under process conditions. vD2 instability and transformation under low energy UV-B was an example to emphasise the potential of radiation energy to transformation of molecules

Author Response

REVIEWER 2

The authors revised the manuscript according to the reviewers’ reports and responded to the comments. The authors’ responses provide satisfactory answers in general but still there are some points to be addressed.  

1 - It was meant about the SEM images that SEM images of the non treated Ti surfaces shall be provided in Figure 2 to express the comparison between non treated and vD3 coated Ti surfaces (there should be 12 images in Figure 2). And the SEM images Does not show a significant change with the coating concentration 1x and 10x.  

RESPONSE: We chose to present only the treated surfaces in Figure 2.

The protocols for preparation and functionalization of titanium disc and implant surfaces for dental applications were optimized by our group, and there are other studies by our group that indicate the morphology of untreated surfaces. We can indicate the following references:

1. Kitagawa, I. L. ; Miyazaki, C. M. ; Pitol-Palin, L. ; Okamoto, R. ; de Vasconcellos, L. M. R. ; Constantino, C. J. L. ; Lisboa-Filho, P. N. . Titanium-Based Alloy Surface Modification with TiO 2 and Poly(sodium 4-styrenesulfonate) Multilayers for Dental Implants. ACS Applied Bio Materials, v. 4, p. 3055-3066, 2021, doi:10.1021/acsabm.0c01348.
2. Santos, A. A. ; Teixeira, J. V. U. ; Pintão, C. A. F. ; Correa, D. R. N. ; Grandini, C. R. ; Lisboa-Filho, P. N. Ti-15Zr and Ti-15Zr-5Mo Biomaterials Alloys: An Analysis of Corrosion and Tribocorrosion Behavior in Phosphate-Buffered Saline Solution. Materials, v. 16, p. 1826, 2023, doi.org/10.3390/ma16051826
3. Albano, C. S. ; Moreira G., A. ; da Silva F., G. ; da Costa F., C. J. ; Trino, L, D. ; Zambuzzi, W. F. ; Lisboa-Filho, P. N. . Biofunctionalization of titanium surfaces with alendronate and albumin modulates osteoblast performance. HELIYON, v. 6, p. e04455, 2020, doi.org/10.1016/j.heliyon.2020.e04455

Regarding the differences between the images at different magnifications, considering that the thickness of each layer of deposited film is close to 100nm [Ref. 1], no significant differences are expected.

 

2 - The information asked about UV-C treatment parameters was the UV-C energy acting on unit area (watts/cm2), distance from UV source together with the treatment period where only treatment period (15 minutes) was given.

RESPONSE: We emphasize that UV-C light exposure serves as a material sterilization protocol and does not play a role in the chemical activation of the molecules used to functionalize the surfaces. The references cited above also support this specific purpose

3 - The comment on UV-C stability was to clarify if the vD3 remains stable during coating and sterilisation under process conditions. vD2 instability and transformation under low energy UV-B was an example to emphasise the potential of radiation energy to transformation of molecules

RESPONSE: As previously mentioned, UV-C light exposure is intended for sterilization purposes. However, it is important to note that there are no reports in the scientific literature indicating that UV-C irradiation compromises the molecular stability of vitamin D