Optimized Size and Distribution of Silver Nanoparticles on the Surface of Titanium Implant Regarding Cell Viability

Round 1

Reviewer 1 Report

Dear authors,

congratulations on your article, this study is very interesting and very complex at the same time.
I don't have many comments to make about it, but I suggest an English proof-reading.
Furthermore, reading the article could be difficult for those who are not particularly familiar with this particular discipline of nanoparticles, I therefore suggest placing more emphasis on the practical/clinical implications of your findings, especially in discussions and conclusions.
What suggestions do you provide to implant manufacturers, how implant surfaces should be integrated according to your findings?
You conclude saying: "physical procedures can be used to modify ...", can't you instead indicate what are the ideal characteristics to provide to these nanoparticles in order to give them the indicated properties?

Thanks and congratulations.

Author Response

Reviewer #1

Thank you very much the Reviewer for the helpful suggestions. The valuable comments are appreciated and we have made the changes according to the suggestions. Below we present the responses to the questions one-by-one. Comments and questions are marked in red, while answers to them are marked in blue:

 

1. I don't have many comments to make about it, but I suggest an English proof-reading.

English was improved by the best knowledge of the authors. The abstract was significantly modified.

1. Furthermore, reading the article could be difficult for those who are not particularly familiar with this particular discipline of nanoparticles, I therefore suggest placing more emphasis on the practical/clinical implications of your findings, especially in discussions and conclusions.

The relevant sections (discussion and conclusion) of the article have been modified. Among the materials resistant to bacterial bonding, especially for the oral cavity, one of the most commonly used implant materials is titanium. In recent years, silver nanoparticles have received much attention [1-6]. They have significant antimicrobial properties when AgNp is used in controlled amounts. Numerous articles have already been published on AgNp, although little is said about their applicability.

[1]        Unosson E, Rodriguez D, Welch K, Engqvist H. Reactive combinatorial synthesis and characterization of a gradient Ag-Ti oxide thin film with antibacterial properties. Acta Biomater. 2015;11(1):503–510. 

[2]        Nakajo K, Takahashi M, Kikuchi M, et al. Inhibitory effect of Ti-Ag alloy on artificial biofilm formation. Dent Mater J. 2014;33(3):389–393.

[3]        Fordham WR, Redmond S, Westerland A, et al. Silver as a bactericidal coating for biomedical implants. Surf Coatings Technol. 2014;253:52–57.

[4]        Cabal B, Cafini F, Esteban-Tejeda L, et al. Inhibitory effect on in vitro Streptococcus oralis biofilm of a soda-lime glass containing silver nanoparticles coating on titanium alloy. PLoS One. 2012;7(8):e42393.

[5]        Gasquères C, Schneider G, Nusko R, Maier G, Dingeldein E, Eliezer A. Innovative antibacterial coating by anodic spark deposition. Surf Coatings Technol. 2012;206(15):3410–3414.

[6]         Liao J, Anchun M, Zhu Z, Quan Y. Antibacterial titanium plate deposited by silver nanoparticles exhibits cell compatibility. Int J Nanomedicine. 2010;5(1):337–342. 

 

1. What suggestions do you provide to implant manufacturers, how implant surfaces should be integrated according to your findings?

Among other things, we sought the answer to how to coat the surface of dental implants with silver nanoparticles to make it as economical as possible. This has several beneficial effects: the potential manufacturing process becomes cheaper and less silver nanoparticles dissolve in human tissues from a dental point of view. Reducing the amount of silver helps in production efficiency from a financial point of view. Because we describe step-by-step the exact process for the production of AgNPs, any implant manufacturer can easily produce the right biomaterials.

1. You conclude saying: "physical procedures can be used to modify ...", can't you instead indicate what are the ideal characteristics to provide to these nanoparticles in order to give them the indicated properties?

It is stated in the re-written abstract, that the ideal characteristics belong to 60 nm nanoparticle size: “Surfaces covered with 60 nm particle sizes proved to be the most hydrophilic and the viability of the cells was comparable to the viability measured on the untreated control surface.”

 

Reviewer 2 Report

1. This is a preclinical paper addressing an interesting topic on the effect of silver nanoparticles on the titanium implant surface and its possible association with cytotoxicity.
2. Silver nanoparticles coating, is an increasingly common application regarding various medical devices and biomaterials and its use on titanium dental implants may be interesting as a research topic because of the antibacterial effect described. Nevertheless, the connection of cytotoxicity with particle size, which is the major finding of the present study, is not a novel finding in the literature.
3. The background build up, presented by the authors in introduction, should become more enlightening, with regard to the biological benefit(s) of implant therapy, silver coating application may offer.
4. Given the fact that titanium dental implants interfere with either bone or connective tissue cells, authors have to explain the rationale of using dental pulp stem cells for the cytotoxicity testing instead of cells being detected in bone (osteocytes) or fibroblasts. From a clinical perspective, a dental implant (with silver nanoparticle coating) is never in contact and there is no interference with dental pulp. On the other hand, possible influence of silver nanoparticles in bone tissue or fibroblasts may be of interest. 
5. Cell cytotoxicity, resulting from silver nanoparticles, is not the only biological aspect that has to be taken into account, when a coating in dental implants is being considered. An implant surface modification with silver particles may affect osseointegration and hence biological and clinical outcomes of Implant therapy. This issue has to be examined as well, or at least discussed. Probable influence of silver nanoparticles on macrophages or PMNs might be crucial and should be discussed too.

Author Response

Many thanks to the Reviewer for the helpful suggestions. Valuable comments are appreciated and changes have been made as suggested. The answers to the questions are presented below. Comments and questions are marked in colour red, while answers are marked in colour blue:

1. This is a preclinical paper addressing an interesting topic on the effect of silver nanoparticles on the titanium implant surface and its possible association with cytotoxicity.

 

Thank you very much for the nice summary of our work, we agree.

 

1. Silver nanoparticles coating, is an increasingly common application regarding various medical devices and biomaterials and its use on titanium dental implants may be interesting as a research topic because of the antibacterial effect described. Nevertheless, the connection of cytotoxicity with particle size, which is the major finding of the present study, is not a novel finding in the literature.

 

Indeed, the connection between cytotoxicity and silver nanoparticle (AgNP) size because it is a well-known phenomenon in the literature. However, the results from the studies regarding AgNps also suggest, that besides the size of the particles, other physico-chemical properties, like shape, concentration, and agglomeration can also affect cytotoxicity [5]. As the method we have used for AgNp deposition is new for the preparation of implant materials, we think that these physico-chemical properties may also be different from the earlier examined AgNps, so the investigation of this connection is not without merit.

 

1. The background build up, presented by the authors in introduction, should become more enlightening, with regard to the biological benefit(s) of implant therapy, silver coating application may offer.

 

We have carefully checked the corresponding literature and we find it the Referee is right in terms on that more applications need to be incorporated into the references. Thank you for the comment, we have enlightened the background of this work with extra-literary references.

 

1. Given the fact that titanium dental implants interfere with either bone or connective tissue cells, authors have to explain the rationale of using dental pulp stem cells for the cytotoxicity testing instead of cells being detected in bone (osteocytes) or fibroblasts. From a clinical perspective, a dental implant (with silver nanoparticle coating) is never in contact and there is no interference with dental pulp. On the other hand, possible influence of silver nanoparticles in bone tissue or fibroblasts may be of interest. 

 

The reviewer has right, dental implants get in contact with osteocytes and fibroblasts so the examination of the possible effects on these cell types is also a very important and interesting matter which has to be investigated to fully clarify the advantages and drawbacks for the application in the dental practice.

In this study, however, we have used dental pulp stem cells as a representative (and also available in our laboratory) for mesenchymal stem cells, which are very important cells in the generation of different tissues including bone [6] and gingiva [7]. Moreover, the goal for the final application of the implant would be only a partial deposition of AgNps to the material surface where its antimicrobial effect may prevail, preventing bacterial infections of the gingiva mainly, and as the particles are anchored hopefully never get in contact with any cell types inside bone.

 

1. Cell cytotoxicity, resulting from silver nanoparticles, is not the only biological aspect that has to be taken into account, when a coating in dental implants is being considered. An implant surface modification with silver particles may affect osseointegration and hence biological and clinical outcomes of Implant therapy. This issue has to be examined as well, or at least discussed. Probable influence of silver nanoparticles on macrophages or PMNs might be crucial and should be discussed too.

 

We agree with the Reviewer. As in the case of the possible effects of the implant surface on the other cell types which can contact it, the investigation of other biological processes that can affect the usefulness of their clinical application is crucial. From the results of our measurements, we can only claim, that the growing size of AgNps reduced the number of viable cells on the material surface. As the poor wettability of the implant surface negatively affects the adhesion of different proteins and / or cells, it may cause the observed reduction in cell number through the decreased interactive material surface, and might have nothing to do with the cytotoxicity of the AgNps, but also may influence bone healing around the implant material [8].To clarify the reasons behind this decrease in cell number, further examinations with other cell types and thorough study of the processes involved would need to be carried out, which could be the topic for a more biology oriented paper. Without the appropriate measurements we would not hypothesise further effects of the silver nanoparticles.

In this investigation, we were focused on the more immediate effect of the AgNps on cell adhesion/viability to narrow down the appropriate circumstances for silver deposition to reduce the cost and time demand of a detailed examination. Moreover, as mentioned in our answer to point 4, the goal for the final application of the implant would be only a partial deposition of AgNps where it may prevent bacterial infections of the gingiva mainly, without any effect on the processes regarding bone healing and osseointegration.

[1]        Gittens RA, Scheideler L, Rupp F, Hyzy SL, Geis-Gerstorfer J, Schwartz Z, et al. A review on the wettability of dental implant surfaces II: Biological and clinical aspects. Acta Biomater 2014;10:2907–18. https://doi.org/10.1016/j.actbio.2014.03.032.

[2]        Milleret V, Lienemann PS, Gasser A, Bauer S, Ehrbar M, Wennerberg A. Rational design and in vitro characterization of novel dental implant and abutment surfaces for balancing clinical and biological needs. Clin Implant Dent Relat Res 2019;21:15–24. https://doi.org/10.1111/cid.12736.

[3]        Webb K, Hlady V, Tresco PA. Relative importance of surface wettability and charged functional groups on NIH 3T3 fibroblast attachment, spreading, and cytoskeletal organization. J Biomed Mater Res 1998;41:422–30. https://doi.org/10.1002/(SICI)1097-4636(19980905)41:3<422::AID-JBM12>3.0.CO;2-K.

[4]        Altankov G, Grinnell F, Groth T. Studies on the biocompatibility of materials: Fibroblast reorganization of substratum-bound fibronectin on surfaces varying in wettability. J Biomed Mater Res 1996;30:385–91. https://doi.org/10.1002/(SICI)1097-4636(199603)30:3<385::AID-JBM13>3.0.CO;2-J.

[5]        Akter M, Sikder MT, Rahman MM, Ullah AKMA, Hossain KFB, Banik S, et al. A systematic review on silver nanoparticles-induced cytotoxicity: Physicochemical properties and perspectives. J Adv Res 2018;9:1–16. https://doi.org/10.1016/j.jare.2017.10.008.

[6]        Bahney CS, Zondervan RL, Allison P, Theologis A, Ashley JW, Ahn J, et al. Cellular biology of fracture healing. J Orthop Res 2019;37:35–50. https://doi.org/10.1002/jor.24170.

[7]        Smith PC, Cáceres M, Martínez C, Oyarzún A, Martínez J. Gingival wound healing: An essential response disturbed by aging? J Dent Res 2015;94:395–402. https://doi.org/10.1177/0022034514563750.

[8]        Shiu HT, Goss B, Lutton C, Crawford R, Xiao Y. Formation of Blood Clot on Biomaterial Implants Influences Bone Healing. Tissue Eng Part B Rev 2014;20:697–712. https://doi.org/10.1089/ten.teb.2013.0709.

 

 

Reviewer 3 Report

Dear authors, 

I found the topic of manuscript interesting, although it has not high level novelty. I think the research experiment and consequent is well organised, but it needs to be revised and modified in several points as follow:

1. In experimental procedure section, authors acceptebaly explained about the post-synthesis heat treatment,but before that the brief explanation about the preparation of Ti layer and AG cluster on it.
2.  Authors should clearly explain their aims from evaluating titanium oxide layer. In current version there is only short presentation of Titania samples but without any proper supportive discussion. 
3. Authors frequently addressed to Ag nanoparticles on the layer. How do they conclude that the Ag clusters formed as nanoparticles? I strongly believe that the nanoparticles phrase is not suitable name for them. What is the mechanism of Ag particles growing? It seems there were several Ag seeds on the layer and then contact to each other via sintering and created the sintered cluster with size of sub microns and inhomogeneous shapes. This problem is very serious scientific issue that should be completely and clearly discussed.
4. Why did authors provide the size and thickness evaluations of small Ag nanoparticles (3-15 nm) while all toxicity and wetting angle measurement were done for sub micron clusters?
5. What is the difference between thickness of small nanoparticles and sub micron clusters?
6. Authors discussed the toxicity and wetting angle based on size of nanoparticles, but size in which direction? The cluster shape is not uniform and it is not easy to measure the size and discuss based on that. I strongly recommend to investigate the toxicity results as a function of percentage of bare and AG covered layer instead of particles size. Authors could easily use sem/eds results to find the covered layer. Also they could employ AFM for surface topology investigation.
7. Please briefly explain the procedure of wetting angle measurement technique. Did authors draw the reference lines of angle manually or automatically by software? Because the angles are to close to each other (63 and 66 degree) and I think they were not correctly fitted into the cluster edge, especially in the case of 66 degree.
8. Authors claimed the logical similarity manners between wetting angle and cytoxity results,but without any meaningful discussion. Please explain the reason of this approach. How did they affect each other?
9. Some minor points: the captions of some figure are vaguely without required information about the measured sample. Please mention in all caption that figure related to which samples and under which condition. 

Author Response

Thank you to the Reviewer for the helpful suggestions. We appreciate your valuable comments and we made changes as suggested. The answers to the questions are presented below. Comments and questions are marked in red, while answers are marked in blue:

1. In experimental procedure section, authors acceptebaly explained about the post-synthesis heat treatment,but before that the brief explanation about the preparation of Ti layer and AG cluster on it.

 

We believe that the sample preparation (section 2.) was well written in the original manuscript however we have reorganized this part of the manuscript to appear in chronological order in the Materials and method section. Maybe the disorder confused the Reviewer.

           

1. Authors should clearly explain their aims from evaluating titanium oxide layer. In current version there is only short presentation of Titania samples but without any proper supportive discussion.

 

According to the Reviewers notes the relevant text was changed in the manuscript in section 2.1.

 

1. Authors frequently addressed to Ag nanoparticles on the layer. How do they conclude that the Ag clusters formed as nanoparticles? I strongly believe that the nanoparticles phrase is not suitable name for them. What is the mechanism of Ag particles growing? It seems there were several Ag seeds on the layer and then contact to each other via sintering and created the sintered cluster with size of sub microns and inhomogeneous shapes. This problem is very serious scientific issue that should be completely and clearly discussed.

 

By the similarity of questions 3 and 5, please let us answer these two in one after question 5.

 

1. Why did authors provide the size and thickness evaluations of small Ag nanoparticles (3-15 nm) while all toxicity and wetting angle measurement were done for sub micron clusters?

 

The thickness of the silver layer sputtered on the surface was 3 nm, 5 nm, 8 nm and 15 nm (not submicron size). These surfaces were heat-treated to form nanoparticles with average diameters of 60 nm, 96 nm, 149 nm and 368 nm (submicron cluster). During heat treatment,  numerous processes take place on the surface of the sample, such as evaporation of silver, surface diffusion, and so on. Due to the superposition of these processes, in our case, a continuous silver surface with a layer thickness of 3 nm formed silver nanoparticles with an average diameter of 60 nm after 1 minute of heat treatment, 96 nm diameter with a thickness of 5 nm, and so on.

 

1. What is the difference between thickness of small nanoparticles and sub micron clusters?

 

We believe that in Chapter 2 (sample preparation) it is clearly described that in all cases the Ag particles were produced by the decay of thin Ag films deposited.  The mechanism of formation of nanoparticles is the decomposition of the continuous Ag layers by a process described by the refined Brandon-Bradshow model (see the sub-Chapter 2.3. and ref. [31] in the original version). In order to make clear, what the basic driving force is of such bead formation, the corresponding text was extended. The process starts at grain boundaries, where the energies of the free surface and the intersecting two grain boundaries are such that the surface/grain boundary tension vectors result in a co-called Mullins-type groove formation, which is also accompanied with formation of “hills” on both sides of the groove (W.W. Mullins, J of Appl. Phys. 28 333 (1957)). After the groove from the free surface reaches the substrate, a hole forms which further grows by surface diffusion, rearranging the structure in order to decrease the surface energies. Separate beads form after the holes overlap during such a process.

The size (and the distribution) of beads of course depend on the initial film thickness as well as on the annealing temperature and time. Fig. 4 illustrates that starting from different film thicknesses (from 3nm to 15 nm) beads, with a radius between 25 and 200 nm, are formed at 600^oC. Although it is true that the radius of such beads can also be in the submicron range, we used the “nano-film” and “nano-particles” terms in order to illustrate the above range of sizes. 

The corresponding text was modified accordingly see section 2.3.

1. Authors discussed the toxicity and wetting angle based on size of nanoparticles, but size in which direction? The cluster shape is not uniform and it is not easy to measure the size and discuss based on that. I strongly recommend to investigate the toxicity results as a function of percentage of bare and AG covered layer instead of particles size. Authors could easily use sem/eds results to find the covered layer. Also they could employ AFM for surface topology investigation.

 

The size of the silver nanoparticles was evaluated from the images captured by SEM. The images were taken in top view, giving a kind of projected image. These SEM images were evaluated using a Visual Assistance program.

 

1. Please briefly explain the procedure of wetting angle measurement technique. Did authors draw the reference lines of angle manually or automatically by software? Because the angles are to close to each other (63 and 66 degree) and I think they were not correctly fitted into the cluster edge, especially in the case of 66 degree.

 

The dosing system of drop shape analyser instrument contains a syringe with a flat-ended needle that is connected to an ultra-pure water containing container. A camera, that is opposing positioned with light source, is focused on the tested area of the surface. One drop of ultra-pure water is dosed on the surface of the tested sample. The dosing position is set and calibration is performed before the measurements. After water drop dosing, the shape of drop appeared in front of camera and picture was taken from it. This shape of the water drop on tested surface can be seen on Fig. 5. After the choosing of fitting method the software automatically fits a curve along the radius of curvature and automatically calculates the contact angle. The angle is between the tangent to the radius of curvature and the plane of the surface. The starting point of the angle is a triple point where air, solid surface and water droplet meet. The software automatically fits the shape of the curvature, the baseline setting can be modified, manually or automatically. In our study the baseline was set manually. 10 drops of water were analysed at different positions. Generally, the 10 drop measurements are accepted in contact angle measurements. The 63 and 66 angles data are very close to each other, but the data differ significantly. However, the final results of the study are not significantly influenced by this. 

Thank you for your comments in connection of incorrect fitting of baseline. We re-evaluated the contact angles by software. The data are not changed by recalculation. We tried to make more precise fitting on the curve of water drop. The representative contact angel pictures are replaced by better fitted contact angle pictures.

 

 

1. Authors claimed the logical similarity manners between wetting angle and cytoxity results,but without any meaningful discussion. Please explain the reason of this approach. How did they affect each other?

 

Thank you very much for pointing out this deficiency. However, we have measured reduced cell numbers on these surfaces, we believe, that these reductions are rather connected to the surface wettability than to the cytotoxic effect of the nanoparticles. The poor wettability of the implant surface negatively affects the adhesion of different proteins and/or cells [1-4], which may cause the observed reduction in the cell number through the decreased interactive material surface.

 

1. Some minor points: the captions of some figure are vaguely without required information about the measured sample. Please mention in all caption that figure related to which samples and under which condition. 

 

The figure captures are modified to be more informative.

 

Reviewer 4 Report

Dear Editor,

Hajdua et al. compared the effect of the size of silver nano particle coating on the titanium implants associated cytotoxicity. Manuscript demonstrated a good quality of science and with in the scope of the journal. My comments are follows:

1. A number of methodology or sample preparation is matching with an author’s previous paper published as Int J Nanomedicine. 2019; 14: 4709–4721. Authors expanded their earlier study, but they should make sure that they are not reporting methodology reported earlier. Also, focus on modification or improvement implemented in the sample preparation than earlier reported results.
2. Recheck the manuscript for grammar by native English speaker. Few sentences do not make any sense.
3. Rewrite this sentence in abstract section ‘Sample surfaces were examined by Scanning Electron Microscopy (SEM, JEOL JSM-IT500HR, Japan) Secondary Neutral Mass Spectrometry (SNMS, SPECS GmbH, Berlin) Drop Shape Analyser (Krüss 26 GmbH, Germany) To investigate how different physical surface characteristics are related to cell viability Alamar Blue assay for dental pulp stem cells (DPSCs) was carried out’.
4. Correct the periim plantitis in key words section.
5. Replace anorganic with inorganic.

Thank you,

A.

Author Response

Thank you very much the Reviewer for the helpful suggestions. The valuable comments are appreciated and we have made the changes according to the suggestions. Below we present the responses to the questions one-by-one. Comments and questions are marked in red, while answers to them are marked in blue:

 

1. A number of methodology or sample preparation is matching with an author’s previous paper published as Int J Nanomedicine. 2019; 14: 4709–4721. Authors expanded their earlier study, but they should make sure that they are not reporting methodology reported earlier. Also, focus on modification or improvement implemented in the sample preparation than earlier reported results.

We paid attention to this, if short repetition was necessary we always cited our previous papers. There is no overlapping between the scientific results in the two papers but the applied experimental techniques were partly similar. The differences were strongly emphasised.

1. Recheck the manuscript for grammar by native English speaker. Few sentences do not make any sense.

English was improved by the best knowledge of the authors.

1. Rewrite this sentence in abstract section ‘Sample surfaces were examined by Scanning Electron Microscopy (SEM, JEOL JSM-IT500HR, Japan) Secondary Neutral Mass Spectrometry (SNMS, SPECS GmbH, Berlin) Drop Shape Analyser (Krüss 26 GmbH, Germany) To investigate how different physical surface characteristics are related to cell viability Alamar Blue assay for dental pulp stem cells (DPSCs) was carried out’.

We have rewritten the above mentioned sentences correctly in abstract.

1. Correct the periimplantitis in key words section.

Sorry for this miss typing, we corrected.

1. Replace anorganic with inorganic.

Revised as requested. We have replaced anorganic to inorganic.

 

 

Round 2

Reviewer 3 Report

The authors' responses are significant and I believe after the modification the manuscript can be accept for publication.