A Novel 3D Titanium Surface Produced by Selective Laser Sintering to Counteract Streptococcus oralis Biofilm Formation

Round 1

Reviewer 1 Report

General comments.

Although the topic is interesting, the manuscript is very confusing and difficult to read. The manuscript would gain from a revision and reorganization. The objective(s) of the work should be clearly identified.

The abstract did not respect the journal guidelines: “The abstract should be a single paragraph and should follow the style of structured abstracts, but without headings: 1) Background: …”

The figures should have a short explanatory title and caption. This is not present in the manuscript. For SEM images is more important to display clearly the scale bar than to write down the magnification used to acquire the image. Consider revising the presentation.

The methods are poorly described and do not allow other groups potentially interested in adopting the same procedure to reproduce it.

Results and discussion could be improved. It would much easier to the reader if the figures legends were clearly separated from the main text. Considering the figures, some axis legends are not legible being very difficult to evaluate the results. Consider revising.

 

Specific comments:

Line 27. The abbreviation DAE is used here but only defined in line 93. Please revise.

Line 55. “Further advantage of AM if the possibility…” Check if you meant if or is.

Lines 85-96. Consider re-writing in order to improve clarity and avoid repetitions.

Line 107. “….fluorhydric acid and the second with nitric acid.” The pure acids or diluted solutions were used?

Line 127. “200 um*200 um” Did you meant micrometres?

Line 144. Explain is a pool saliva sample was used or the saliva of each patient was used individually. Justify your choice.

Line 154. “Then the bacteria were inoculated on the discs…” Specify the inoculum concentration.

Line 164. “The mixture was observed at the microscope through viable staining…” Either describe the procedure or say that it will be described in another section.

Lines 166-167. Re-write this part. It is confusing.

Line 309-311. This sentence is highly speculative and not based on the data presented. Please revise or show results for ROS.

Lines 311-313. How do you explain this?

Line 331: “…and antibiofilm activity…”. This contradicts the previous comment (lines 311-313). Consider revising.

Author Response

Although the topic is interesting, the manuscript is very confusing and difficult to read. The manuscript would gain from a revision and reorganization. The objective(s) of the work should be clearly identified.

AUTHOR’S ANSWER: Thank you very much for your comment. The objectives of this study have been clearly specified.

“The primary objective of this study is to characterize the chemical composition, the nano, and micro-topography of a novel 3D surface produced in Ti6 Al4V through the SLS. The secondary objective is to evaluate the Streptococcus oralis interaction with this novel surface, in comparison with two traditionally titanium surfaces already used in dental implantology: the machined and the titanium double acid etching (DAE). The third objective is to correlate the topographical features of the surfaces with the microbiological results, in order to identify those parameters that are more influential for bacterial growth”.

The abstract did not respect the journal guidelines: “The abstract should be a single paragraph and should follow the style of structured abstracts, but without headings: 1) Background: …”

AUTHOR’S ANSWER: Thank you very much for your comment. We have removed the heading from the abstract

The figures should have a short explanatory title and caption. This is not present in the manuscript. For SEM images is more important to display clearly the scale bar than to write down the magnification used to acquire the image. Consider revising the presentation.

AUTHOR’S ANSWER: Thank you very much for your comment. The bar of SEM figure has been modified in order to be more clear and evident. The title of the figures has been written in bold.

 

The methods are poorly described and do not allow other groups potentially interested in adopting the same procedure to reproduce it.

AUTHOR’S ANSWER: Thank you very much for your comment. We better explained the M&M section.

Results and discussion could be improved. It would much easier to the reader if the figures legends were clearly separated from the main text. Considering the figures, some axis legends are not legible being very difficult to evaluate the results. Consider revising.

AUTHOR’S ANSWER: Thank you very much for your comment. The whole manuscript has been revised and most of the figures have been modified in order to make more clear and visible the bars and the axes. The legend has been left in the text, in order to follow the style requested by the journal.

Specific comments:

Line 27. The abbreviation DAE is used here but only defined in line 93. Please revise.

AUTHOR’S ANSWER: Thank you very much for your comment. The DAE acronym has been defined early in the text.

Line 55. “Further advantage of AM if the possibility…” Check if you meant if or is.

AUTHOR’S ANSWER: Thank you very much for your comment. The sentence has been corrected in:

“A further advantage of AM is the possibility to plan the production of infinite complex geometries characterized by different porosities, in order to mimic the natural tissues”.

Lines 85-96. Consider re-writing in order to improve clarity and avoid repetitions.

AUTHOR’S ANSWER: Thank you very much for your comment, the sentence has been rewritten.

Line 107. “….fluorhydric acid and the second with nitric acid.” The pure acids or diluted solutions were used?

AUTHOR’S ANSWER: Thank you very much for your comment. These data are covered by a patent, however, we have specified the Company that produced the discs. (Resista, Omegna (VB), Italy)

Line 127. “200 um*200 um” Did you meant micrometres?

AUTHOR’S ANSWER: Thank you very much for your comment. Yes, we meant micrometers. We have changed the sentence in “200 µm*200 µm”.

Line 144. Explain is a pool saliva sample was used or the saliva of each patient was used individually. Justify your choice.

AUTHOR’S ANSWER: Thank you very much for your comment.  We used a pool saliva, in order to use the same saliva mixture for all discs.

Line 154. “Then the bacteria were inoculated on the discs…” Specify the inoculum concentration.

AUTHOR’S ANSWER: Thank you very much for your comment. We better specified this aspect

Line 164. “The mixture was observed at the microscope through viable staining…” Either describe the procedure or say that it will be described in another section.

ANSWER: Thank you very much for your comment. We added the required explanation.

Lines 166-167 “Then, the count of CFU/mL was obtained by culture on Tryptic Soy Agar (TSA) plates of selected 10-fold dilutions followed by overnight incubation at 37 °C”. Re-write this part. It is confusing.

AUTHOR’S ANSWER: Thank you very much for your comment. According to your suggestion, we re-write this sentence.

Line 309-311. This sentence is highly speculative and not based on the data presented. Please revise or show results for ROS.

AUTHOR’S ANSWER: Thank you very much for your comment. We agree with your opinion that this sentence was highly speculative, so we preferred to remove from the manuscript, and considering the quantification of ROS, for further studies.

Lines 311-313. How do you explain this?

As reported in our previously work (Ref. n° 15), the double acid-etching surface is labeled by higher wettability and cellular adhesion increasing bacterial proliferation. Diomede et al. showed that DAE discs are characterized by increased cell growth, cell adhesion, and improved osteogenic and angiogenic events, as well as the osseointegration process, with respect to machined samples.

Diomede, F.; Marconi, G.D.; Cavalcanti, M.F.X.B.; Pizzicannella, J.; Pierdomenico, S.D.; Fonticoli, L.; Piattelli, A.; Trubiani, O. VEGF/VEGF-R/RUNX2 upregulation in human periodontal ligament stem cells seeded on dual acid etched titanium disk. Materials 2020, 13, 706.

Line 331: “…and antibiofilm activity…”. This contradicts the previous comment (lines 311-313). Consider revising.

As reported in the Result section and Discussion section, with DAE there were a significant incrementation of biofilm biomass. In any case, we modified the sentence in conclusion section.

Reviewer 2 Report

.pdf file

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

 

thank you very much for your comments. We have added a point-to point answer to each question. Kind Regards,

 

Morena Petrini

 

The Manuscript (ID: APPLSCI-1456611) entitled A novel 3D titanium surface produced by Selective Laser Sintering to counteract the biofilm formation and the bacterial colonization takes up a needed and an interesting issue – a comparison the chemical composition of a novel 3D titanium surface, (obteined by SLS) with machined and DAE discs and their evaluate with bacterial interaction. The undertaken topic is justified and interesting in relation to the more and more common use of 3D surfaces (3D print), with particular emphasis on the application potential in medicine.

Below, I present my thoughts and suggestions on the Manuscript submitted for review.

Introduction

The Introduction perfectly introduces the subject matter and explains the sublimity of the concept of the research direction. It presents the current knowledge and shortcomings, arguing the need for such research as those proposed by the Authors. All the criteria of a good Introduction have been met, which should be emphasized – briefly.

Materials and methods

The research concept and the used analyzes are comprehensive and complete, planned with great care. According to the reviewer knowledge, the methods used for chemical characterization (atomic force microscopy, scanning electron microscopy and energy dispersive X-ray spectrometry) are accurate, justified, properly described and allow to obtain a broad knowledge of the analyzed surfaces.

Line 154 : I miss information about the amount of inoculum (concentration of bacteria used for inoculation, log value – I hope the Authors understand what I mean. Including an inoculum value in evaluation of bacterial cells adhesion/interaction with the surface is needed.

AUTHOR’S ANSWER: Thank you very much for your comment. We better specified the bacteria concentration and the amount of inoculum.

This section is not objectionable. It is prepared very carefully.

Results

The Results section describes in detail the redundant information that we can see in the Figures and in the Tables – especially the statements that the Tables contain information with standard deviation etc. We can see this data perfectly in the graphical presentation of the results. Too many unnecessary details. Please limit yourself to the results. Of course, the descriptions of the Figures are necessary, but in the case of tables, I consider some elements unnecessary (eg. Line 203-204). I suggest slightly revising this section. Nevertheless, I appreciate the thoughtful way of presenting the results – both in the content of the Manuscript and the quality of the graphic presentation, with a kind of clear and narrow "summary" of each part of the research.

AUTHOR’S ANSWER: Thank you very much for your comment. We removed table 1 and 2, but we preferred to maintain Table 3 (that now is table 1), because it shows the Pearson Analysis.

 

Discussion

The Discussion meets all standards, is prepared at a high level, presents the "background" of the research, confronts the results obtained by the Authors. I appreciate the brief organization of this section.

Conclusions

The Conclusions are presented in a clear and lucid way, without unnecessary deliberations and explanations (ie the typical "manner" of contemporary publications – congratulations !

I consider the Manuscript to be of a very high quality, carefully planned conceptually, methodically and in terms of publication. My suggestions are cosmetic and do not alter the perception of the Manuscript.

Reviewer 3 Report

A novel 3D titanium surface produced by Selective Laser Sintering to counteract the biofilm formation and the bacterial colonization – the title of this manuscript is misleading and should be changed as only one bacteria strain was examined.

In the Introduction section authors have focused mainly on the technology and more information about surfaces, bacterial contamination and the role of biofilm should be added.

Based on what authors claim that this material/surface is novel, it needs to be explained (surface characteristics) and references to the literature are needed.

Why hydrogen peroxide was used? What is the reason behind this procedure as per literature we know that it can destroy the titanium oxide layer and change the biological and physico-chemical   properties of the material.

lines 85-86 are confusing, what is the real aim and scientific importance of this article? Simple topography and chemical composition depends on the used materials, printing device and design parameters.

sub-section 2.4- how was the process controlled, what were the inclusion/exclusion criteria for the donors?

sub-section 2.5: Why only Streptococcus oralis was used? This should be explained.

line 146: what dose it mean “sterilized through a 0.2 μm filter”

line 159- why there was no negative control?

More up to date literature about biofilm formation on 3D- printed implants is required for proper introduction and discussion sections, for example, the authors should refer to articles like  “Susceptibility to biofilm formation on 3D-printed titanium fixation plates used in the mandible: a preliminary study” or  “Manual polishing of 3D printed metals produced by laser powder bed fusion reduces biofilm formation”, “Bacterial Biofilm Growth on 3D-Printed Materials” or  “Toward Bactericidal Enhancement of Additively Manufactured Titanium Implants”  as some of the literature is in contrast with the results obtained by the authors. At this point the conclusion pointed by the authors dose not have enough support in the conducted research.

In the present form the manuscript is not ready for publishing and misleading. In order to gain some scientific value, novelty and justification for publication it needs to be significantly improved. Also, please remove some self-citations as there are definitely too many.

 

Author Response

Thank you very much for your comment. We have added a point to point answer to each comment.

 

Kind Regards,

 

Morena Petrini 

A novel 3D titanium surface produced by Selective Laser Sintering to counteract the biofilm formation and the bacterial colonization – the title of this manuscript is misleading and should be changed as only one bacteria strain was examined.

• AUTHOR’S ANSWER: Thank you very much for your comment. The title has been modified in:

A novel 3D titanium surface produced by Selective Laser Sintering to counteract the Streptococcus oralis biofilm formation

 

In the Introduction section authors have focused mainly on the technology and more information about surfaces, bacterial contamination and the role of biofilm should be added.

• AUTHOR’S ANSWER: Thank you very much for your comment. We preferred to focus on the technology that permitted the production of the samples, because it is the novelty of this study. For what concerning the bacterial contamination on titanium surfaces, according to your suggestion, we added new sentences in the new version of MS.

“ Peri-implantitis is associated with complex microbiota profiles and the process of biofilm formation on the implant surface is comparable to biofilm formation on natural teeth. The species Streptococcus oralis and Streptococcus sanguinis followed by Neisseria pharyngis and Gemella haemolysans are considered to be the first colonizers of the implant surface. Changes in the biofilm composition of Streptococci species are observed between the 4^th and 8^th hour of biofilm formation. The first colonizers correlate positively with each other and influence the colonization of bacteria of the orange and red complex during the development of peri-implantitis.“

Pokrowiecki, R.; Mielczarek, A.; Zaręba, T.; Tyski, S. Oral microbiome and peri-implant diseases: Where are we now? Ther. Clin. Risk Manag. 2017, 13, 1529–1542.

 

Based on what authors claim that this material/surface is novel, it needs to be explained (surface characteristics) and references to the literature are needed.

• AUTHOR’S ANSWER: Thank you very much for your comment. To our knowledge, no references results about this novel surface, however, The surface has been largely characterized by the Scanning electron microscopy (SEM), the Energy Dispersive X-ray Spectrometry (EDX), the Atomic force microscopy (AFM) and the Measurement of wettability of the discs.

Why hydrogen peroxide was used? What is the reason behind this procedure as per literature we know that it can destroy the titanium oxide layer and change the biological and physico-chemical   properties of the material.

• AUTHOR’S ANSWER: Thank you very much for your comment. The use of hydrogen peroxide for implant decontamination is very common in the postproductive phase of AM titanium devices, as confirmed by the literature. As explained in the text, this process permits to eliminate the titanium spherical non-adherent particles from the surfaces of the discs
• • Pingueiro J, Piattelli A, Paiva J, Figueiredo LC, Feres M, Shibli J, Bueno-Silva B. Additive manufacturing of titanium alloy could modify the pathogenic microbial profile: an in vitro study. Braz Oral Res. 2019 Sep 30;33(suppl 1):e065. doi: 10.1590/1807-3107bor-2019.vol33.0065. PMID: 31576949.
  • Guan B, Wang H, Xu R, Zheng G, Yang J, Liu Z, Cao M, Wu M, Song J, Li N, Li T, Cai Q, Yang X, Li Y, Zhang X. Establishing Antibacterial Multilayer Films on the Surface of Direct Metal Laser Sintered Titanium Primed with Phase-Transited Lysozyme. Sci Rep. 2016 Nov 8;6:36408. doi: 10.1038/srep36408. PMID: 27821857; PMCID: PMC5099579.

 

lines 85-86 are confusing, what is the real aim and scientific importance of this article? Simple topography and chemical composition depends on the used materials, printing device and design parameters.

AUTHOR’S ANSWER: Thank you very much for your comment. We agree with your comment, and for this reason, considering that the novel surface presented in this study has been never described in literature, the present study had two objectives:

The first was to characterize the chemical composition, the nano and micro-topography of the novel 3D surface

• The second was to evaluate the bacterial interaction with this novel surface.

sub-section 2.4- how was the process controlled, what were the inclusion/exclusion criteria for the donors?

AUTHOR ANSWER: Thank you very much for your comment, the following sentence has been added  in the text:

“The inclusion criteria were: age > 18 years old, and absence of chronic systemic diseases. Exclusion criteria were antibiotic treatments within 1 month prior to the study [23]”.

 

sub-section 2.5: Why only Streptococcus oralis was used? This should be explained.

• AUTHOR’S ANSWER: Thank you very much for your comment. According your suggestion, we specified the use of oralis and we added new sentence in discussion section in new version of MS.

“In this study S. oralis was used because it is an early colonizer during the process of biofilm formation on implant surface and provides the basis for the subsequent colonization of facultative and obligate anaerobes.” We also added the use of a single bacteria as a limitation of the study: “This study should be considered as preliminary observation, because it is based on a bacterial mono-specie model”.

 

line 146: what dose it mean “sterilized through a 0.2 μm filter”

• AUTHOR’S ANSWER: Thank you very much for your comment. The human saliva is composed of organic and inorganic components and in particular harbors a wide spectrum of microorganisms, proteins/peptides, nucleic acids, electrolytes, and hormones that originate from multiple local and systemic sources.  Salivary proteins together with proteins, peptides, lipids and other macromolecules adhere to the tooth surface to form the acquired salivary film. Acquired salivary pellicle influences the initial colonizer of oral biofilm.

In this study we sterilized the human saliva through a 0.2 μm filter in order to eliminate the microorganisms and preserve the compounds necessary to form the acquired salivary pellicle.

 

line 159- why there was no negative control?

• AUTHOR’S ANSWER: Thank you very much for your comment. We apologize but we did not understand the comment. As indicated in the manuscript, for all tests “ Non-inoculated titanium discs were used as negative controls.”

More up to date literature about biofilm formation on 3D- printed implants is required for proper introduction and discussion sections, for example, the authors should refer to articles like  “Susceptibility to biofilm formation on 3D-printed titanium fixation plates used in the mandible: a preliminary study” or  “Manual polishing of 3D printed metals produced by laser powder bed fusion reduces biofilm formation”, “Bacterial Biofilm Growth on 3D-Printed Materials” or  “Toward Bactericidal Enhancement of Additively Manufactured Titanium Implants”  as some of the literature is in contrast with the results obtained by the authors. At this point the conclusion pointed by the authors dose not have enough support in the conducted research.

AUTHOR’S ANSWER: Thank you very much for your comment. We inserted the following sentence in introduction section and the new references:

“The surfaces structure and hydrophobicity influence the capability of the bacteria to colonize and to form biofilm. In particular, the presence of concave features such as valleys or depression enhance the bacterial colonization [17]. McGaffey et al. demonstrated that manual polishing of 3D printed surfaces reduced biofilm formation, with preparation-specific relationships between surface roughness and biofilm growth suggesting that metallic implants produced by laser powder bed fusion should be polished [18].”

 

In the present form the manuscript is not ready for publishing and misleading. In order to gain some scientific value, novelty and justification for publication it needs to be significantly improved. Also, please remove some self-citations as there are definitely too many.

AUTHOR’S ANSWER: Thank you very much for your comment. The whole manuscript has been enriched, following your suggestions. The following manuscripts have been removed from the reference list:

D’Ercole, S.; Di Campli, E.; Pilato, S.; Iezzi, G.; Cellini, L.; Piattelli, A.; Petrini, M. Streptococcus oralis biofilm formation on titanium surfaces Superficial characterization of chemical-, nano- and microscopical level. Int. J. Oral Maxillofac. Implants.

Di Giulio, M.; D’Ercole, S.; Zara, S.; Cataldi, A.; Cellini, L. Streptococcus mitis/human gingival fibroblasts co-culture: the best natural association in answer to the 2-hydroxyethyl methacrylate release. Apmis 2012, 120, 139–146.

                  Spoto, G.; De Iuliis, V.; Petrini, M.; Flati, V.; Di Gregorio, J.; Vitale, D.; Caruso, M.; Dadorante, V.; Ciarmoli, M.; Robuffo, I.; et al. Effect of low energy light irradiation by light emitting diode on U937 cells. J. Biol. Regul. Homeost. Agents 2016, 30.

Scarano, A.; Lorusso, F.; Inchingolo, F.; Postiglione, F.; Petrini, M. The Effects of Erbium-Doped Yttrium Aluminum Garnet Laser (Er: YAG) Irradiation on Sandblasted and Acid-Etched (SLA) Titanium, an In Vitro Study. Materials (Basel). 2020, 13, 4174, doi:10.3390/ma13184174.

 

 

Round 2

Reviewer 3 Report

First of all, I'd like to see the design of the disc the authors mention in the paper and know the reason for the chosen parameters of the surface. After submitted corrections, it seems to me that the authors have confused dental implants and peri-implantitis and medical implants (plates/meshes) and their bacterial contamination which may cause infection. They did not discuss the differences in the roughness and wettability parameters of the surface and their impact on the cells and proteins adhesion. They claim that the surface was designed to promote cellular interaction and osseointegration but failed to support this statement. If the surface is novel why there is no information on the patent pending? I had asked the authors to refer to specific articles in the discussion especially to those describing similar materials and study design as they reported different results from those mentioned in the reviewed paper but the authors completely ignored this. 

Author Response

REV2  

1)First of all, I'd like to see the design of the disc the authors mention in the paper and know the reason for the chosen parameters of the surface. 

-Author answer: Dear reviewer, thank you very much for your comment. The sample that we have described and tested in this manuscript on this paper, has been selected by a group of surfaces produced with SLM, on the basis of the best cellular interaction, as previously shown by Gallorini et al. (2021). This study has been published on September 2021, so we cannot cite on our first submission, but now we have cited in the text and discussed:     Gallorini M, Zara S, Ricci A, Mangano FG, Cataldi A, Mangano C. The Open Cell Form of 3D-Printed Titanium Improves Osteconductive Properties and Adhesion Behavior of Dental Pulp Stem Cells. Materials (Basel). 2021 Sep 15;14(18):5308. doi: 10.3390/ma14185308. PMID: 34576532; PMCID: PMC8467079.       2)After submitted corrections, it seems to me that the authors have confused dental implants and peri-implantitis and medical implants (plates/meshes) and their bacterial contamination which may cause infection.  -Author answer: Dear reviewer, thank you very much for your comment. We have not confused "dental implants and peri-implantitis and medical implants (plates/meshes) and their bacterial contamination “; We are aware that they are different infections, that could also be sustained by a different microbial ecosystems; however, we believe that a titanium surface, that show antibacterial properties, could be a first step for the prevention of both diseases. As we have previously specified, we need more in vitro and in vivo studies to confirm this, indeed this is only a preliminary study.     3)They did not discuss the differences in the roughness and wettability parameters of the surface and their impact on the cells and proteins adhesion. -Author answer: Dear reviewer, thank you very much for your comment, the aim of this study was not to investigate the interaction between this surface with mammalian cells and proteins, it has been largely described in our previous work (Gallorini et al 2021). We have insert this study and discussed.  The following text has been insert in the discussion session:   "3D was chosen, from a group of other titanium ones, produced by SLM and tested on a previous study[21]. In particular, it showed not only biocompatibility but also the ability to increase the Dental Pulp Stem Cells (DPSCs) proliferation paralleled by the decrease of LDH released in a time-dependent manner[21]. 3D surface showed a behavior resembling the inner structure of the native bone, allowing cells to better adhere inside the specimen, being proteins related to cell adherence highly expressed. It showed also osteoconductive properties, being the profile of osteogenic markers improved compared to titanium samples[21]. Gallorini M, Zara S, Ricci A, Mangano FG, Cataldi A, Mangano C. The Open Cell Form of 3D-Printed Titanium Improves Osteconductive Properties and Adhesion Behavior of Dental Pulp Stem Cells. Materials (Basel). 2021 Sep 15;14(18):5308. doi: 10.3390/ma14185308. PMID: 34576532; PMCID: PMC8467079.     -They claim that the surface was designed to promote cellular interaction and osseointegration but failed to support this statement. If the surface is novel why there is no information on the patent pending? I had asked the authors to refer to specific articles in the discussion especially to those describing similar materials and study design as they reported different results from those mentioned in the reviewed paper but the authors completely ignored this.
-Author answer: Dear reviewer, thank you very much for your comment. I have to apologize, but you are right...I have asked about the patent-pending, and I was wrong...however, all the procedures for the production of the samples, comprised the interaction with cell has been described in the manuscript   Gallorini M, Zara S, Ricci A, Mangano FG, Cataldi A, Mangano C. The Open Cell Form of 3D-Printed Titanium Improves Osteconductive Properties and Adhesion Behavior of Dental Pulp Stem Cells. Materials (Basel). 2021 Sep 15;14(18):5308. doi: 10.3390/ma14185308. PMID: 34576532; PMCID: PMC8467079.   That we have cited in the text. We did not insert all the description, in order to not repeat and information that was already published. However, if you believe, that we should add other information about the process of production of the samples, please let us know.