Examination of the Quality of Particulate and Filtered Mandibular Bone Chips for Oral Implants: An In Vitro Study
Round 1
Reviewer 1 Report
The manuscript entitled “Examination of the Quality of Particulate and Filtered Mandibular Bone Chips for Oral Iimplants: An in vitro study.” submitted to Applied Sciences aims to investigate particulate and filtered bone chips harvested during oral surgery procedures with regard to the osteogenic potential and the extent of microbial contamination to evaluate the usefulness for jawbone reconstruction.
The manuscript appears interesting, nevertheless the topic is wide documented in literature.
I have some suggestions to improve the quality of the manuscript, enriching the text with further notions.
- English language: Minor spell check is required.
- Abstract: Please structure the abstract to attract the reader's attention.
- In the introductory part of the stem cells of the oral cavity I suggest referring to the periodontal ligament stem cells [PMID: 30369260 - PMID: 33086890].
- Methods: please, specify in a better way the randomisation process.
- Methods: did you calculate a simple size?
- Methods: specify the possible use of drugs in the exclusion criteria
- Methods: why did you choose such a large age range?
do you think this could affect the results of the study?
I strongly suggest adding this part as limitations of the study at the end of the discussion.
The osteogenetic potential of a young patient is different from that of an older patient.
- Why is there such a high discrepancy between the sample numbers taken from the different sites?
I suggest adding this information as well within the scope of the study.
- Discussion: I suggest to introduce a more clinical part on the role of bone regeneration in implant surgery [PMID: 21504476]. Sometimes, in fact, the use of autologous materials could help to reach parameters such as the primary stability of the implant of fundamental importance in the rehabilitation of the prosthetic implant [PMID: 30025006 - PMID: 32475099]
- I strong suggest to add an important part on the study limitations at the end of the discussions.
On my opinion the article is interesting, however the manuscript must be improved.
I am available for a second round of review.
Author Response
Dear Professores Roman and Soanca,
We appreciate the time and effort you and each of the reviewers have dedicated to refine our paper. Thank you very much.
We have edited the manuscript addressing the suggestions of the reviewers for improvements. The revisions are highlighted and can be followed by Microsoft Word “Track Changes” function. To facilitate your review of our revisions, the following is a point-by-point response to the questions and comments of the reviewers.
Reviewer 1:
The manuscript entitled “Examination of the Quality of Particulate and Filtered Mandibular Bone Chips for Oral Iimplants: An in vitro study.” submitted to Applied Sciences aims to investigate particulate and filtered bone chips harvested during oral surgery procedures with regard to the osteogenic potential and the extent of microbial contamination to evaluate the usefulness for jawbone reconstruction.
The manuscript appears interesting, nevertheless the topic is wide documented in literature.
I have some suggestions to improve the quality of the manuscript, enriching the text with further notions.
- English language: Minor spell check is required.
We have revised the whole manuscript and corrected several typing errors and improved some phrases. Thank you for pointing it out to us.
- Abstract: Please structure the abstract to attract the reader's attention.
The abstract is written according to the rules given by MDPI and thus structured into (1) Background, (2) Methods, (3) Results) and (4) Conclusions. We also checked if another structure can be now found in recent publications of the journal “Applied Sciences”. However this is not the case. No further structure such as e.g. dividing the abstract into paragraphs was found. Most often even the structure (1) Background, (2) Methods, (3) Results) and (4) Conclusions is not given. Thus we stick to this to meet MDPI rules and the suggestion of the referee.
- In the introductory part of the stem cells of the oral cavity I suggest referring to the periodontal ligament stem cells [PMID: 30369260 - PMID: 33086890].
Within the introduction part as an overview, we present indeed several types of stem cells, which have a high osteogenic capacity, among which are also periodontal ligament stem cells, which we now added. Of course, there are hundreds of publications for this topic. We went through the suggested publications and added the first one additionally into the introduction. In our opinion, the second one is too detailed on bisphosphonates, the most commonly used drug in osteoporosis treatment. As periodontal ligament stem cells are not the topic of the paper presented, and bisphosphonates is only one possible molecule of several used to increase osteogenesis, we feel that the second suggested paper is too far from the main topic of our manuscript.
- Methods: please, specify in a better way the randomisation process.
The two major groups of samples were cortical bone chips collected during wisdom tooth removal and cortical-cancellous bone chips collected during dental implant surgery. These two subgroups were divided into antibiotic pre-treatment and no pre-treatment. Consequently, there have been four groups.
The randomization was done by the dental surgeon, Dr. Winter, from whom the scientists, working on cell culture and microbiological experiments, got only numbered samples. Only after the experiments were finalized, there was a meeting with Dr. Winter to distribute the results in the respective four groups.
We have added this information in the manuscript.
- Methods: did you calculate a simple size?
Yes, we did calculate a simple size. In detail, we determined the volume of each sample and also weighted them. This data was used to adjust the amount of material for outgrowth experiments. We now added this information in methods.
For the wisdom tooth removal samples, the amount of material was always quite similar per tooth, whereas the amount of material from dental implants differed per patient, mainly due to the number of implants and thus drills.
- Methods: specify the possible use of drugs in the exclusion criteria
Only healthy patients were used as donors for samples. Patients with systemic diseases and drug treatment such as anti-rheumatic drugs, cortisone therapy, or known high blood pressure treatment were excluded from the study.
We have added this information in the manuscript.
- Methods: why did you choose such a large age range?
The large age range for the donors was mainly given by the large age range of donors receiving dental implants. Even if older people need an implant more often than younger donors, the necessity is given in all age groups. On the other hand the age range of patients for wisdom tooth removal is usually very narrow because patients are teenagers or at the beginning of their 20th’s. This is reflected in the age range of the groups. We have added this information in the manuscript.
However, we know from previous data for bone chips from way more than hundred donors, that the differentiation capacity varies more between individuals than between groups of different ages. There is however a difference in the differentiation capacities of bone chips from the head area compared to bone chips from the body region due to the developmental origin. This we have published already in 2011 (Zippel et al., 2011). But this is not affecting data of this manuscript as material of the four groups is only from the head area. Also, there is a difference in the doubling times of the out grown stem cells over longer time periods which is linked to the age of the donor. But this is not affecting in the time periods of the experiments performed for this manuscript.
do you think this could affect the results of the study?
We are quite certain that the ages does not affect the results of our study above what is mentioned in the manuscript. Next to our data, there are other publications as well which showed that donor age does not affect osteogenesis (e.g. PMID: 29113460). And as stated, before, especially not in the time frame of the experiments performed for this manuscript.
I strongly suggest adding this part as limitations of the study at the end of the discussion.
We have discussed this now in more detail in the conclusions.
Why is there such a high discrepancy between the sample numbers taken from the different sites?
I suggest adding this information as well within the scope of the study.
Donors of nearly all age groups receive dental implants regularly with or without antibiotic pre-treatment. Thus, it is not very difficult to get enough numbers of donors for these two groups. On the other hand antibiotic pretreatment is not recommended in the treatment plan for wisdom tooth removal in Germany. Only if a patient is demanding this i.e. in hope of a better wound healing, or due to a patients trepidation of a possible infection in the up-coming surgery, antibiotics will be given. Consequently, this group is smaller because of the very limited availability of such samples. Whereas the group containing bone chips from wisdom tooth removal without antibiotics can grow easily to great numbers as this is a very common surgery in Germany. We have added this information, as suggested, within the scope of the study (Material & Methods: Patient groups).
- Discussion: I suggest to introduce a more clinical part on the role of bone regeneration in implant surgery [PMID: 21504476]. Sometimes, in fact, the use of autologous materials could help to reach parameters such as the primary stability of the implant of fundamental importance in the rehabilitation of the prosthetic implant [PMID: 30025006 - PMID: 32475099]
Thank you for the suggestion. We added a paragraph on the use of autologous material in clinical applications within the discussion.
- I strong suggest to add an important part on the study limitations at the end of the discussions.
Thank you for the suggestion. We have re-written the discussion with emphasis on arguing the pros and cons of the cell growth experiments as well as by discussing the pros and cons of the microbiological experiments.
On my opinion the article is interesting, however the manuscript must be improved.
Thank you for your effort to improve our manuscript. We hope to meet your expectations.
Bonn, January 26th, 2022
Date, Signature
Prof. Dr. Edda Tobiasch
Professor for Genetic Engineering and Cell Culture
University of Applied Sciences, Department of Natural Sciences, Germany
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In this work, the authors approach a very relevant topic from a clinical point of view. It is a very complete study, with interesting results that deserves to be published.
There are some typos to correct.
After reading the article, there are some questions that are not completely clear:
- In the introduction It would be preferable to use the term guided bone regeneration (GBR).
- How was randomization done?
- Why the selection of clindamycin ?
- In the cortico-cancellous group, was the bone removed directly from the bur or was it aspirated and removed from the filter, as in the other group?
- The medium for the isolation of stem cells contains 3 antibiotics. To what extent can it influence the results when trying to assess the influence of pre-op antibiotics?
- In the microbiological analysis the samples were only incubated for 12H (overnight)?
- It would be important to present the number of species found in the groups with and without antibiotics.
- Authors do not discuss study limitations. For example the limitations of the methods used for the characterization of bacterial species.
- The last paragraph of the discussion could be deleted. The conclusion at the end of the discussion does not derive from this study.
- In the conclusion authors state that "pre-operative antibiotic treatment of the patients reduced the microbial contamination of the collected bone chip material" but quantification was not performed, as i understand.
Congratulations for this work
Author Response
Dear Professores Roman and Soanca,
We appreciate the time and effort you and each of the reviewers have dedicated to refine our paper. Thank you.
We have edited the manuscript addressing the suggestions of the reviewers for improvements. The revisions are highlighted and can be followed by Microsoft Word “Track Changes” function. To facilitate your review of our revisions, the following is a point-by-point response to the questions and comments of the reviewers.
Reviewer 2:
In this work, the authors approach a very relevant topic from a clinical point of view. It is a very complete study, with interesting results that deserves to be published.
There are some typos to correct.
We thank the reviewer for the kind comment.
We have revised the whole manuscript and corrected typos and improved some phrases.
After reading the article, there are some questions that are not completely clear:
- In the introduction It would be preferable to use the term guided bone regeneration (GBR).
We extended the introduction by adding the term guided bone regeneration without going too much into detail for the membranes as this is mainly in periodontology.
- How was randomization done?
The two major groups of samples were cortical bone chips collected during wisdom tooth removal and cortical-cancellous bone chips collected during dental implant surgery. These two subgroups were divided into antibiotic pre-treatment and no pre-treatment. Consequently, there have been four groups.
The randomization was done by the dental surgeon, Dr. Winter, from whom the scientists, working on cell culture and microbiological experiments, got only numbered samples. Only after the experiments were finalized, there was a meeting with Dr. Winter to distribute the results in the respective four groups.
We have added information on the randomization in methods.
- Why the selection of clindamycin?
Clindamycin was reported to incorporate into the bone very well (PMID: 17052892, PMID: 18755618). And was thus considered to be the best choice. However since the jointed discussion while writing the paper, we noticed that due to discovered side effects in the intestine the use of Amoxicillin or Ciprofloxacin might have been an interesting alternative.
- In the cortico-cancellous group, was the bone removed directly from the bur or was it aspirated and removed from the filter, as in the other group?
No, it was aspirated and removed from the filter in all surgical procedures to reduce differences in the process. The picture of the bur with the material was chosen for illustration purposes with respect to potential readers not working in the dental area, but e.g. in microbiology, or on bone replacement strategies, and thus not being familiar with the procedure.
- The medium for the isolation of stem cells contains 3 antibiotics. To what extent can it influence the results when trying to assess the influence of pre-op antibiotics?
We are not certain to which part of the results the reviewer is referring to. We assume the outgrowth of the cells (and not the microbiology part) is the most likely data meant, hoping to meet the question.
The culture medium and the differentiation medium contained penicillin, streptomycin and amphotericin. The former two are standard antibiotics and the last is a common antimycoticum in cell culture. All three are used in abundance in cell culture without known negative side effects on stem cells which would have impacted on our results. As it can be expected that scientists working on bone replacement strategies will do so as well, we found this to be the most reasonable approach.
The major goal was to look for differences in the four groups of dental materials. As all four groups have been treated the same with respect to these additions to the media, we can still see the impact of the pre-op antibiotic on our results. In detail we found that cortical-cancellous material group without antibiotic pre-treatment had a significantly lower outgrowth time (P < 0.02) compared to the other groups. We also found that the outgrowth probability is significantly higher (P < 0.05) in both groups (cortical and cortical-cancellous) with antibiotic pre-treatment. Thus, the pretreatment with Clindamycin seems to positively affect stem cells outgrowth probability but hampers to some extent the time needed for it. We discussed now the effect of the antibiotics in more detail in the conclusion.
- In the microbiological analysis the samples were only incubated for 12H (overnight)?
Yes, the samples were incubated for 12 hours. Although overnight, thus approx. 16 hours, is more common, we decided otherwise, as we did not know how many colonies of bacteria we should expect and we did not want the plates to be overgrown. After starting with this time period, we did not want to change the protocol, as some of the samples were rare (cortical with antibiotic pre-treatment) and thus difficult to get.
- It would be important to present the number of species found in the groups with and without antibiotics.
Next to the given data for numbers of microorganism found and percentages of species, a quantification as suggested was unfortunately not conducted. We agree that this would be interesting. However Germany had a catastrophic flooding (see picture) in the middle of July which greatly affected our campus (60 Mio € damage). The labs have been flooded and all material was lost. Thus we cannot add these data now. The main goal was to see if the material can be used further for bone replacement strategies, or if it is too contaminated with bacteria. To do so, we found it would be enough to look for Gram staining, aerob or anaerob species. But of course we agree that having numbers would be beneficial.
- Authors do not discuss study limitations. For example the limitations of the methods used for the characterization of bacterial species.
Since the oral cavity harbors a broad spectrum of microorganism including bacteria, yeast and sometimes fungi, not all possible occurring species could be detected by the used tests. The individual personal oral flora is usually handed over from a mother to her child after birth and thus, there is a difference in every person. The tests only recognize a certain number of species recorded in the database of the manufacturer. Additionally, not all pathogenic microorganisms can be detected by the used API® tests from Biomérieux. We conducted these tests because we wanted to get an overview on the occurring microorganisms of the samples and their possible influence on outgrowth and differentiation experiments, which was the main goal. Further and more detailed characterization such as a sequencing of the bacterial DNA, investigation of the 16s RNA or even Maldi-TOF would have demand long-term investigations, which we only wanted to do, if a detrimental effect of the bacteria would have been found. Which was not the case.
We added comments on the testing of the bacteria and on the limitations of the used tests now in detail in the discussion.
- The last paragraph of the discussion could be deleted. The conclusion at the end of the discussion does not derive from this study.
We rewrote the whole paragraph taking into account the limitations of the performed experiments, embedding our data in the current knowledge and giving an outlook in the end. We hope this meets the expectations of the referee.
- In the conclusion authors state that "pre-operative antibiotic treatment of the patients reduced the microbial contamination of the collected bone chip material" but quantification was not performed, as i understand.
We did calculate a simple size of the material by determining volume and weight of each sample. Since the material is particulate, a more precise adjustment of the amount is nearly impossible. The so balanced material samples were used for outgrowth experiments but also for the microbiological study part. While clearly enough similar for the former, it might be not enough for the latter to evaluate precise numbers without a huge standard deviation. Since Clindamycin was reported to incorporate into the bone very well (PMID: 17052892, PMID: 18755618) and was thus chosen, we did expect to see a reduced microbial contamination of bone chip material in the groups with pre-operative antibiotic treatment of the patients. As this was expected and is a known feature, we found it worthwhile to mention the difference but not to quantify it as the main goal of the study was the usability of the stem cells for future bone replacement strategies.
Congratulations for this work
Thank you very much. We do appreciate the comment and your kind help to improve the publication.
Bonn, January 26th, 2022
Date, Signature
Prof. Dr. Edda Tobiasch
Professor for Genetic Engineering and Cell Culture
University of Applied Sciences, Department of Natural Sciences, Germany
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Although the article is not totally innovative, the authors have made improvements in the text of the manuscript.
I suggest a close review of the English form before proceeding with further steps.
Author Response
Dear Professores Roman and Soanca,
We appreciate the time and effort you and the reviewers have dedicated again to refine our paper. Thank you.
We have edited the manuscript addressing the suggestions of the reviewer for improvements. The revisions are highlighted and can be followed by Microsoft Word “Track Changes” function.
Reviewer 1:
Although the article is not totally innovative, the authors have made improvements in the text of the manuscript.
I suggest a close review of the English form before proceeding with further steps.
We thank the reviewer for the recognition of our additional work. As suggested, the English was checked again in detail. Grammar and spelling was further improved. Changes can be followed by the tracking function of Microsoft Word.
Bonn, February 10th, 2022
Prof. Dr. Edda Tobiasch
Professor for Genetic Engineering and Cell Culture
University of Applied Sciences, Department of Natural Sciences, Germany
Author Response File: Author Response.pdf
