Combining High-Resolution Hard X-ray Tomography and Histology for Stem Cell-Mediated Distraction Osteogenesis

Round 1

Reviewer 1 Report

This study deals with complex problem - combination of different visual datasets. The methodology is well prepared and presented. There are some minor flaws.

The "Discussion" section is missing. There should be noted if there are some different method of scans registrations used by another authors.

Why weren't performed also  µCT scans after decalcification and SRµCT before decalcification? Comparison of pre- and post- decalcification scans by some imaging method could clearly elucidate the deformations caused by the decalcification process.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

 

The manuscript is an interesting study if considered in relation to the definition of a methodology for combining the evaluation on data from conventional micro-CT, synchrotron and histology. The application of the methodology is limited to a single case study.

1.    Title (line 2): The title of the manuscript does not seem very appropriate and it should be revised. Not only X-ray (conventional and synchrotron) has been considered but also histology and, the focus of the study should be kept on the defined methodology to combine the acquired data. Moreover, as the authors also stated in the conclusions, the animal study is just a single case not allowing to evaluate the clinical application and the impact of MSCs. 
2.    Abstract: It should mention that the jaw (line 31) is referred to the one of a single animal-rat underwent the surgical treatment.
3.    Introduction, lines (69-74) and (85-86): the application of Micro-CT combined with other techniques, particularly histology, is common and its relevance has already been discussed in literature reviews also focused on maxillofacial surgery, which are not mentioned here and should be.
4.    Introduction, line 73 [… only allows for post mortem imaging]: What about in vivo micro-CT instruments? Animal studies are usually assessed by performing in vivo analyses to overcome the mentioned limitation.
5.    Paragraph 2.8 (line 222): Please, indicate the format (DICOM, etc.) used to import the images in Itk-Snap.
6.    I suggest the authors to revise the entire paragraph3 limiting it just to the obtained results. Considerations and discussions should be better reported adding a separated section “Discussions”.

Author Response

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This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Remarks to the authors:

 

Sigron et al. presented a study of stem cell-mediated distraction osteogenesis in nude rat mandibles. They investigated the new bone formation and the differentiation of hMSC using micro-CT and histological analyses. There is a major concern about the methods used and the conclusions drawn from flawed methods. The rationale of the experimental design and the characterizations methods must be restated entirely. The flow of the results and the link between experimental results and conclusions must be presented more clearly. The data representation must be improved. The manuscript is very difficult to read because of English typographical mistakes. The manuscript cannot be accepted for publications at its current state. 

 

Comment 1:

 

The authors should clearly emphasize the novelty of the study. The authors presented the introduction section and background information without giving importance to the state of the art. The authors should discuss the state of the art related to the topic of the study and demonstrate the novelty of the study in detail.

Comment 2:

 

There is a major concern about the experimental study. It is not acceptable to draw conclusions from an experimental group that has n=1 animal.

Comment 3:

There is a major concern about the results obtained from animal 2, 3, and 4. None of these results were shown in the manuscript.

The authors mentioned: “In the control animal 3 where no hMSC had been infused osseous bridging of the left hemi mandible was incomplete and a small unmineralized interzone remained (Figure 3f).” However, there is not figure 3f in the manuscript.

Comment 4:

The manuscript must be revised thoroughly for English mistakes. There are a lot of missing articles, punctuation marks, and many structure and composition major mistakes. The manuscript is very difficult to read and contains a lot of formatting errors and grammar mistakes. The authors must rephrase many sentences/paragraphs in the manuscript to improve the readability.

 

Reviewer 2 Report

In the manuscript entitled: “Stem cell-mediated distraction osteogenesis in nude rat mandibles studied by micro computed tomography and histology: A preliminary study” the authors evaluated the influence of injection of human mesenchymal stromal cells (hMSC) in the distraction gap before distraction on osseous bridging and bone mineral density (BMD), custom made distraction devices were bilaterally placed in mandibular angle of 4 nude rats.

In their study, Group 1 was distracted with regular speed with (Animal 1) and without (Animal 3) application of hMSC, Group 2 with rapid speed with (Animal 2) and without (Animal 4) hMSC. After consolidation phase the animals were sacrificed and studied by micro computed tomography and histology.

The authors found that under regular distraction speed injection of hMSC in distraction gap before distraction increased the bone mineral density in comparison with a control group without injection of hMSC. Application of hMSC alone do not allow a rapid distraction or shortening of consolidation phase, 27 because osseous bridging and stability stay away.

The authors concluded that hMSC can differentiate in vivo into human osteoblasts in a nude rat during distraction osteogenesis and that these osteoblasts are functional. The existence of hMSC in the distraction gap at the beginning of distraction alone does not allow to accelerate the distraction velocity. Also, they stated that further studies are needed to investigate factors, which influence the migration of MSCs in the distraction gap for complete bridging of the interzone

 

 

Major comments:

In general, the idea and innovation of this study, regards the analysis of Stem cell-mediated distraction osteogenesis is interesting, because the role of the stem cells in bone disease is validated but further studies on this topic could be an innovative issue in this field could be open an innovative matter of debate in literature by adding new information. Moreover, there are few reports in the literature that studied this interesting topic with this kind of study design.

The study was well conducted by the authors; However, there are some concerns to revise that are described below.

The introduction section resumes the existing knowledge regarding the bone marrow cells.

However, as the importance of the topic, the reviewer strongly recommends to update the literature through read, discuss and cites in the references with great attention all of those recent interesting articles, that helps the authors to better introduce and discuss the aim of the study in light of the bone marrow cells in some other oral conditions: 1) Isola G, Matarese G, Cordasco G, Rotondo F, Crupi A, Ramaglia L. Anticoagulant therapy in patients undergoing dental interventions: a critical review of the literature and current perspectives. Minerva Stomatol. 2015 Feb;64(1):21-46. 2) Suehiro F, Ishii M, Asahina I, Murata H, Nishimura M. Low-serum culture with novel medium promotes maxillary/mandibular bone marrow stromal cell proliferation and osteogenic differentiation ability. Clin Oral Investig. 2017 Dec;21(9):2709-2719. 3) Isola G, Cicciù M, Fiorillo L, Matarese G. Association Between Odontoma and Impacted Teeth. J Craniofac Surg. 2017 May;28(3):755-758.

The authors should be better specified, at the end of the introduction section, the rational of the study and the aim of the study with the null hypothesis. In the material and methods section, should better clarify how was performed the immune-histochemical preparation and the 3D micro-CT analyses. Moreover, specify if was performed, the intra-examiner agreement for the analyses and if the data obtained were normalized or not. Please specify if was used a test unit.

The discussion section appears well organized with the relevant paper that support the conclusions, even if the authors should better discuss the role of bone marrow cells in periodontal disease. The conclusion should reinforce in light of the discussions.

In conclusion, I am sure that the authors are fine clinicians who achieve very nice results with their adopted protocol. However, this study, in my view does not in its current form satisfy a very high scientific requirement for publication in this journal and requests a revision before publication.

 

Minor Comments:

 

Abstract:

-          Better formulate the introduction section by better describe the aim of the study

 

Introduction:

-          Page 1, Line 40: please add the relative sentence

 

Discussion

-          Please add a specific sentence that clarifies the results obtained in the first part of the discussion

-          Page 8 last paragraph (lines 226-238): Please reorganize this paragraph that is not clear

Reviewer 3 Report

Manuscript Review Comments

Title: “Stem cell-mediated distraction osteogenesis in nude rat mandibles studied by micro computed tomography and histology: A preliminary study” (applsci-470647)

 

Comment to the authors

 

General comments

-          The manuscript needs a grammar and style revision, maybe by a native English speaker. Several errors have found throughout the text (e.g. verbal times).

-          The article presents an a serious overlap in certain sections with another article from the same authors “Combined micro computed tomography and histology study of bone augmentation and distraction osteogenesis”

(available in: https://www.spiedigitallibrary.org/conference-proceedings-of-spie/8506/85060M/Combined-micro-computed-tomography-and-histology-study-of-bone-augmentation/10.1117/12.929616.short?SSO=1)

Also figure 7 from that article presents the same image than the one represented in figure 3. Since the article is from 2012 and the topic is similar but using different materials and protocol, it presents a risk for plagiarism and duplication of results that are already published and raises doubts on the reliability of the presented data.

 

Specific comments:

Title page and abstract:

-          Abstract is not correctly written. Since it is an unstructured abstract avoid making different paragraphs.

-          Abstract should be more descriptive (e.g. distraction time).

-          Please ensure that the keywords are correct MeSH terms. Use the appropriate terms as specified in MeSH for better indexation.

 

Introduction

-          Authors need to better justify the rationale of their study since they state that the injection of mesenchymal stem cells has been already showed in the available evidence.

-          Authors speak about determining the ideal moment for the MSC application, but in their design

-          Introduction section should end by stating the study objectives.

 

Materials and Methods

-          Please comment on the study design and follow an appropriate guideline for the manuscript preparation and specify this in the methods section. In this case, ARRIVE guidelines are the appropriate for animal studies.

-          Please give details on the obtention method and the origin of the donor human mesenchymal cells.

-          I do not think that table 1 is necessary, since the study design is quite simple and easy to understand.

-          No information on the statistical test of quantification of the histological images is provided.

 

Results

-          Line 175: Figure 3f does not correspond to any section of figure 3.

-          No table with the numeric results of the analyses is presented.