The Role of Extracellular Vesicles in Bone Regeneration and Associated Bone Diseases

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript entitled “The Role of Engineered Extracellular Vesicles in Bone Regeneration and Associated Bone Diseases”deals with a very topical issue in the field of bone regeneration. The paper is well structured, with an introduction to the different classifications of extracellular vesicles, the influence of EVs on the different cell types in bone and then a detailed description of the use of EVs in the treatment of different diseases. The authors have written a very good review and made use of the current literature. As a small comment, it would be good if the authors included a materials and methods section at the end of the paper, indicating the sources, where they searched, when the literature search took place, what search terms were entered and how many results were obtained. 

After this addition, the paper can be published from the reviewer's point of view. 

Author Response

Dear Reviewer 1：

Thank you very much for reviewing our manuscript and for your valuable suggestions. After carefully reading your comments, we have made revisions, which are marked in red in the manuscript. Below are our detailed responses to each issue raised. We sincerely appreciate your recognition of the strengths of our manuscript.

Materials and Methods: An initial literature search was conducted on February 21, 2024 at Chongqing University School of Medicine. Prior to submission of the manuscript, we re-ran the literature search (August 16, 2024) to determine if any new potential articles were included. The following databases were used for the literature search: PubMed, Web of Science, and Scopus. Our search terms were “extracellular vesicles AND bone regeneration”, “exosomes AND bone regeneration”, “extracellular vesicles AND biogenesis”, (extracellular vesicles) AND (osteoporosis OR osteoarthritis OR fracture). The numbers after searching were 714, 553, 2238, and 944. Review articles were read mainly published in the last five years, while experimental articles did not clearly differentiate the time.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The authors deal with the role of extracellular vesicles with regard to bone regeneration and associated bone diseases. They introduce EVs and their general role in phyhsiology and pathophysiology and with regard to bone regeneration. Additonally, the use of EVs in recent therapeutical approaches is reviewed. The manuscript is generally interesting and well written and fits the scope. However, I added an extra file with my comments which should be adressed in the following review process.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Only minor changes needed

Author Response

Dear Reviewer 2：

Thank you very much for reviewing our manuscript and for your valuable suggestions. After carefully reading your comments, we have made extensive revisions, which are marked in red in the manuscript. Below are our detailed responses to each issue raised.

General: Thank you. We have made the changes in the original article as you requested and marked them in red.

Title: Thanks for the suggestion, it has been modified.

Line 17/18:I agree, it seems more reasonable.

 

Line 29: 1. Climate change may affect the production and availability of food, leading to deficiencies in the intake of key nutrients, such as vitamin D and calcium, which may indirectly lead to an increased risk of osteoporosis and bone-deficiency-related diseases; 2. Extreme weather events (e.g., floods, tornadoes, global warming, etc.) brought about by climate change may affect people's lifestyles and activity levels, e.g., high temperatures may lead to a reduction in outdoor activities, which in turn may affect bone health. in turn affecting bone health.

 

Lines 30-40: Thank you for your meticulous scrutiny, I've checked the citation order of the references and made the modifications.

 

Line 34-35: I agree, it makes the sentence more complete.

 

Line 40 et seq.: Very good advice to give coherence to the article.

 

Line 48: I mean, as a type of exosome, microvesicles are a larger body size with a regular shaped structural appearance.

 

Figure 1: The content shown in the figure has been added and a brief narrative has been provided. The method of production has also been attached.

Extracellular vesicles are categorized as exosomes, microvesicles, and apoptotic vesicles based on their diameter, origin, and shape. The biogenesis of exosomes and microvesicles, as the main carriers for transporting cargos, is very complex and involves the regulation of multiple cytokines and signaling pathways. Primary endosomal vesicles fuse to form early endosomes, followed by EEs carrying a large cargo of biomolecules to form backbone proteins, and then EEs selectively undergo two pathways: one pathway is to become “regenerating endosomes” and the other is to form “late endosomes” (LEs ). Microvesicle generation is a complex process in which the release is mainly regulated by factors such as GTPases (e.g. Rho family), ARF and other cytoskeletal elements. This figure was created by biorender (https://app.biorender.com)

 

Line 154: Your comments are very original and have been reworked to elaborate on the latest concepts of macrophages.

 

Line 159: Osteoarthritis is prevalent in middle-aged and elderly people and its incidence is positively correlated with age, while youthful growth is accompanied by bone loss, which leads to microfractures of subchondral cortical bone, thus affecting the normal structure of articular cartilage and leading to the development of osteoarthritis. Indeed, the main features of osteoporosis are the decrease in bone density and the deterioration of bone tissue structure, and the loss of bone mass will lead to the changes in the bone structure mentioned above, and at the same time, the changes in the structure will further aggravate the loss of bone mass, which will in turn form a vicious cycle, thus bone loss plays an important role in osteoporosis.

 

Line 170: Modified.

 

Line 195-196: The implication of this statement is that primary osteoblast-derived EVs derived from mice have a gene expression profile similar to that of osteogenesis-related genes, which are mainly RUNX2, COL1A1, ALP, and OSX, and which have been used as biomarkers of osteogenic differentiation in the clinic as well as in scientific research.

 

Line 217-218: At the onset of a trauma-induced fracture event, the pre-inflammatory phase disrupts constitutive cellular homeostasis in the skeletal microenvironment such as macrophages, osteoblasts and osteoclasts, which in turn accelerates bone loss.

 

Line 219: Thank you very much. In line 219, 'related cytokines' refers to cytokines associated with the pro-inflammatory response process triggered by traumatic fractures. These cytokines are activated during the inflammatory response at the fracture site and are involved in regulating the differentiation process of precursor cells.

 

Line 221: Thank you. We have a problem with the expression. The exosomes (EVs) themselves do not directly interact with each other. We have revised the sentence: moreover, EVs produced by different cells can interact with other cells, transferring information and influencing each other.

 

Line 228: Thank you. Yes, blood vessels themselves do not usually actively produce paracrine factors. The primary function of blood vessels is to transport blood and the nutrients, oxygen, and other biomolecules (including paracrine factors) in it. These paracrine factors are usually produced by other types of cells (e.g., endothelial cells, smooth muscle cells, etc.) and are transported through the blood flow to target cells or tissues where they play a regulatory role. Blood vessels can indirectly influence cell behavior by regulating blood flow and distributing these factors, but they do not produce them themselves. We have modified the sentence in the text to make it more academic.

 

Line 241: Thank you. We carefully reviewed the relevant literature. Relevant proteins were added, mainly VEGF, HGF, and TGF-β.

 

Line 241: Thank you. We read the literature and found areas where our expression was problematic. We have modified it to : Additionally, overexpression of HIF-1α in human dental pulp MSCs promotes EV release, carrying large amounts of HIF-1α and enhancing angiogenesis through interaction with the Notch signaling ligand Jagged1.

 

Line 254: Thank you. We clarified the concept and made the following changes. During bone regeneration, hypertrophic cartilage is crucial in secondary bone healing with endochondral ossification, as it undergoes mineralization, vascularization, and eventual remodeling into bone after tissue disruption and healing. This process is specific to endochondral ossification and does not apply to intramembranous ossification, where bone formation occurs directly from mesenchymal tissue without the formation of a cartilage intermediate.

 

Figure 2: Thank you. We have made detailed changes to all the inappropriate representations in Figure 2.

Extracellular vesicles act on monocytes, mesenchymal stromal, osteocytes and osteoclasts, which in turn activate signaling pathways such as Wnt, TGF-β and RANKL, and ultimately promote bone regeneration.

This figure was created by biorender (https://app.biorender.com)

 

Line 275: Thank you. We have reviewed a lot of relevant literature to express this statement in more detail and make it more scholarly and logical. Osteoporosis (OP) is increasingly recognized as a major global public health issue, particularly as the population ages. The World Health Organization (WHO) has acknowledged osteoporosis as a common disease, and the growing proportion of elderly individuals is expected to exacerbate this concern, leading to higher incidence rates and increased healthcare burdens associated with osteoporosis.

 

Line 278-279: Thank you. We have scrutinized our manuscript and made the following changes. OP is primarily associated with the disruption of bone homeostasis. It arises from an imbalance in the processes of bone formation and resorption, leading to a net loss of bone mass. This condition is largely due to the dysregulation of osteoblast and osteoclast activities, which disrupts the dynamic equilibrium necessary for maintaining bone health.”

 

Line 284-285: Thank you. We have carefully read the manuscript and have revised inappropriate expressions. Pluripotent stem cells such as embryonic stem cells (ESC) and mesenchymal stromal have been used in bone regeneration-related fields in a few countries, and it is a very promising direction in the future.

 

Line 315 et seq.: Thank you. These studies are tests done on mice.

 

Line 351: Thank you. We replaced words that were inaccurately expressed. Use Physicians.

 

Line 356 et seq.: Thank you. We fixed this improper formatting issue.

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

Comments and Suggestions for Authors

The manuscript entitled "Advances in Extracellular Vesicles in Bone Regeneration and Related Bone Diseases" is a review that brings to the fore the exceptional potential of extracellular vesicles (EV), in an important medical field, namely bone regeneration.

EVs and the biomolecules carried by them can regulate cell functions and signaling, having a key role in the development, diagnosis and treatment of various diseases, being also used as alternatives to cell transplantation, including promoting bone regeneration.

The paper shows the mechanisms by which EV-mediated cells influence bone regeneration and their role in the treatment of several bone diseases.

The review is well written, with a fluid coherence of the presentation.

 

I recommend the publication of this work in the journal "Current Issues in Molecular Biology" after a minor revision.

 

I suggest the following to the authors:

1. In chapter 2 we discussed exosomes, microvesicles. A short paragraph about apoptotic vesicles should be added here.

2. Since they are missing, the legends of the two figures must be added (Fig.1 and Fig.2).

3. The legends of the two tables (Table 1 and Table 2) are missing and must be added.

4. In the chapter "Clinical diagnostic tools", the role of EV in the diagnosis of diseases related to bone regeneration could be presented distinctly, using bullet points, not as a list in the text, to be easier for readers to follow.

5. Several case studies (in vitro or in vivo) could be presented here regarding the role of EVs, as well as the results obtained in bone regeneration.

6. Citations from the text and from the reference chapter must be cited in accordance with the journal's requirements.

 

Author Response

Comments1: In chapter 2 we discussed exosomes, microvesicles. A short paragraph about apoptotic vesicles should be added here.

Response1: Agree.Considering the completeness of the exosome description. We have taken your suggestion to add a short paragraph about apoptotic vesicles.

Comments2:Since they are missing, the legends of the two figures must be added (Fig.1 and Fig.2).

Response2: Agree.We have added two figures (Fig.1 and Fig.2).  Fig.1 The main biogenesis of EV and Fig.2 Different cell-mediated mechanisms of EV in bone regeneration.

Comments3: The legends of the two tables (Table 1 and Table 2) are missing and must be added.

Response3: We have added two tables(Table 1 and Table 2). Table 1 The role of EV in bone regeneration diseases and Table 2 Clinical trial of EV in bone regeneration related diseases.

Comments4: In the chapter "Clinical diagnostic tools", the role of EV in the diagnosis of diseases related to bone regeneration could be presented distinctly, using bullet points, not as a list in the text, to be easier for readers to follow.

Response4: I agree with your suggestion and have used bullet points to let readers easier to find the points.

Comments5: Several case studies (in vitro or in vivo) could be presented here regarding the role of EVs, as well as the results obtained in bone regeneration.

Response5: At your suggestion ,we have added several case studies of EV therapy for bone regeneration-related diseases.

Comments6: Citations from the text and from the reference chapter must be cited in accordance with the journal's requirements.

Response6: Agree. We have modified the citation format of the references according to the journals requirements

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript under review addresses the application of extracellular vesicles (EVs) in bone-related therapies. However, it fails to introduce significant innovative developments in the field. The overall aim of the manuscript is confusing, as it mixes diverse topics such as bone regeneration, fracture healing, and bone-related pathologies without clearly distinguishing between them. This lack of focus detracts from the overall impact and clarity of the review. In addition, previous reviews on the topic have already been published, including but not limited to :

https://doi.org/10.3390/cells13110904; https://doi.org/10.3390/ijms25063480; https://doi.org/10.3390/ijms25010568; https://doi.org/10.3390/jfb14040193; https://doi.org/10.3390/cimb44120433; https://doi.org/10.3390/pharmaceutics14081684; https://doi.org/10.3390/membranes12070716;

The manuscript attempts to cover a broad spectrum of bone-related applications of EVs, yet it does so in a manner that lacks coherence. The amalgamation of various themes—regeneration, fracture healing, and bone pathologies—results in a diluted narrative that fails to provide a clear direction or purpose. It is crucial for a review to have a well-defined scope and aim to effectively contribute to the field, which this manuscript does not achieve.

One of the significant shortcomings of the manuscript is its inadequate disclosure of relevant mechanistic approaches. The review merely presents the major outcomes of the articles it covers, without delving into the underlying mechanisms by which EVs exert their effects on bone tissue. Understanding these mechanisms is essential for advancing the application of EVs in bone therapies, and the manuscript's failure to address this aspect significantly limits its contribution to the field.

Overall, the manuscript does not present any significant advancements or innovative contributions to the existing literature on the use of EVs for bone applications. Its broad and confusing aim, lack of detailed mechanistic insights, and mere presentation of major outcomes render it a superficial overview rather than a critical and insightful review. Authors are encouraged to enhance the focus, providing detailed mechanistic discussions, and offer novel insights or syntheses to advance the field meaningfully.

 

In addition, the quality of English needs to be significantly improved to enhance readability and clarity.

Comments on the Quality of English Language

In addition, the quality of English needs to be significantly improved to enhance readability and clarity.

Author Response

Comments1: The manuscript under review addresses the application of extracellular vesicles (EVs) in bone-related therapies. However, it fails to introduce significant innovative developments in the field. The overall aim of the manuscript is confusing, as it mixes diverse topics such as bone regeneration, fracture healing, and bone-related pathologies without clearly distinguishing between them. This lack of focus detracts from the overall impact and clarity of the review. In addition, previous reviews on the topic have already been published, including but not limited to :

https://doi.org/10.3390/cells13110904; https://doi.org/10.3390/ijms25063480; https://doi.org/10.3390/ijms25010568; https://doi.org/10.3390/jfb14040193; https://doi.org/10.3390/cimb44120433; https://doi.org/10.3390/pharmaceutics14081684; https://doi.org/10.3390/membranes12070716;

Response1:  Firstly, this review provides a summary of the diseases most commonly associated with bone regeneration, with the aim of providing possible clinical treatments for bone regeneration. So, we introduce bone regeneration, fracture healing, and bone-related pathologies, which are commonly encountered in clinical practice, and bone regeneration plays a key role in all of  the above. Here, we searched PubMed for articles with associations between bone regeneration and facture healing and bone-related diseases:

https://doi.org/10.1038/nrrheum.2014.164

https://doi.org/10.1007/s13346-022-01222-6

https://doi.org/10.3390/ijms24010171

https://doi.org/10.3390/bioengineering10101187

Our review begins with an introduction to the classification of exosomes, with the extracellular vesicles in exosomes as the focus of the summary. The cell-derived EVs associated with bone regeneration are described first, and previous articles have described the cell classification, as we have done, but we consider the issue of the interaction of cell-derived EVs, which is not mentioned. We believe this aspect has a few contributions to make in this filed. And provide some ideas for future research.

Comments2: The manuscript attempts to cover a broad spectrum of bone-related applications of EVs, yet it does so in a manner that lacks coherence. The amalgamation of various themes—regeneration, fracture healing, and bone pathologies—results in a diluted narrative that fails to provide a clear direction or purpose. It is crucial for a review to have a well-defined scope and aim to effectively contribute to the field, which this manuscript does not achieve.

Response2: Agree.We've removed the irrelevant sections and add some new, which has been marked in red ,more relevant content to make this review became more coherence.

Commnets3: One of the significant shortcomings of the manuscript is its inadequate disclosure of relevant mechanistic approaches. The review merely presents the major outcomes of the articles it covers, without delving into the underlying mechanisms by which EVs exert their effects on bone tissue. Understanding these mechanisms is essential for advancing the application of EVs in bone therapies, and the manuscript's failure to address this aspect significantly limits its contribution to the field.

Response3:  Specific mechanisms by which EV affects bone regeneration have been added. We have added concrete mechanistic in chapter 3.2,4.3,5.1and 5.2,which have marked in red. However, there are some specific mechanisms that have not yet been fully explored, and we will follow up with a targeted, in-depth discussion of those that have not yet been elucidated.

Comments4:Overall, the manuscript does not present any significant advancements or innovative contributions to the existing literature on the use of EVs for bone applications. Its broad and confusing aim, lack of detailed mechanistic insights, and mere presentation of major outcomes render it a superficial overview rather than a critical and insightful review. Authors are encouraged to enhance the focus, providing detailed mechanistic discussions, and offer novel insights or syntheses to advance the field meaningfully.

Response4: As research continues, EVs are becoming more widely available as treatments and some are already in clinical use, but some EVs as potential treatments are not fully understood in terms of their specific mechanisms and some are in clinical trials. We will follow the progress of the research as we continue to deepen our insights into EV as a therapeutic area. Furthermore, this review concludes with an outlook on the homeostasis of the environment surrounding bone regeneration and analyses what limitations remain for EV as a future treatment method in summary.

Comments5: In addition, the quality of English needs to be significantly improved to enhance readability and clarity.

Response5:We actually need to improve our English writing, which is our weakness. We've reworked the English language to make it more understandable. Like chapter 5.1,

we have rewritten the paragraph to list the diseases associated with clinical bone regeneration according to their degree of prevalence, as well as chapter 4.1and 6. Taken together, thank you very much for your careful review and give us precise suggestion.

Author Response File: Author Response.pdf