Three-Dimensional Human Liver Micro Organoids and Bone Co-Culture Mimics Alcohol-Induced BMP Dysregulation and Bone Remodeling Defects

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Some concerns should be considered as follows:

1. Abstract: The aim of this study should be clearly described. Line 33: Please revise it.
2. Intro: Line 48: The authors mentioned different characterizations of Hepatic osteodystrophy and cited one reference, but I think it does not cover this statement. Line 49-52: Please support the statement with pertinent references since the authors mentioned growing evidence ……….. line 58-62: it should be moved to the end of the intro to justify the novelty of this study. Line 105-110: it should be added as a conclusion of this study based on the findings obtained in this study. Several statements in the intro lack references, please revise.
3. Methods: Please mention the country of each kit or products, such as line 148, 149, and …… Line 212: model of the omega plate reader. Line 231 and 232, pleas revise the spaces and the end of each sentence.
4. Results: It is not important to mention the exact p-value in the text. Fig. 8b: it should be improved. The results include several references; please cite only the necessary references.
5. Discussion: Please spell out the full scientific expressions when mentioning them for the first time in the manuscript. It is too lengthy; please remove redundant information. The limitations should be discussed, showing future perspectives.

6. Conclusion: key findings should be highlighted.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review report for Manuscript ID cells-4052635

 

Journal: Cells

Title: 3D Human Liver Micro Organoids and Bone Co-culture Mimic  Alcohol Induced BMP Dysregulation and Bone Remodeling Defects

 

The paper presents the results of an original study conducted on cell cultures that consists of long-term human three-dimensional liver-bone co-culture model that integrates hepatocytes (HepaRG), hepatic stellate cells (LX-2), and human umbilical vein endothelial cells (HUVECs) with bone scaffolds seeded with osteoblast precursors (SCP-1) and osteoclast precursors (THP-1). This human liver micro-organoid co-culture which reproduces alcohol-induced hepatic BMP dysregulation and downstream bone defects, offering an organoid-centric, microengineered platform for mechanistic studies and BMP-targeted therapeutic screening in HOD has revealed a decrease in BMP2 and an increase in BMP13, without affecting BMP9. The paper is comprehensive and well-designed. The introduction part is informative with clearly defined aim of the study.  The methodology is explained in details, the results are presented in a visually adequate manner with excellent Figures. The discussion is well-written and the conclusion is valid. The authors should address the following minor comments before the paper being considered for publication:

1. Provide in the discussion why there is decrease in the levels of CYP1A2 and CYP3A4 while increase in CYP 2C9 activity (Figure 2)
2. Give more details about the alcohol effect on mineral density (Figure 6)
3. Provide limitations and strengths of the study before the conclusion part.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I congratulate the authors for this very interesting study. The article is worth being published, provided some changes are made:

• decrease the overall length of discussion section of the article, and make sure that all relevant information is properly cited.
• from a methodological point of view, there are some major issues:
  • you have provided insufficient data to support the statement that your model closely mimic the in vivo situation (no direct comparisons of BMP levels, cellular responses with samples from patient with FAD, the 50mL concentration is too high, the time is too low (28 days) to properly represent chronic alcoholism;
  • you state that alcohol does not directly affect bone cells, but you failed to include critical controls;
  • the BMP2/BMP13 axis is incompletely characterized;
  • some cell lines do not accurately represent primary cell biology that is relevant here: HepaRG cells are tumor-derived, and may not fully mimic hepatocyte responses to alcohol; LX-3 cells may have altered activation profiles compared to primary stellate cells
  • the absence of Kupffer cells in the model is a fundamental limitation, as inflammation (to which these cells are critical in the liver), is essential to both liver fibrosis and subsequent bone loss
• the WB bands from fig. 8b show inconsistent loading, despite normalizatio claims;
• some error bars are hard to distinguish.
•  

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

    This manuscript presents a well-designed and biologically meaningful study that fits very well within the scope of Cells, particularly given the journal’s interest in advanced in vitro systems, inter-organ communication, and mechanistic signalling pathways. The authors introduce an elegant and technically demanding 3D human liver–bone co-culture model that convincingly recapitulates key aspects of alcohol-induced hepatic osteodystrophy. The concept is timely, the experimental platform is sophisticated, and the biological questions are addressed in a logical and coherent manner.

The Introduction is generally strong and well referenced, offering a solid overview of hepatic osteodystrophy, alcohol-related liver disease, and the role of BMP signalling in liver–bone crosstalk. Minor improvements could be achieved by slightly sharpening the final paragraphs to more explicitly highlight the specific knowledge gap addressed by this study—namely, the lack of long-term human in vitro systems capable of reproducing hepatic BMP dysregulation and its downstream skeletal consequences. Clarifying this point would further strengthen the rationale and originality of the work.

The Materials and Methods section is detailed, transparent, and reproducible, which is particularly important given the complexity of the co-culture system. The description of liver micro-organoid generation, bone scaffold preparation, and their integration is clear and sufficiently thorough. One small suggestion would be to improve readability by harmonizing tense usage and sentence structure in several subsections (e.g., cell culture descriptions and enzymatic assays), but no methodological gaps were identified. Statistical analyses are appropriate, clearly described, and consistent with the data presented.

The Results section is logically structured and easy to follow. Each subsection builds convincingly on the previous one, guiding the reader from model validation through mechanistic exploration. The long-term stability and functionality of the liver micro-organoids are well demonstrated, and the authors appropriately justify the choice of alcohol concentration. Importantly, the distinction between direct alcohol effects on bone and indirect liver-mediated effects is a major strength of the study and is handled very clearly.

The findings related to BMP2 downregulation and BMP13 upregulation are particularly compelling and biologically plausible, especially when combined with the downstream activation of canonical and non-canonical BMP signalling pathways and the observed lineage shift toward chondrogenesis. The use of functional readouts such as mineral density and scaffold stiffness adds significant translational value and strengthens the conclusions. Figures are generally clear, well-labelled, and appropriately referenced in the text.

The Discussion is thorough, well contextualised, and demonstrates a strong command of the relevant literature. The authors successfully integrate their findings with existing animal and clinical data while clearly articulating the added value of their human in vitro model. The discussion around the absence of overt osteoclast activation is balanced and thoughtful, and the limitations of the system—such as the absence of Kupffer cells and inflammatory components—are appropriately acknowledged. As a minor suggestion, the final paragraphs could be slightly streamlined to reduce redundancy and to emphasise more clearly the platform's translational and screening potential.

Comments on the Quality of English Language

Regarding language and style, the manuscript is generally well written and understandable, but it would benefit from moderate English editing, particularly to improve sentence flow, correct minor grammatical inconsistencies, and standardise terminology throughout the text. These issues do not detract from the scientific quality, but addressing them would improve clarity and readability for an international audience.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors responded to all comments properly; thus, I recommend accepting the current version of this work.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have provided the required changes.