Anterior Cruciate Ligament Tissue Engineering: Biological Principles, Engineered Substitutes, and Preclinical Outcomes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The title of the paper addresses an important topic in orthopedic tissue engineering. However, the manuscript requires substantial improvements as it has significant issues with organization, analytical depth, critical evaluation, methodological rigor, and technical clarity.

Most fundamentally, the title misrepresents the content. The paper purports to focus on “tissue engineering” of the ACL, yet large portions of the text are devoted to general knee anatomy, ACL biomechanics, and conventional surgical treatments, essentially reproducing textbook material. As a result, the title is overly broad and somewhat misleading. In scientific writing, titles should accurately reflect the aim and scope of the work. Here, a more specific title or a narrowed focus is warranted. For example, if the intent is to review only tissue-engineering approaches to ACL repair, the introduction and title should clearly state that narrower focus. Currently, the mismatch between title and content confuses the reader about the paper’s true scope.

The organization and focus of the review are also problematic. A high-quality literature review should be organized around clear themes or questions, not simply a sequence of disjointed topics. The manuscript’s structure is diffuse: Sections on basic anatomy, collagen biology, and clinical reconstruction occupy many pages with little connection to the core theme of tissue engineering. In a well-structured review, the introduction would explicitly outline the review’s question and scope, for example, which aspects of ACL tissue engineering are being surveyed, and would identify gaps in current knowledge. Instead, the introduction largely describes ACL injury prevalence and standard grafts, then briefly mentions tissue engineering. The boundaries of the review are not clearly defined, and readers cannot discern a specific organizing principle. As one guide notes, reviewers should check that the “title and approach of the work” are closely aligned and that the authors have a clear research question for their narrative. This paper lacks such framing: the transition from general background to tissue engineering is abrupt, and later sections (especially on artificial ligaments and artificial intelligence) feel like loosely connected appendices rather than integral parts of a coherent review.

In terms of content, much of the material is superficial and lacks novelty. The anatomy and biomechanics sections mostly repeat well-known facts (e.g. knee joint structure, ACL collagen composition) without adding new insights or critical synthesis. Good reviews should synthesize existing literature to advance understanding, rather than simply recount common knowledge. Here, basic information is presented in encyclopedic detail (with numerous older references), but little critical perspective is offered. By contrast, a review of tissue engineering ought to emphasize recent studies, technologies, and remaining challenges in ACL repair. The core tissue-engineering section (Section 4) outlines various biomaterials and techniques (synthetic, natural, hybrid scaffolds, decellularization, cells), but its depth is uneven. Some subsections read like lists of materials with minimal discussion of how each specifically advances ACL repair. Cross‐references to ACL context are sparse. For example, it is unclear how the many mentioned biomaterials have performed in ACL-specific experiments. The narrative fails to tie these subtopics together into a focused discussion. In fact, it reads as if the authors describe general soft-tissue engineering concepts rather than ACL-specific strategies. Thus the section lacks thematic continuity and fails to highlight what is novel or particularly promising about ACL tissue engineering.

The figures and illustrations are also problematic. Figure 1 (knee anatomy) is said to be created in BioRender, but in style and execution it does not appear to match typical BioRender outputs. Regardless, it simply shows basic ligaments without any labeling of magnitudes or references; as drawn, it adds no information beyond what the text already states. Figures in scientific papers must be informative and precise. They should be understandable on their own, with captions that describe all elements and highlight the main message. In this paper, the figure captions are often too brief or vague. For example, Figure 3’s caption merely lists “patient-derived cells, culture media, scaffold techniques” in generic terms. An effective caption should “describe everything in the graph, draw attention to its important features, and (when practical) describe the main conclusions to be drawn from it”. None of the figures meets this standard. Figure 2 (“Tissue engineering workflow”) is especially disappointing. It is visually cluttered and includes non-scientific elements (the reviewer notes a label like “Healthy and happy patient doing sport”), which is inappropriate in a professional manuscript. Flowcharts in reviews should use clear schematic symbols and standardized scientific language. Adding cartoonish or subjective phrases undermines the paper’s credibility. The caption for Figure 2 is not self-contained and fails to explain key steps; readers must guess what the icons mean. Moreover, Figures 4 and 5 (reused from the authors’ prior work, see below) include histological images but lack scale bars or calibration. In microscopy and tissue images, including scale bars is essential so that readers can interpret sizes and dimensions. As one source emphasizes, “scales give the reader the key for aligning the image content with reality”. Without scale bars or detailed legends, the histology panels in Figures 4–5 cannot be properly understood or compared. The figures in this manuscript do little to support the discussion; in some cases they merely duplicate known results (or even earlier publications by the same authors) without new interpretation, which per guidelines would be considered redundant or “confirmatory” data that adds little new understanding.

The most striking issue is the dominant presence of Dr. Francine Goulet and the Goulet group's work throughout the manuscript. The entire Section 5 ("Example of a successfully goat-implanted bioartificial anterior cruciate ligament") is essentially a detailed exposition of the Goulet group's research spanning decades. This creates several problematic patterns: the paper reads less as a balanced review and more as a technical report promoting one research program; Section 5 is disproportionately detailed about fabrication steps and staging of integration; and the extensive acknowledgment stating Dr. Goulet "chose not to be listed as an author" despite being the focus of an entire section raises questions about authorship transparency and conflicts of interest.

Section 4, which addresses tissue engineering approaches, lacks rigorous comparative analysis. Tables 2-4 present preclinical data but offer minimal critical interpretation. The statement "typically ≤ 20-40% of native strength in the mid-term" appears without supporting data synthesis or analysis of whether this represents a meaningful clinical threshold. Most critically, there is no systematic evaluation of critical parameters, mechanical property targets, degradation kinetics matching, integration timelines, across approaches, nor any quantitative framework for comparing synthetic/hybrid, natural non-decellularized, and decellularized materials.

Several important topics receive insufficient or no adequate treatment: immune response and graft rejection are mentioned but underdeveloped; mechanical testing standards and what constitutes clinically meaningful performance lack discussion; vascularization and revascularization kinetics are glossed over; proprioceptive/sensory nerve integration is addressed in anatomy but never integrated into tissue engineering strategies; regulatory pathways and clinical trial considerations are nearly absent; cost-effectiveness and manufacturing scalability receive no attention; and long-term in vivo outcomes beyond 12 months are rarely discussed.

Section 7 feels like an add-on rather than an integral part of tissue engineering discussion. Most AI applications discussed (surgical planning, tunnel placement, kinematics prediction) apply to conventional ACL reconstruction rather than bioengineered substitutes. Missing is discussion of AI applications directly relevant to tissue engineering: computational modeling of scaffold design, optimization of degradation kinetics, prediction of integration outcomes, or personalized graft design. The section reads more like a literature scan than critical analysis.

The review presents information comprehensively but rarely critically evaluates fundamental barriers. Why hasn't any tissue-engineered ACL reached clinical practice after decades of research? What are the specific bottlenecks? Are current mechanical targets realistic? The review notes constructs achieve "up to 40%" of native strength but doesn't discuss whether achieving higher percentages is necessary or possible. The apparent biological-mechanical tradeoff (natural materials support integration but are weak; synthetic materials are strong but trigger inflammation) is presented but never analyzed as potentially inherent vs. solvable.

Section 2.8 addresses mechanoreceptors and proprioceptive function well, but its implications are inadequately integrated into tissue engineering discussion. Few constructed designs address proprioceptive restoration. The implication that restoring structural ligament function automatically restores proprioceptive function is unsupported.

The preclinical trial tables present data inconsistently, different studies report different parameters, making cross-study comparison impossible. No critical appraisal distinguishes well-controlled from poorly controlled studies. Mechanical properties lack effect sizes or confidence intervals. The statement "Decellularized Materials: Decellularized grafts offer excellent integration" is based on only two studies. No discussion addresses why some approaches work better than others.

Manufacturing process validation, GMP compliance, FDA/EMA regulatory pathways, scale-up feasibility, cost per implant, sterilization requirements, quality control, and commercial viability are largely absent, yet these factors are as critical as biological function for clinical translation.

The Conclusions section is brief and generic, restating problems without offering specific recommendations about which approaches warrant investment or clear guidance on experimental priorities. No timeline prediction for clinical translation is provided.

Several passages contain typos (e.g., "trilpe-helix"), tangential discussions (lactate release concerns in oncology for an ACL review), and overly specific technical details about one group's methodology. The discussion of post-translational modifications in collagen biosynthesis appears excessive for a broad tissue engineering audience.

Figure 1 is basic; Figure 3 lacks quantitative information; Figures 4-5 are Goulet group-specific, reinforcing bias; missing are comparative mechanical property trajectories

"Ligamentization," need explicit definition

One author affiliated with Christie Innomed; no disclosure of intellectual property; no conflicts of interest statement

Missing discussion of species-specific considerations and animal model translatability to humans

The manuscript currently lacks the novelty, depth, and organization expected of a scholarly review. It reads more like a descriptive report than a critical literature synthesis.

Comments on the Quality of English Language

The language requires extensive editing.

Author Response

As indicated in the letter to the Editor, we have chosen not to provide a point‑by‑point response to Reviewer 1. We acknowledge that several technical comments were considered during the revision process, but given the overall tone and nature of the report, we do not believe that further direct exchange would be productive. The revised manuscript integrates significant improvements, and detailed responses have been provided for Reviewers 2 and 3, whose comments were constructive and directly oriented toward strengthening the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors report, “Tissue Engineering of the Anterior Cruciate Ligament”.

I recommend minor revisions as outlined in the comments below.

1. The abstract should be revised to include the clear aim of the article.
2. The novelty of the article should be clearly discussed in the Introduction (last paragraph).
3. The copyright statement for the figures does not meet the standard requirements, as it lacks information about the publisher and corresponding reference citations.
4. All abbreviations in the manuscript should be defined upon their first occurrence.
5. Resolution of Figures should be increased.
6. Some referenced studies are outdated. Can the authors incorporate more recent reports from the last 2–3 years to strengthen the scientific relevance?
7. The author is encouraged to enhance Section 4, by incorporating relevant figures and illustrations from reported studies to strengthen the scientific rigor and impact of the chapter
8. More reports should be added to Table 2
9. Section 7 (‘Artificial Intelligence to Improve ACL Reconstruction’) should be discussed in more detail, including recent studies and practical applications
10. The conclusions section needs to be improved and better structured.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Major issues:

1. Some sentences seem not to be written by human. If AI was used in writing or polishing the manuscript, according to “MDPI’s Updated Guidelines on Artificial Intelligence and Authorship” (https://www.mdpi.com/about/announcements/5687?utm_source=chatgpt.com), authors are required to be fully transparent, within the “Acknowledgments” section, about which tools were used, and to describe in detail how the tools were used, in the “Materials and Methods” section.
2. A very big problem about this manuscript is numerous inappropriate citations. Some references are not accurate, and a lot of references are not supporting authors’ claims. I will show some examples here but not every one because it is authors’ duty to check carefully every reference in the whole manuscript: (1) Line 39: “no universally optimal long‑term reconstructive strategy has yet been established [5,6]” is inaccurate. Ref 5 and 6 only talk about methods based on tissue engineering or bioengineering, but tissue engineering or bioengineering is not mentioned in the sentence on line 39. The current sentence implies autograft is not a long-term reconstructive strategy, which could be controversial. (2) Line 68-70: “allogeneic constructs made of silk fibers, chitosan, collagen, or other fibrous biomaterials, sometimes used in combination and seeded with various cell types [16]”. Ref 16 only is not sufficient to support this sentence, because it only talks about silk fibers and more references are needed to support the usage of other biomaterials. (3) Line 71-73: “Supplementing scaffolds with growth factors may help restore a microenvironment conducive to cell proliferation, collagen synthesis, and early ligamentization in vitro and in vivo [13,18]”. Ref 13 does not relate to growth factors at all. (4) Line 78-79: “Restoration of key ultrastructural properties of the native ACL is essential for long‑term function [4,19]”. Ref 4 only mentions a bit about ultrastructure and in the long-term recovery, they did not emphasize on the restoration of ultrastructural properties. (5) Line 133: “The ACL is composed chiefly of type I collagen fibrils, with smaller amounts of type III collagen [27]”. Ref 27 is a paper about collagen family, so it only cares about collagen. ACL composition is chiefly collagen, but it also contains many more other types of ECMs.
3. Line 58-59: “Owing to its anatomical and biomechanical similarities to the human knee, the ovine model is highly relevant for studying post‑injury OA mechanisms”. This sentence is too weird to be here. It is not closely related to the former sentences and cannot support the conclusion “taken together, these challenges highlight the need for improved surgical strategies to restore and preserve long‑term knee function.”
4. Can authors confirm Figure 1 is created with BioRender? I checked every diagram in the library of BioRender and cannot find anything exactly like Figure 1. Another concerning issue is that 2 big tendons in the knee, quadriceps tendon and patellar tendon, are missing in this figure. I also suggest that in the legends it should be explained which one of right or left knee is shown here because that would be more friendly to readers.
5. The knee icons in Figure 2 are inconsistent. In the knee icon above the text “ACL torn/rupture” has quadriceps tenson, but the other 3 knee icons do not, which is not consistent.
6. Mechanical stimulus is also a very important tool for getting better ligament tissue in vitro. Related papers should be cited and discussed, for example, “Intermittent cyclic stretch of engineered ligaments drives hierarchical collagen fiber maturation in a dose- and organizational-dependent manner (https://doi.org/10.1016/j.actbio.2024.07.025)”.

 

Minor issues:

1. In Figure 5G and H, what do the green and red lines stand for should be explained in the legends.
2. References still need to be checked carefully. Ref 99 misses the name of the journal (bioengineering), and “(Basel)” should not be included in the title. Ref 149 mistakenly has a “)” in the end.
3. Line 153: what “X” and “Y” stand for should be explained in “Gly-X-Y”.
4. The format should be consistent in the whole text. PCL on line 443 and PGA… on line 450 do not have a newline, but collagen on line 483 and elastin on line 497 have.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the opportunity to review the revised version of the manuscript. The authors have clearly undertaken substantial revisions, and the overall quality, clarity, and organization of the manuscript have improved compared to the original submission. I acknowledge and commend the effort invested in strengthening the work.

However, I must express concern regarding the authors’ decision not to provide a point-by-point response to my review comments. While authors are, of course, free to disagree with a reviewer’s opinions or interpretations, the standard practice in scholarly peer review is to provide a structured, itemized response indicating how each comment was addressed, revised, or respectfully rebutted. The absence of such a response makes it difficult to systematically assess which specific concerns were considered, how they were resolved, and whether certain issues remain unaddressed.

It is unfortunate that the authors perceived the tone or nature of my previous report as unproductive. My review was written with a two-phase intent: first, to provide analytical observations grounded in the current state of the field and comparable literature; and second, to identify specific areas requiring clarification, strengthening, or correction. The former naturally includes professional opinions and comparative assessments, which authors may legitimately contest. The latter consists of constructive suggestions aimed at improving rigor, clarity, and impact. In both cases, scholarly dialogue, through reasoned response is the mechanism by which manuscripts are refined.

Disagreement with a reviewer’s perspective is entirely acceptable in academic discourse. However, declining to engage with comments through a point-by-point response departs from established peer-review norms and limits transparency in the revision process. Even where authors choose not to implement a suggested change, a brief explanation is both customary and professionally appropriate.

In its current form, the manuscript appears improved, but without a structured response, it is not possible to clearly determine how the previously raised concerns were addressed. I therefore recommend that the authors provide a concise, point-by-point response to my previous comments, indicating for each point whether it has been incorporated, partially addressed, or not adopted (with justification). This would ensure fairness, transparency, and alignment with standard editorial practice.

I remain supportive of constructive scientific dialogue and believe that addressing this procedural issue would further strengthen the integrity of the review process.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all the questions raised before, therefore the manuscript can be accepted in its present form

Author Response

Thank you very much for your help us to improve our manuscript

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the detailed response letter and for engaging systematically with my comments. I agree with the perspective of the authors, and the manuscript can be accepted in the current form. I would like to point out some minor comments before publication:

1. Please include a high-definition image for Figure 4. Currently, it looks pixelated.
2. Table 1 description can be modified to "Modified from [27] under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)". No need to add a full reference as it is already cited in the reference list.
3. Follow the same pattern for image reuse. No full reference is required in the figure legend.
4. The first character in the text included within Figure 6 has to be capitalized. 

Author Response

Please see the attachment

Author Response File: Author Response.pdf