Aging Biology of Bone-to-Tendon Healing and the Epigenetic Clock: A Biological-Age Readout of Rotator Cuff Healing Capacity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This narrative review offers an original and intellectually stimulating hypothesis for rotator cuff healing, introducing the idea of the "hidden biological age" of tendon-bone interfaces and presenting the epigenetic clock as a possible integrative marker of healing ability. The manuscript is logically structured; the illustrations are very good and the literature review is very extensive and up-to-date. The offered theoretical framework is very original and manages to integrate different biological mechanisms underlying tendon-bone healing into a consistent hypothesis.

Nevertheless, despite all these strengths, some concerns should be resolved prior to publication.

The manuscript includes a number of speculative ideas presented with language more powerful than the scientific evidence behind them. Even though the authors point out that the hypothesis of the "hidden biological age" is still hypothetical, there are some parts of the paper which convey a message of future applicability of this hypothesis in the clinic without any experimental support at the moment

It would be useful to see a separate paragraph devoted to discussing practical limitations of epigenetic clocks in orthopaedics at present. Problems with assay standardization, costs, access to tissues, reproducibility, platform variability, and even possibility of assessing methylation specific for tendon/enthesis would need to be clarified prior to suggesting any clinical application.

One other limitation is that this review pays practically no attention to well-known mechanical parameters affecting rotator cuff healing despite the fact that biological age is mostly considered throughout the paper. Such parameters like tear size, tendon retraction, repair construct, tendon quality, rehabilitation procedures, and compliance could not be ignored in order to obtain a more balanced view.

It is interesting to discuss possible clinical scenarios made feasible with the help of epigenetic clock, however, the authors should present these ideas much more cautiously, considering them long-term goals rather than immediate perspectives.

Graphical depiction of the proposed translational path, from biological mechanisms of aging to epigenetic clock determination, regeneration treatments, and possible clinical decision making, could add even more clarity to the concept.

Author Response

#Reviewer 1

Comments and Suggestions for Authors

This narrative review offers an original and intellectually stimulating hypothesis for rotator cuff healing, introducing the idea of the "hidden biological age" of tendon-bone interfaces and presenting the epigenetic clock as a possible integrative marker of healing ability. The manuscript is logically structured; the illustrations are very good and the literature review is very extensive and up-to-date. The offered theoretical framework is very original and manages to integrate different biological mechanisms underlying tendon-bone healing into a consistent hypothesis.

Nevertheless, despite all these strengths, some concerns should be resolved prior to publication.

The manuscript includes a number of speculative ideas presented with language more powerful than the scientific evidence behind them. Even though the authors point out that the hypothesis of the "hidden biological age" is still hypothetical, there are some parts of the paper which convey a message of future applicability of this hypothesis in the clinic without any experimental support at the moment
It would be useful to see a separate paragraph devoted to discussing practical limitations of epigenetic clocks in orthopaedics at present. Problems with assay standardization, costs, access to tissues, reproducibility, platform variability, and even possibility of assessing methylation specific for tendon/enthesis would need to be clarified prior to suggesting any clinical application.

Response: We thank the reviewer for this constructive suggestion, with which we fully agree. Two sets of changes address this comment. First, the language conveying near-term clinical applicability has been moderated throughout the revised manuscript: Section 8 has been retitled "Long-Term Clinical Scenarios: What the Epigenetic Clock Could Ultimately Enable," an explicit caveat paragraph now precedes the scenarios stating that none is achievable with present technology or evidence, the declarative mood has been replaced by the conditional throughout, and the hypothesis framing has been strengthened in the Abstract, Introduction, and Section 5 (please also see our responses to Comment 4 below and to Reviewer 3). Second, we have added a new paragraph to the Discussion (Section 9), immediately following the paragraph acknowledging that the central construct remains unvalidated, that addresses each of the six barriers the reviewer identified: (1) incomplete assay standardization and the absence of a clinically certified workflow; (2) the current cost of array-based profiling, which precludes routine preoperative use; (3) asymmetric tissue access, as the enthesis is sampled only intraoperatively while the envisioned decisions are preoperative; (4) imperfect test–retest reproducibility, only partially mitigated by principal-component–based clocks (new Ref. [51]); (5) platform variability across Illumina 450K/EPIC/EPICv2 arrays and sequencing; and (6) the absence of any clock trained or validated on tendon/enthesis tissue, with the skeletal-muscle clock (MEAT; new Ref. [52]) cited as a precedent showing that a tissue-specific clock is achievable but must precede clinical application. The paragraph closes by restating that the clinical scenarios in Section 8 are long-term research goals rather than near-term practice.

Lines 434~460: “Beyond this conceptual limitation, the practical barriers of current epigenetic-clock technology must also be weighed before any clinical application is contemplated. Assay standardization remains incomplete: methylation values are sensitive to batch effects, bisulfite-conversion efficiency, and normalization pipelines, and no clinically certified workflow for clock estimation yet exists in musculoskeletal care, while array-based genome-wide profiling still costs on the order of several hundred US dollars per sample, feasible for research cohorts but not for routine preoperative testing. Tissue access is likewise asymmetric to the clinical question: the enthesis can be sampled only intraoperatively, whereas the decisions this framework ultimately envisions—patient selection, surgical timing, and preoperative rejuvenation—are preoperative, so blood-based clocks or imaging surrogates, which may underestimate the true aging state of the enthesis given the tissue-specificity of aging discussed above, would first need to be validated as intermediaries. Reproducibility and comparability add further constraints: technical noise at individual CpG probes can shift first-generation clock estimates by several years between technical replicates, a variability that principal-component–based reformulations reduce but do not eliminate [51], and probe content differs among the Illumina 450K, EPIC, and EPICv2 arrays and sequencing-based approaches, so clocks trained on one platform do not transfer to another without recalibration. Most fundamental to this review, no epigenetic clock has yet been trained or validated on tendon or enthesis tissue; existing clocks derive from blood or multi-tissue panels that did not include tendon, and the marked cellular heterogeneity of the fibrocartilaginous transition zone means that bulk methylation signals conflate shifts in cell composition with intrinsic cellular aging. The precedent of a dedicated skeletal-muscle clock, which outperformed the pan-tissue clock within muscle [52], indicates that a tendon/enthesis-specific clock is achievable in principle, but it must first be constructed and validated before any of the applications discussed in Section 8 can proceed. Taken together, these constraints reinforce that the clinical scenarios proposed in this review are long-term research goals rather than near-term clinical tools.” was added.

51. Higgins-Chen, A.T.; Thrush, K.L.; Wang, Y.; et al. A computational solution for bolstering reliability of epigenetic clocks: Implications for clinical trials and longitudinal tracking. Nat. Aging 2022, 2, 644–661.
52. Voisin, S.; Harvey, N.R.; Haupt, L.M.; et al. An epigenetic clock for human skeletal muscle. J. Cachexia Sarcopenia Muscle 2020, 11, 887–898.

One other limitation is that this review pays practically no attention to well-known mechanical parameters affecting rotator cuff healing despite the fact that biological age is mostly considered throughout the paper. Such parameters like tear size, tendon retraction, repair construct, tendon quality, rehabilitation procedures, and compliance could not be ignored in order to obtain a more balanced view.

Response: We agree with the reviewer that the original manuscript, in focusing on the biology of aging, gave insufficient weight to the well-established mechanical and structural determinants of rotator cuff healing. In the revised manuscript, we have added a dedicated paragraph at the end of Section 2 that explicitly acknowledges these parameters—tear size and tendon retraction (Refs. [15,47]), tendon quality (Refs. [15,16,33]), repair construct and time-zero fixation mechanics (Refs. [44,48]), and postoperative rehabilitation and patient compliance (Ref. [44])—as validated predictors of structural outcome. The paragraph then clarifies the intended relationship between the two axes: mechanical parameters define the load environment and the technical quality of the repair, whereas the aging domains reviewed in Section 3 determine the intrinsic capacity of the interface to heal within that environment. The biological-age framework is thus explicitly positioned as complementary to—not a replacement for—the established mechanical predictors, and we note that any future predictive model incorporating an epigenetic clock would need to be validated against, and integrated with, these structural variables. We believe this addition provides the balanced view the reviewer requested.

Lines 147~164: “It must be emphasized, however, that the healing of this interface does not occur in a biological vacuum: it is powerfully conditioned by well-established mechanical and structural parameters. Tear size and tendon retraction remain among the strongest predictors of structural failure [15,47], and tendon quality [15,16,33], the repair construct and its time-zero fixation mechanics [44,48], and postoperative variables such as rehabilitation and patient compliance [44] all measurably influence whether the repaired tendon remains apposed to bone long enough for healing to proceed. These parameters and the aging biology reviewed below are best understood as two complementary axes of a single multifactorial model: the mechanical axis defines the load environment and the technical integrity of the repair, whereas the biological axis, the aging domains detailed in Section 3, determines the intrinsic capacity of the interface to heal within that environment. A small, minimally retracted tear repaired with an optimal construct may still fail in a biologically aged interface. Conversely, a biologically young interface cannot compensate for an overtensioned or mechanically insufficient repair. Accordingly, the biological-age framework proposed in this review is intended to complement, not replace, the established mechanical predictors, and any future predictive model incorporating an epigenetic clock will need to be validated against and ultimately integrated with these structural variables.” was added.

It is interesting to discuss possible clinical scenarios made feasible with the help of epigenetic clock, however, the authors should present these ideas much more cautiously, considering them long-term goals rather than immediate perspectives.

Response: We agree entirely. In the revised manuscript, Section 8 has been systematically reframed as a set of long-term conceptual scenarios rather than imminent clinical applications: the section title now reads "Long-Term Clinical Scenarios: What the Epigenetic Clock Could Ultimately Enable"; an explicit caveat has been inserted before the scenarios stating that none is achievable with present technology or evidence, that each presupposes a validated tendon/enthesis-specific clock, and that all should be read as long-term research goals; the captions of Figure 4 and Table 4 have been revised accordingly; and the declarative mood has been replaced by the conditional (could/would) throughout the section. These changes are consistent with the limitations paragraph added to Section 9 in response to Comment 2, which closes with the same framing, and they also address the related concern raised by Reviewer 3. We believe the revised section now presents these ideas with the caution the reviewer appropriately requested, while preserving their heuristic value for defining a research agenda.

Lines 316~318: “8. Clinical Translation: What the Epigenetic Clock Makes Possible -> 8. Long-Term Clinical Scenarios: What the Epigenetic Clock Could Ultimately Enable” was changed.

Lines 354~367: “The crucial point is that the clock does not merely make existing variables “a little more accurate.” Because it is an age that is both quantitative and reversible, it opens surgical strategies that are, in principle, impossible with any current tool. What follows are not the predictable uses such as risk stratification or rehabilitation tuning, but scenarios that simply cannot exist without the clock (Figure 4, Table 4). à The conceptual point is that the clock would not merely make existing variables "a little more accurate." Because it is an age that is both quantitative and, in principle, reversible, it could ultimately open surgical strategies that no current tool can support. We emphasize at the outset that none of the scenarios below is achievable with present technology or evidence: each presupposes a validated tendon/enthesis-specific clock, which does not yet exist, and all should therefore be read as long-term research goals—the conceptual end-points of a research program—rather than immediate clinical perspectives. With this caveat, what follows are not near-term uses such as risk stratification or rehabilitation tuning, but scenarios that could not even be formulated without such a clock (Figure 4, Table 4).” was changed.

Lines 369~376: “Figure 4. Scenarios that become possible only with a quantitative, reversible tissue-age readout. The headline paradigm is a shift from “repair, then hope it heals” to a closed loop of “rejuvenate first, then repair”; each of the four cards below contrasts what is impossible today with what the clock enables. à Long-term conceptual scenarios that would become possible only with a validated, quantitative, reversible tissue-age readout. The headline paradigm is a shift from "repair, then hope it heals" to a closed loop of "rejuvenate first, then repair"; each card contrasts what is not feasible today with what a validated clock could enable. All scenarios are hypothetical long-term goals, not current clinical options.” was changed.

Lines 377~382: “Table 4. Scenarios the epigenetic clock uniquely enables: why each is impossible today, and how the clock makes it possible. These are not incremental refinements such as risk stratification; none can even begin without an age that is quantitative and reversible. à Table 4. Long-term scenarios that a validated tissue-specific epigenetic clock could ultimately enable: why each is not feasible today, and how such a clock could make it conceivable. None of these scenarios is supported by current clinical evidence; all are presented as long-term research goals rather than immediate perspectives.” was changed.

Lines 384~387: “The most fundamental shift is from a surgery that "repairs and then waits for healing" to one that "makes the tissue younger first, then repairs." à The most fundamental long-term shift would be from a surgery that "repairs and then waits for healing" to one that "makes the tissue younger first, then repairs." was changed.

Lines 401~403: “Third, the partial reprogramming—now entering the clinic can be precisely titrated: ... à Third, if partial reprogramming—now entering early-phase trials in other organ systems—were ever applied locally, it could in principle be titrated: ...” was changed.

Lines: 412~415: “What these scenarios share is that none can even begin without a readout of a molecular age that is both quantitative and reversible. à “What these scenarios share is that none can even begin before a molecular-age readout that is both quantitative and reversible has been developed and validated for the enthesis.” was changed.

Graphical depiction of the proposed translational path, from biological mechanisms of aging to epigenetic clock determination, regeneration treatments, and possible clinical decision making, could add even more clarity to the concept.

Response: We thank the reviewer for this excellent suggestion. A new Figure 5 has been added at the beginning of Section 10 (Future Directions), depicting the proposed translational path as a four-stage sequence—(1) biological mechanisms of aging (the eight domains of Section 3), (2) epigenetic clock determination (systemic and tissue-resolved), (3) regenerative interventions mapped to the domains they target, and (4) possible clinical decision-making (patient selection, surgical timing, intervention matching, and treatment monitoring). Consistent with the cautious framing adopted in response to Comments 2 and 4, the figure explicitly marks the validation checkpoints that must be crossed between stages—development of a tendon/enthesis-specific clock, prospective outcome validation, and interventional evidence—and visually distinguishes the currently evidence-supported segment from the hypothetical downstream segments. A sentence introducing the figure has been added to the opening of Section 10. We believe this figure improves the clarity of the overall concept exactly as the reviewer envisioned, while reinforcing rather than undermining the manuscript's distinction between established evidence and long-term goals.

Lines 462~464: “The translational path from the aging biology reviewed here to eventual clinical decision-making is summarized in Figure 5, which also marks the validation checkpoints that must be crossed at each transition.” was added.

Lines 478~490: Figure 5

“Figure 5. Proposed translational path of the hidden-biological-age framework. Stage 1, biological mechanisms of aging (the eight domains governing tendon-to-bone healing capacity); Stage 2, epigenetic clock determination (systemic blood-based and, ultimately, tissue-resolved enthesis-specific measurement); Stage 3, regenerative interventions categorized by the aging domains they target; Stage 4, possible clinical decision-making (patient selection, surgical timing, intervention matching, and treatment monitoring). Arrows between stages indicate the validation checkpoints required for each transition: development and validation of a tendon/enthesis-specific clock (1→2), prospective cohort validation against repair outcomes (2→3), and interventional evidence that shifting the clock improves healing (3→4). Solid outlines denote currently evidence-supported elements; dashed outlines denote hypothetical elements awaiting validation. The entire downstream path remains a long-term research goal.” was added.

Submission Date

16 July 2026

Date of this review

06 Aug 2026 08:27:56

Reviewer 2 Report

Comments and Suggestions for Authors

This paper explores the interesting concept that epigenetic age (rather than chronological age) plays an important role in rotator cuff tendon healing. The clinical issue is certainly important, as rotator cuff tendon healing is a slow and imperfect process.  Furthermore, it is well-established that simple chronological age is not an accurate predictor of tendon healing.  This manuscript nicely explains how DNA methylation patterns are a standard measure of epigenetic aging. They relate DNA methylation/epigenetic changes to fundamental aspects of healing biology, including inflammation, matrix synthesis, cellular senescence, macrophage polarization, angiogenesis, etc.  This manuscript proposes the DNA-methylation– based epigenetic clock as a biological-age readout. In this way this may be a valuable biomarker that can be measured as a possible predictor of rotator cuff repair outcomes. 

 The authors also briefly discuss the concept of "partial reprogramming", where the "Yamanaka factors" can partially reverse some of the age-related effects. 

It is also established that epigenetic age varies amongst different tissues, and the authors propose that perhaps the rotator cuff enthesis has more advanced epigenetic age than other tissues in the body. However, little evidence currently exists to support this hypothesis. 

Overall the basic thesis of this manuscript is reasonable, but the major limitation is that much of this is speculation. The authors review literature demonstrating how epigenetic aging affects other tissues and clinical conditions, and then propose that similar relationships may exist for the rotator cuff.  All reasonable but speculative. 

Author Response

Reviewer 2

Comments and Suggestions for Authors

This paper explores the interesting concept that epigenetic age (rather than chronological age) plays an important role in rotator cuff tendon healing. The clinical issue is certainly important, as rotator cuff tendon healing is a slow and imperfect process.  Furthermore, it is well-established that simple chronological age is not an accurate predictor of tendon healing.  This manuscript nicely explains how DNA methylation patterns are a standard measure of epigenetic aging. They relate DNA methylation/epigenetic changes to fundamental aspects of healing biology, including inflammation, matrix synthesis, cellular senescence, macrophage polarization, angiogenesis, etc.  This manuscript proposes the DNA-methylation– based epigenetic clock as a biological-age readout. In this way this may be a valuable biomarker that can be measured as a possible predictor of rotator cuff repair outcomes.

Response: We sincerely thank the reviewer for this accurate and generous summary of the manuscript's aims and for recognizing the clinical importance of the problem and the potential value of the epigenetic clock as a biomarker of rotator cuff repair outcomes. No specific changes were requested in this comment.

 The authors also briefly discuss the concept of "partial reprogramming", where the "Yamanaka factors" can partially reverse some of the age-related effects. 

It is also established that epigenetic age varies amongst different tissues, and the authors propose that perhaps the rotator cuff enthesis has more advanced epigenetic age than other tissues in the body. However, little evidence currently exists to support this hypothesis.

Response: We agree completely. The revised Section 5 now separates what is established from what is proposed: tissue-to-tissue variation in epigenetic aging is documented [2], but no study has directly measured the methylation age of the human enthesis, so the proposition that the enthesis "runs ahead" of blood is explicitly labeled an untested hypothesis resting only on indirect plausibility (chronic mechanical loading, hypovascularity, and degenerative burden of the footprint). One sentence has been added to the Figure 2 caption stating the same, and direct methylation profiling of enthesis tissue is now named as the first requirement in Future Directions (Section 10), consistent with the limitations paragraph added to Section 9 (Response to Reviewer 1), which states that no clock has been trained or validated on tendon/enthesis tissue.

Lines 238~240: “The proposition in (B) that enthesis biological age runs ahead of the systemic clock is a hypothesis of this review; no direct measurement of enthesis methylation age currently exists.” was added.

Lines 245~258: “Furthermore, the pace of aging differs among tissues, so within the same individual the biological age of local tissue may run ahead of blood [2]. This means that a blood-based clock alone risks underestimating the true aging of the enthesis, and it argues for tissue-resolved measurements such as spatial transcriptomics or imaging-based surrogates [28]. à Furthermore, it is established that the pace of aging differs among tissues within the same individual [2]. Whether the rotator cuff enthesis in particular carries a more advanced epigenetic age than other tissues, however, has never been directly measured; this proposition is an untested hypothesis of the present review, supported only by indirect plausibility from the chronic mechanical loading, hypovascularity, and degenerative burden of the footprint. If the hypothesis holds, a blood-based clock alone would risk underestimating the true aging of the enthesis, which argues for tissue-resolved measurements such as spatial transcriptomics or imaging-based surrogates [28], and direct methylation profiling of enthesis tissue is accordingly the first requirement of the research agenda in Section 10.” was changed.

Overall the basic thesis of this manuscript is reasonable, but the major limitation is that much of this is speculation. The authors review literature demonstrating how epigenetic aging affects other tissues and clinical conditions, and then propose that similar relationships may exist for the rotator cuff.  All reasonable but speculative. 

Response: We agree that this is the central limitation of the manuscript, and the revision addresses it in two ways. First, the extrapolative structure of the argument is now stated openly at the outset: a sentence added to the final paragraph of the Introduction declares that the evidence reviewed derives from other tissues and clinical conditions, that no direct evidence yet links epigenetic age to rotator cuff healing, and that the framework is therefore presented as an extrapolative hypothesis to be tested rather than an established mechanism. Second, the caution requested here is implemented throughout the manuscript by the changes described in our responses to Reviewer 1: a dedicated paragraph on the practical limitations of current clock technology in Section 9, the reframing of Section 8 as long-term scenarios with conditional language, and the balancing paragraph on established mechanical predictors in Section 2. We believe the revised manuscript now maintains a consistent separation between evidence and hypothesis while preserving the heuristic value of the framework the reviewer found reasonable.

Lines 108~111: “We state at the outset that the evidence assembled here derives from other tissues and clinical conditions; no study has yet directly linked epigenetic age to rotator cuff healing, and the framework of this review is therefore an extrapolative hypothesis to be tested, not an established mechanism.” was added.

Submission Date

16 July 2026

Date of this review

06 Aug 2026 13:33:42

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for this interesting and timely review. The manuscript presents a novel perspective on biological aging and tendon-to-bone healing; however, several points should be clarified or moderated.

The central concept of a “hidden biological age of bone-to-tendon healing” is interesting but remains entirely hypothetical and has not been validated in rotator cuff patients. This distinction should be emphasized more consistently throughout the manuscript.

The manuscript repeatedly suggests that epigenetic clocks integrate the eight proposed aging domains into a single biologically meaningful measure. However, the cited evidence does not directly demonstrate such integration specifically for the rotator cuff enthesis. This claim should be presented as a hypothesis rather than an established mechanism.

Statements suggesting that rotator cuff repair failure is fundamentally a tendon-to-bone healing problem “rather than a problem of muscle” appear too categorical. Muscle atrophy and fatty infiltration are well-established prognostic factors and should not be positioned merely as peripheral modifiers.

The sections proposing “rejuvenate-then-repair,” molecular-age–timed surgery, aging-signature–matched biologics, and clock-guided reprogramming are highly speculative. These concepts are interesting but should be clearly separated from clinically supported applications.

The manuscript would benefit from a brief description of the literature search and article selection methodology. At present, it is unclear how the evidence included in this narrative review was identified and selected.

There is an important authorship inconsistency. Multiple authors are listed on the title page, whereas the Author Contributions section states that “This manuscript has a single author.” This should be corrected.

Overall, the manuscript introduces an original and potentially valuable conceptual framework, but the distinction between established evidence and future hypothesis should be strengthened.

Author Response

#Reviewer 3

Comments and Suggestions for Authors

Dear Authors,

Thank you for this interesting and timely review. The manuscript presents a novel perspective on biological aging and tendon-to-bone healing; however, several points should be clarified or moderated.

The central concept of a “hidden biological age of bone-to-tendon healing” is interesting but remains entirely hypothetical and has not been validated in rotator cuff patients. This distinction should be emphasized more consistently throughout the manuscript.

Response: We agree, and the revised manuscript now maintains this distinction consistently from the Abstract to the Conclusions. The hypothetical status of the framework is stated at every stage of the argument: the Abstract presents the clock as a "candidate" readout and closes by stating the concept "remains an unvalidated hypothesis requiring prospective validation"; the Introduction declares the framework "an extrapolative hypothesis to be tested, not an established mechanism"; the Figure 1 and Figure 2 captions label the integrative readout and the enthesis-aging proposition as hypotheses of this review; Section 5 is retitled "The Epigenetic Clock as a Hypothesized Integrative Readout" and states that integration at the enthesis "has never been tested"; Section 8 is reframed as long-term scenarios behind an explicit evidence boundary ("Everything beyond this point is hypothesis"); Section 9 acknowledges the unvalidated status and details the practical barriers; and the Figure 5 caption states that "the entire downstream path remains a long-term research goal" (please see our responses to Comments 2 and 4, to Reviewer 1, and to Reviewer 2 for the individual edits). In direct response to the present comment, two further edits were made: the statement of non-validation in Section 9 now specifies rotator cuff patients, exactly as the reviewer notes, and the one remaining declarative phrasing of the integration claim in the Conclusions has been changed to the hypothesized form for full consistency.

Lines 426~427: “This integrative metric, however, has not been validated in any prospective cohort and remains an explicit hypothesis. à This integrative metric, however, has not been validated in any prospective cohort of rotator cuff patients and remains an explicit hypothesis.” was changed.

Lines 498~503: “The epigenetic clock, as a readout that integrates these domains into a single biological age, holds the potential to capture the inter-individual variation in aging that chronological age misses. à The epigenetic clock, as a candidate readout hypothesized to integrate these domains into a single biological age, holds the potential to capture the inter-individual variation in aging that chronological age misses.” was changed.

The manuscript repeatedly suggests that epigenetic clocks integrate the eight proposed aging domains into a single biologically meaningful measure. However, the cited evidence does not directly demonstrate such integration specifically for the rotator cuff enthesis. This claim should be presented as a hypothesis rather than an established mechanism.

Response: We agree, and we thank the reviewer for identifying precisely where the manuscript's language exceeded its evidence. The revised manuscript now presents the integration claim as a hypothesis at every location where it previously appeared as established: the Abstract now positions the clock as a "candidate" integrative readout and states that its integration of the eight domains at the enthesis is hypothesized rather than demonstrated; the title of Section 5 has been changed to "The Epigenetic Clock as a Hypothesized Integrative Readout"; a clarifying sentence added to Section 5 states explicitly that the supporting evidence—including the twin study [13]—demonstrates prediction of bone-aging outcomes, not integration of the proposed domains at the rotator cuff enthesis, which remains untested; the Figure 1 caption now reads "hypothesized as their integrative readout"; and the declarative phrasing in Section 7 has been conditioned accordingly. These changes are consistent with the existing acknowledgment in Section 9 ("It does not establish that these domains integrate into a single biological-age index that predicts repair outcome") and with the extrapolative-hypothesis sentence added to the Introduction in response to Reviewer 2, so the hypothesis framing is now maintained uniformly from Abstract to Conclusions.

Lines 26~30: “-and position it not as one more factor but as the quantitative integrative readout of these domains. à -and propose it as a candidate quantitative readout of these domains; whether it truly integrates them into a single biologically meaningful measure at the enthesis is a hypothesis of this review, not an established mechanism.” was changed.

Lines 129~130: “...with the epigenetic clock positioned as their integrative readout and a set of modifiable levers. à ...with the epigenetic clock hypothesized as their integrative readout and a set of modifiable levers.” was changed.

Lines 217~218: “5. The Epigenetic Clock as an Integrative Readout à 5. The Epigenetic Clock as a Hypothesized Integrative Readout” was changed.

Lines 219~232: “Each of these eight domains can be measured and targeted, but assessing all of them separately is impractical in clinical care. The epigenetic clock is attractive as an integrative readout precisely because it does not merely sum these as parallel factors but integrates the cumulative aging state into a single biological age (their generational development and tissue-specificity are summarized in Table 2 and Figure 2). à Each of these eight domains can be measured and targeted, but assessing all of them separately is impractical in clinical care. The epigenetic clock is attractive because, in principle, it would not merely sum these as parallel factors but capture the cumulative aging state in a single biological age (their generational development and tissue-specificity are summarized in Table 2 and Figure 2). We emphasize, however, that this integrative capacity at the rotator cuff enthesis is a hypothesis of the present review: the evidence cited below demonstrates that epigenetic clocks predict bone-aging outcomes, not that they integrate the eight proposed domains into a single biologically meaningful measure for the enthesis, which has never been tested.” was changed.

Lines 285~289: “The key insight is that each regenerative strategy targets a specific clock-measurable aging domain, and that the clock serves as a common metric of how far a given strategy rejuvenates the tissue. à The key insight is that each regenerative strategy targets a specific aging domain, and that the clock could serve as a common metric of how far a given strategy rejuvenates the tissue—contingent on the integrative hypothesis above being validated.” was changed.

Statements suggesting that rotator cuff repair failure is fundamentally a tendon-to-bone healing problem “rather than a problem of muscle” appear too categorical. Muscle atrophy and fatty infiltration are well-established prognostic factors and should not be positioned merely as peripheral modifiers.

Response: We agree that the original dichotomy was overstated, and we thank the reviewer for pressing this point. Muscle atrophy and fatty infiltration are indeed among the best-validated prognostic factors for both structural and functional outcome after rotator cuff repair [16,33], and the revised manuscript no longer positions them as merely peripheral. The categorical "rather than a problem of muscle" framing has been removed or reworded at every occurrence: the Abstract and Conclusions now state that repair failure is multifactorial and that this review focuses on the enthesis because it is the anatomical site at which structural failure occurs—without ranking it above muscle factors; the Introduction paragraph has been rewritten to describe muscle quality and interface healing as interdependent determinants acting on the same repair; and the phrase "peripheral modifiers" has been deleted from the Figure 1 caption, which now describes muscle fatty infiltration and systemic metabolism as established codeterminants interacting with the interface milieu. These changes complement the paragraph on established mechanical and structural predictors added to Section 2 in response to Reviewer 1, so the revised manuscript consistently presents the biological-age framework as one axis within a multifactorial model rather than a replacement for validated muscle-based predictors.

Lines 14~17: “The "unexplained failure" of rotator cuff repair is, in essence, a failure of the tendon-to-bone interface (enthesis) to heal rather than a problem of muscle. à The "unexplained failure" of rotator cuff repair is multifactorial, but its structural end-point is anatomically consistent: the failure of the tendon-to-bone interface (enthesis) to heal.” was changed.

Lines 85~99: “Crucially, this failure does not arise simply because "the muscle is poor"; it originates in the failure of the repaired tendon to re-anchor to bone—that is, in the failure of the tendon-to-bone interface (enthesis) to heal. Fatty infiltration and atrophy of the muscle do not recover well after repair and correlate with poor function [16,33], but these are factors that degrade the healing environment rather than the direct outcome substrate of the repair. à Muscle atrophy and fatty infiltration are well-established, independent prognostic factors for both structural and functional outcome: they recover poorly after repair and correlate with poor function [16,33]. Yet the anatomical event of structural failure itself—the loss of continuity between the repaired tendon and bone—occurs at the tendon-to-bone interface (enthesis). Muscle quality and interface healing are therefore interdependent determinants of the same repair: a degenerated muscle imposes an unfavorable mechanical and biological environment on the healing interface, while a failed interface in turn perpetuates muscle degeneration. This review focuses on the enthesis because it is the site at which failure ultimately manifests and the substrate on which regenerative interventions act—not because muscle factors are secondary.” was changed.

Lines 131~133: “Muscle fatty infiltration and systemic metabolism are positioned as peripheral modifiers of the milieu. à Muscle fatty infiltration and systemic metabolism, both established prognostic factors in their own right, are shown as codeterminants interacting with the interface milieu.” was changed.

Lines 493~498: “The "unexplained failure" of rotator cuff repair is, at its core, a failure of bone-to-tendon healing rather than a problem of muscle, and this healing capacity is governed by the biology of multiple aging domains. à The "unexplained failure" of rotator cuff repair is multifactorial, and its structural end-point is the failure of bone-to-tendon healing—a capacity governed by the biology of multiple aging domains and conditioned by established muscle-based and mechanical prognostic factors.” was changed.

Lines 504~510: “We hope this review can serve as a starting point for shifting the outcome variable in rotator cuff prognosis research from "muscle" to "bone-to-tendon healing," and the measurement variable from "calendar age" to "biological age." à We hope this review can serve as a starting point for adding "bone-to-tendon healing" alongside established muscle-based predictors as an outcome variable in rotator cuff prognosis research, and "biological age" alongside "calendar age" as a measurement variable.” was changed.

The sections proposing “rejuvenate-then-repair,” molecular-age–timed surgery, aging-signature–matched biologics, and clock-guided reprogramming are highly speculative. These concepts are interesting but should be clearly separated from clinically supported applications.

Response: We agree. Two sets of changes implement the separation the reviewer requests. First, as detailed in our response to Reviewer 1, Section 8 has been retitled "Long-Term Clinical Scenarios: What the Epigenetic Clock Could Ultimately Enable," an explicit caveat paragraph now precedes the scenarios stating that none is achievable with present technology or evidence, the Table 4 caption has been revised accordingly, and the declarative mood has been replaced by the conditional throughout. Second, specifically in response to the present comment, we have added an evidence-boundary paragraph within Section 8 that explicitly demarcates where clinical support ends: the clinically supported evidence to date—the CALERIE randomized trial [43], the TRIIM study [42], and the regulatory clearance of a first-in-human reprogramming trial [41]—is entirely systemic and non-orthopaedic, and no epigenetic-clock-based application of any kind is currently supported in rotator cuff care. The four concepts the reviewer names ("rejuvenate-then-repair," molecular-age–timed surgery, aging-signature–matched biologics, and clock-guided reprogramming) are now introduced only after this boundary is drawn, so they cannot be read as extensions of the supported evidence. The overstated sentence on clinical accessibility in the industrialization paragraph has also been tempered. We believe the revised section now separates hypothesis from clinically supported application as clearly as the reviewer intended.

Lines 324~329: “This momentum matters for orthopaedics, because it means standardized, scalable, quantitative biological-age measurement is becoming progressively accessible in the clinic. à This momentum matters for orthopaedics because it suggests that standardized, scalable, quantitative biological-age measurement may become progressively accessible, although, as discussed in Section 9, substantial technical and validation barriers remain.” was changed.

Lines 330~339: “The boundary of clinical support must be drawn explicitly at this point. The evidence summarized above—an RCT slowing a methylation clock through caloric restriction [43], a small human study observing epigenetic-age reversal [42], and the regulatory clearance of a first-in-human reprogramming trial in vision loss—is entirely systemic or non-orthopaedic [41]. No epigenetic-clock-based application of any kind—diagnostic, prognostic, or therapeutic—is currently supported by clinical evidence in rotator cuff care, and none of the concepts that follow, including "rejuvenate-then-repair," molecular-age–timed surgery, aging-signature–matched biologics, and clock-guided reprogramming, has been tested in any musculoskeletal patient population. Everything beyond this point is hypothesis.” was added.

The manuscript would benefit from a brief description of the literature search and article selection methodology. At present, it is unclear how the evidence included in this narrative review was identified and selected.

Response: We agree. A paragraph describing the literature search and selection approach has been added at the end of the Introduction, specifying the databases searched (PubMed, Scopus, Web of Science), the principal search terms, the prioritization criteria for the aging-biology and orthopaedic literature, and the English-language restriction. It also states explicitly that this is a narrative not a systematic review, that no formal protocol (e.g., PRISMA) was followed, and that a potential for selection bias should be considered. The paragraph was placed in the Introduction rather than a separate Methods section to preserve the existing section numbering and cross-references.

Lines 112~125: “That evidence was identified by searching PubMed, Scopus, and Web of Science, supplemented by manual searches of reference lists and of recent issues of major shoulder and aging-biology journals, using combinations of the terms "rotator cuff," "enthesis," "tendon-to-bone (bone-to-tendon) healing," "epigenetic clock," "DNA methylation," "biological age," "epigenetic age acceleration," "cellular senescence," "inflammaging," "partial reprogramming," and "regenerative medicine." For the aging-biology and epigenetic-clock literature, we prioritized randomized controlled trials, systematic reviews and meta-analyses, and landmark mechanistic studies; for the orthopaedic literature, we prioritized preclinical and clinical studies directly addressing tendon-to-bone interface healing and validated prognostic factors of rotator cuff repair. Only English-language publications were included. This is a narrative review: no systematic protocol (e.g., PRISMA) was followed, article selection reflects the authors' judgment of relevance to the proposed framework, and a potential for selection bias should accordingly be considered when interpreting the synthesis.” was added.

There is an important authorship inconsistency. Multiple authors are listed on the title page, whereas the Author Contributions section states that “This manuscript has a single author.” This should be corrected.

Response: We thank the reviewer for catching this error, and we apologize for the oversight. The sentence was a remnant of an earlier draft. The Author Contributions section has been rewritten to state the individual contributions of all seven listed authors.

Lines 512~518: “This manuscript has a single author. J.P.Y.: conceptualization, literature review, writing—original draft preparation, and writing—review and editing. The author has read and agreed to the published version of the manuscript. à Conceptualization, J.P.Y.; methodology, J.P.Y. and S.J.P.; investigation (literature search and analysis), J.P.Y., S.J.P., D.H.K., Y.Y. and H.N.; writing—original draft preparation, J.P.Y.; writing—review and editing, S.J.P., D.H.K., C.H.C., Y.Y., H.N. and S.W.C.; visualization, S.J.P. and D.H.K.; supervision, S.W.C.; project administration, S.W.C. All authors have read and agreed to the published version of the manuscript.” was changed.

Overall, the manuscript introduces an original and potentially valuable conceptual framework, but the distinction between established evidence and future hypothesis should be strengthened.

Response: We thank the reviewer for this positive overall assessment. The distinction between established evidence and future hypothesis has been strengthened throughout the revised manuscript: the Abstract, Figure 1 and Figure 2 captions, and Section 5 now present the integrative claim as a hypothesis; an extrapolative-hypothesis statement and a description of the literature search were added to the Introduction (Reviewer 2); Section 8 was reframed as long-term scenarios with an explicit evidence-boundary paragraph; and a paragraph on the practical limitations of current clock technology was added to Section 9 (Reviewer 1). We believe the revised manuscript now maintains this distinction consistently from Abstract to Conclusions.

Submission Date

16 July 2026

Date of this review

15 Aug 2026 19:01:55

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for your careful and comprehensive revision. The authors have adequately addressed my previous comments, and the manuscript has been substantially improved. I have no further comments.