Review Reports - Anti-Inflammatory and Angiogenic Effects of Stem Cell Secretome

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Abstract

Abstract highlights rationale to look at MSCs, a sentence linking to clinical use would be of interest for context i.e use in OA treatment.
Can the n number for each MSC source be included in the abstract.
Can n number for ex vivo model be included.

Introduction

Appropriate rationale for EVs.
A short paragraph explaining OA pathophysiology and the need for better therapeutic options is needed.

Methods

Include n number for donor human cells and explants, as well as donor information- i.e/ sex, age, any co morbidities?

Results

EV characterisation – very detailed, it should be noted that surface marker characterisation only characterises exosome populations.
Very detailed figure legends.
Figure 7- No significance bars included on graphs despite appearing to have significance?

Discussion

Fair balanced conclusions.
Discussion- more detailed linking of findings to OA and capacity as a treatment. More evaluation of results in the context of other literature exploring MSC secretome and EVs in OA research.

Author Response

We would like to thank the reviewer for their constructive review of this work. We have addressed each comment and indicate how we have altered the revised text to accommodate these points.

Comments and Suggestions for Authors

Comment 1:

Abstract

Abstract highlights rationale to look at MSCs, a sentence linking to clinical use would be of interest for context i.e use in OA treatment.
Can the n number for each MSC source be included in the abstract.
Can n number for ex vivo model be included.

Author Response:

We have updated the last sentence in the abstract to “These findings support the bioactivity and therapeutic potential of stem cell-derived secretomes in OA.”

We have added the number of cell sources and ex vivo tissue sources as requested in the abstract and the main text.

Response 1:

We have updated the last sentence in the abstract to “These findings support the bioactivity and therapeutic potential of stem cell-derived secretomes in OA.”

We have added the number of cell sources and ex vivo tissue sources as requested in the abstract and the main text.

Comment 2:

Introduction

Appropriate rationale for EVs.
A short paragraph explaining OA pathophysiology and the need for better therapeutic options is needed.

Response 2:

We thank the reviewer for this point. We have now included a short statement to introduce osteoarthritis , the treatment options and how MSC secretomes may be one of these.

New text:

“Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage loss, synovial inflammation, and subchondral bone remodeling that collectively led to chronic pain and functional disability. Current surgical and pharmacological treatments alleviate symptoms but do not halt or reverse disease progression (Scerif et al. 2025 PMID: 41418993), which highlights the need for disease-modifying and regenerative therapeutic options.”

Comment 3:

Methods

Include n number for donor human cells and explants, as well as donor information- i.e/ sex, age, any co morbidities?

Response 3:

We thank the reviewer for this point. We have now added the number of donor human cells and explants, as well as donor information. ES-MSC were derived from an embryonic cells line (see Tannenbaum et al. 2012 PMID: 22745653 and Grogan et al. 2023 PMID: 36458467). The IPFP tissues were isolated from patients undergoing total knee arthroplasty, co-morbidities to the patient are associated with that procedure rather than isolation of the fat pad tissues per se.

Comment 4:

Results

EV characterisation – very detailed, it should be noted that surface marker characterisation only characterises exosome populations.

Response 4:

We have updated the methods that these makers only target exosomes in our CCM fractions.

Modified text:

” Streptavidin-coated paramagnetic beads with biotinylated antibodies specific to tetraspanins CD9, CD63 and CD81 were used to isolate exosomal EVs from CCM, following manufacturer instructions (EXOFLOW, System Biosciences, Palo Alto, CA).”

Comment 5:

Very detailed figure legends.

Response 5:

We thank the reviewer for this kind remark.

Comment 6:

Figure 7- No significance bars included on graphs despite appearing to have significance?

Response 6:

We thank the reviewer for this point. We have now updated Figure 7 and the text to present statistical comparisons.

Comment 7:

Discussion

Fair balanced conclusions.

Response 7:

We thank the reviewer for this kind remark.

Comment 8:

Discussion- more detailed linking of findings to OA and capacity as a treatment. More evaluation of results in the context of other literature exploring MSC secretome and EVs in OA research.

Response 8:

Author Response:

We thank the reviewer for the recommendations. We have extended the discussion of our data in relation to other published work on the MSC secretome and linked to OA as a target.

Modified text:

Treatment with ES-MSC-derived CCM had a significant chondroprotective and anti-inflammatory effect on osteoarthritic human cartilage explants, as evidenced by reduced glycosaminoglycan (GAG) release and downregulation of catabolic genes (IL-1b, MMP-1, MMP-3), respectively. These findings indicate that ES-MSC secretome counteracts key inflammatory and catabolic pathways in human OA cartilage, supporting its potential as a disease-modifying, cell-free therapeutic (Wu et al. 2022 PMID: 35927232).

Our findings are consistent with other ex vivo explant studies using human and animal tissue, which have shown that the MSC secretome or EV can preserve tissue morphology, reduce catabolic marker expression, and attenuate damage induced by inflammatory cytokines [47]. For example, adipose- and bone marrow–derived MSC secretomes or EVs reduce IL-1β–induced MMPs and ADAMTS5, preserve proteoglycan content, and maintain collagen II in cartilage explants (Woo et al. 2020 PMID:32284824; Colombini et al. 2021 PMID:34066077; Wang et al. 2017 PMID:28807034), which parallels the reduced GAG loss and catabolic gene expression observed in our human OA explants. By demonstrating similar chondroprotective responses in clinically relevant human tissue, this study strengthens the translational bridge from preclinical explant data to potential intra-articular use in OA patients. Comparative analyses of EVs derived from bone marrow (BMSC), adipose tissue (ADSC), and umbilical cord (UMSC) MSCs indicate MSC secretomes exert anti-inflammatory and chondroprotective effects in both explant and in vivo models. BMSC- and UMSC-derived EVs were effective in suppressing inflammation (reduced MMP-13, IL-6, TNF-α, and COX-2) and preserving cartilage integrity [48–50]. Collectively, these data suggest that modulation of common OA effectors (IL-1β, TNF-α, MMPs, ADAMTS5) is a shared mechanism across MSC sources, and our ES-MSC-derived CCM similarly targets this network in human OA cartilage, implying that ES-MSC secretome could provide efficacy comparable to adult-tissue–derived products (Woo et al. 2020 PMID:32284824; Jin et al. 2020 PMID:32122385; Sun et al. 2025 PMID:40881706). Notably, ESC-MSC exosomes have been shown in a mouse OA model to maintain chondrocyte phenotype, increase collagen II, and reduce ADAMTS5, closely aligning with the anti-catabolic profile seen in our explant system and reinforcing the relevance of ES-MSC derived therapies for OA (Wang et al. 2017 PMID:28807034).

Intra-articular injections of EVs reduced some of the arthritic changes in mouse joints after medial meniscus destabilization, presumably via decreased expression of ADAMTS5 [51]. Across multiple OA models, MSC-EVs lessen synovial inflammation, cartilage erosion, and pain behaviors while restoring anabolic–catabolic balance, supporting their potential as candidate disease-modifying OA drugs (DMOADs) (Luo et al. 2024 PMID: 38292826; Hejazaian et al.2025 PMID: 41509067; Wang et al. 2017 PMID:28807034).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study explores the anti-inflammatory and angiogenic effects of mesenchymal stem cell (MSC) secretome derived from human embryonic stem cell-derived MSCs. Using xeno-free and serum-free media, the secretome was isolated and characterized using ultrafiltration and size-exclusion chromatography. The secretome demonstrated bioactivity in promoting cell proliferation, stabilizing endothelial networks, and reducing cartilage degradation in an ex vivo osteoarthritis (OA) model. Hypoxic conditioning of MSCs enhanced the regenerative properties of the secretome, particularly in preserving endothelial networks. The study highlights the therapeutic potential of MSC-derived secretome as a cell-free regenerative treatment for musculoskeletal and inflammatory disorders, emphasizing the need for further research to optimize production, dosing, and clinical applications. However, the data must be confirmed by other methodologies.

Major points:

Results. The percentage shown in the dot plot did not correspond to Figure 1. For example, the percentage probably varies between 20-60%, please verify. Please also show the Forward Scatter data. In the futures studies, the use of different channels for this market will be necessary.

MTT is a preliminary methodology to prove proliferation. Please provide methodologies such as cellular cycle analysis (e.g., BrdU or similar) and western blotting of cell cycle proteins.

Please quantify HUVEC network formation.

Please provide data at protein level of cytokines.

Author Response

We would like to thank the reviewer for their constructive review of this work. We have addressed each comment and indicate how we have altered the revised text to accommodate these points.

Comment 1:

Response 1:

We thank the reviewer for their succinct overview of the study and the clinical goal.

Comment 2:

Major points:

Response 2:

We thank the reviewer for this comment. In this bead-based assay, the dot plots are intended solely to demonstrate the presence of fluorescently stained exosome populations bound to paramagnetic beads via CD9, CD63, or CD81 antibodies. The positive FITC signal (red events) represents >95% marker-positive bead–exosome complexes relative to the isotype/negative control (blue events), which is why the percentages in the dot plots do not correspond directly to the proportions shown in Figure 1, where a different analysis and gating strategy were used. Because this is a bead-based assay, the forward- and side-scatter parameters primarily reflect the uniform bead size and do not provide meaningful information on exosome size or presence. For clarity, we have now added a brief explanation of this in the Methods/figure legend. We can provide a representative FSC/SSC plot of the bead population in the supplementary material if the reviewer considers it helpful.

Comment 3:

MTT is a preliminary methodology to prove proliferation. Please provide methodologies such as cellular cycle analysis (e.g., BrdU or similar) and western blotting of cell cycle proteins.

Response 3:

We agree that complementary assays such as BrdU incorporation, flow‑cytometric cell‑cycle analysis, or western blotting of cell‑cycle regulators are useful in providing additional mechanistic insight. Our intent in this study was to quantify the effect of different secretomes and concentrations on cell number and viability. The MTT assay is a widely used colorimetric method that measures mitochondrial reduction of tetrazolium to formazan and is routinely applied as a surrogate of viable cell number and overall cell growth/proliferation in response to treatments such as growth factors and conditioned media. We agree that resolving detailed cell‑cycle dynamics would be extremely useful in future studies to identity mechanistic relationships between secretome cargo and target cell cycle and cellular proliferation pathways.

Comment 4:

Please quantify HUVEC network formation.

Response 4:

We thank the reviewer for this suggestion. We quantified HUVEC network formation using the Angiogenesis Analyzer plugin in Fiji/ImageJ, extracting standard morphometric indices including total network length, total segment length, total branch length, node/junction counts, and mesh index. By day 7, untreated controls showed marked regression across multiple network metrics, whereas secretome‑treated cultures exhibited less regression. Among all groups, only hypoxic ES‑MSC conditioned medium produced a consistent, statistically significant preservation of several pre‑specified network parameters at day 7 relative to untreated controls. For completeness, we now provide the full set of analyzed network metrics (means, variability, and key statistical comparisons for all conditions and time points) in Supplemental Data Angiogenesis Analysis.

The new legend for Figure 4.

Figure 4. Quantitative analysis of endothelial network morphology on Day 7 HUVEC in fibrin gels with and without ES-MSC hypoxic CCM. All metrics were significantly increased by treatment with hypoxic CCM (p<0.05). The bar graph shows total network length, total segment length, total branch length, and mesh index for hypoxic CCM–treated versus control cultures at Day 7, while Day 7 measurements for normoxic and IPFP‑MSC CCM did not differ significantly from controls (see Supplemental Data Angiogenesis Analysis).

The new Legend created:

Supplemental Data Angiogenesis Analysis. Original data generated using the Angiogenesis Analyzer plugin in Fiji/ImageJ. Images of HUVEC cultured in fibrin gels alone (control) or with normoxic or hypoxic CCM from ES-MSC or IPFP-MSC over 3 and 7 days. The extracted morphometric indices measured were: Total Network length, Total Segment length, Total Branch length and Mesh index.

Comment 5:

Please provide data at protein level of cytokines.

Response 5:

We agree that quantifying secreted cytokines at the protein level would complement our functional readouts. However, cytokine profiling of the conditioned media and explant supernatants was beyond the scope of the present study. In this study, we focused on functional outcomes (GAG release) and on gene expression of key inflammatory and catabolic mediators in cartilage explants (IL‑1, IL‑6, MMP‑1, MMP‑3), which together demonstrate the anti‑inflammatory and anti‑catabolic effects of the ES‑MSC secretome.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This paper describes the effects of the MSC conditioned medium (CCM) which includes exosomes (EVs) or purified EVs on two types of MSC and HUVEC cells, as well as on the cells obtained from human femoral condyles and patellar tissue (disks). Close studies demonstrated some effects of EVs at intervertebral disc degeneration (doi: 10.1016/j.jot.2023.10.004; doi: 10.1002/advs.202404275). Both not cited.

There are major remarks both on the methods and results.

Abstract

It should be shorten, undeeded abbriviations removed, cytometry results should be thought of, results more clealy put, conclusion shorten.

Results

Fig 1 represents four TEM images, two must be removed. The size of EVs is evidently different (D and F). Why?
Results for CCM in A, C and D, F do not coincide. Why?
The authors included CCM obtained in hypoxia conditions possibly basing on some papers such as doi: 10.1002/advs.202404275, which demonstrated a better effect of hypoxic EVs. However the data in Fig 3 do not provide confidence in the conclusion. Please include this citation instead of 11-12 which are not related to disc degeneration. If possible include data on normapoxic CCM.
Fig 4. Please include normopoxic CCM and both from ES-MSC and IPFP-MSC CCM.
The authors used two types of MSC: one from human embryonic mesenchymal stem cells (ES-MSC) and the other from fat pad mesenchymal stem cells (IPFP-MSC). What kind of ES-MSC? Why at all the IPFP-MSC were selected for this work? Some paper demonstrate that EVs from that cells are toxic to nucleus pulposus tissues [Zhou Z et al. Osteoarthritic infrapatellar fat pad aggravates cartilage degradation via activation of p38MAPK and ERK1/2 pathways. Inflamm Res. 2021 Dec;70(10-12):1129-1139. doi: 10.1007/s00011-021-01503-9].
Fig 5 describes the protocol with cell incubation, marks are not clear, please replace with the table.
Fig 6 shows GAG release by cartilage disks under CCM and EVsm what about IPFP-MSC CCM and EVs?
Fig 7 shows GAG release and gene expression under EVs treatment. Which EVs and doses?

Methods

The authors prepared CCM by concentrating super on 100 kDa cartridges loosing small molecules and enriching by EVs. Most papers use unconcentrated super [Kay AG et al. Mesenchymal Stem Cell-Conditioned Medium Reduces Disease Severity and Immune Responses in Inflammatory Arthritis. Sci Rep. 2017;7: 18019 10.1038/s41598-017-18144-w; Suteja RC et al. In Vitro and In Vivo Potential of Human Stem Cell-Derived Conditioned Medium (Secretome) and Exosomes as a Novel Treatment for Osteoarthritis: A Systematic Review of Experimental Studies. Clin Orthop Surg. 2025 Oct;17(5):797-814. doi: 10.4055/cios25023; Chen W et al. Conditioned medium of mesenchymal stem cells delays osteoarthritis progression in a rat model by protecting subchondral bone, maintaining matrix homeostasis, and enhancing autophagy. J Tissue Eng Regen Med. 2019 Sep;13(9):1618-1628. doi: 10.1002/term.2916]. Why the authors decided to use CCM?
Funny that all the preparations ES-MCK-CCM, IPFP-MSC-CCM, ES-MSC-EVs (called by SEC), and IPFP-MSC-EVs gave the same amount of EVs (49 billions per mL). Probably the authors adjusted to that amount, but stated as it.

Comments for author File: Comments.pdf

Author Response

We would like to thank the reviewer for their constructive review of this work. We have addressed each comment and indicate how we have altered the revised text to accommodate these points.

Comment 1:

Response 1:

We thank the reviewer for highlighting these important studies on EVs in intervertebral disc (IVD) degeneration. The present work focuses on articular cartilage and vascular/endothelial models and serves as a proof‑of‑concept that ES‑MSC and IPFP‑MSC secretomes/EVs, produced under xeno‑free, scalable conditions, are biologically active by enhancing proliferation, stabilizing angiogenic networks, and exerting anti‑inflammatory, anti‑catabolic effects in osteoarthritic cartilage explants. We agree that EV‑based therapies are also highly relevant to IVD degeneration, and EV applications across all musculoskeletal tissues may be clinically relevant.

Comment 2:

There are major remarks both on the methods and results.

Abstract

It should be shorten, undeeded abbriviations removed, cytometry results should be thought of, results more clealy put, conclusion shorten.

Response 2:

We thank the reviewer for these suggestions regarding the Abstract. In the revised manuscript, we i) reviewed the use of abbreviations and removed those not needed for understanding the main message, (ii) clarified the flow cytometry results to make them easier to follow, and (iii) made a reduction in length and tightened the wording of the conclusion.

Comment 3:

Results

Fig 1 represents four TEM images, two must be removed. The size of EVs is evidently different (D and F). Why?

Response 3:

We thank the reviewer for these observations. Figure 1 was intentionally designed to show, for each secretome source, both a lower‑magnification field illustrating the overall distribution of vesicles and a higher‑magnification view highlighting the typical exosomal morphology (round, membrane‑bound vesicles). We believe that keeping these paired images for ES‑MSC and IPFP‑MSC provides a more representative visualization of the preparations than a single panel per source. Regarding the apparent size differences between panels D and F, extracellular vesicles—particularly exosomes/small EVs—are well known to form a heterogeneous population spanning approximately 30–150 nm, and even within a single sample the size distribution is broad. The range observed in our TEM images is consistent with this expected heterogeneity and with the size profiles obtained by nanoparticle tracking analysis in our study. We have clarified this point in the Figure 1 legend to emphasize that the panels depict different magnifications and illustrate the normal size variability of small extracellular vesicles.

The modified text legend:

Figure 1. Extracellular vesicle (EV) characterization with nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and flow cytometry. Conditioned medium from human embryonic-derived mesenchymal stem cells (ES-MSC) or human Infrapatellar fat pad MSC (IPFP-MSC) in 2D culture concentrated via ultrafiltration to make concentrated conditioned medium (CCM). CCM was subjected to size exclusion chromatography (SEC) for selection of exosome-sized particles. A-B: NTA was used to analyze particle size (nm) and concentration (PSD = particle size density distribution). Typical size distribution plots for CCM (50–300 nm) and SEC (50–180 nm). Typical yields averaged 49±13 billion particles per ml. C-D: The bar graph shows total network length, total segment length, total branch length, and mesh index for hypoxic CCM–treated versus control cultures at Day 7, while Day 7 measurements for normoxic and IPFP‑MSC CCM did not differ significantly from controls (see Supplemental Data: Angiogenesis Analysis)

E-F. TEM images of IPFP-MSC–derived SEC fractions shown at corresponding low and high magnifications to illustrate vesicle abundance and morphology. G-H. Flow cytometry was used to detect exosomes. Streptavidin coated paramagnetic beads were combined with biotinylated antibodies for tetraspanins (CD9, CD63 or CD81) and a lipophilic dye to stain lipid membranes (PKH67). G. Scatter plots show control (non-conjugated) beads (blue) and a positive shift (red) for each tetraspanin marker in ES-MSC derived SEC fractions. H. Scatter plot of SEC fraction from IPFP-MSC positive for CD63.

Comment 4:

Results for CCM in A, C and D, F do not coincide. Why?

Response 4:

We appreciate the reviewer’s careful evaluation of the proliferation data. Panels A–F show results from independent MTT assays with different cell types. Each OD is normalized to its own internal control within the experiment, so absolute OD values are not directly comparable across panels. In ES‑MSC and IPFP‑MSC, concentrated conditioned medium (CCM) consistently produced a dose‑dependent increase in proliferation relative to the corresponding untreated control in each experiment, whereas HUVEC responses to CCM and SEC were more variable and did not reach statistical significance, as reported in the figure legend and text. Thus, while the numerical OD ranges differ between experiments, the relative patterns (enhanced MSC proliferation with CCM, lack of a robust proliferative effect in HUVEC) and the statistical conclusions are concordant. For clarity, we can add a brief note to the figure legend stating OD values are normalized to each experiment and that comparisons should be made within, rather than between, panels.

Modified Figure 2 legend:

Figure 2. Cell proliferation of ES-MSC, IPFP-MSC, and HUVEC after exposure to ES-MSC secretome. Cells were seeded in 96 well plates (5,000 per well) and subjected to ES-MSC CCM at different concentrations (N=3). A. ES-MSC cultured with 10B particles showed significantly greater (p<0.005) proliferation rate compared to control. B. IPFP-MSC responded to all concentrations examined (0.5B to 10B) in comparison to control (p<0.002). C. A significant reduction in HUVEC proliferation was observed with 0.5B particle exposure and with TGFβ1 treatment (p<0.04). D-F. In separate experiments, we compared the response to CCM and SEC fractions from ES-MSC secretomes (N=3). CCM exposure resulted in a dose-dependent increase in proliferation of ES-MSC and IPFP-MSC (p<0.002) but no significant change in HUVECs. SEC treatment significantly increased proliferation of IPFP-MSC (p<0.001) and ES-MSC (p<0.003) but without a clear dose response. OD values from MTT assays were normalized within each independent experiment; therefore, comparisons should be restricted to panels A–C and separately to panels D–F.

Comment 5:

The authors included CCM obtained in hypoxia conditions possibly basing on some papers such as doi: 10.1002/advs.202404275, which demonstrated a better effect of hypoxic EVs. However the data in Fig 3 do not provide confidence in the conclusion. Please include this citation instead of 11-12 which are not related to disc degeneration. If possible include data on normapoxic CCM.

Response 5:

We thank the reviewer for these comments. Hypoxic conditioning was included in our study because multiple prior reports have shown that culturing MSCs under reduced oxygen enhances the release of pro‑angiogenic and pro‑regenerative factors and improves the functional activity of their secretomes in various tissue contexts (e.g., skin, bone, and muscle), rather than specifically in intervertebral disc models. In line with these observations, our angiogenesis data show that hypoxic ES‑MSC CCM preserves endothelial networks over 7 days more effectively than normoxic CCM or no CCM, with significant improvements across several quantitative network metrics (Figure 3–4, Supplemental Table 2 and Supplemental Data Angiogenesis Analysis). We respectfully feel that the cited IVD‑specific EV papers, while important for disc degeneration, are outside the scope of our vascular/endothelial model. Instead, we have cited more directly relevant work on hypoxia‑enhanced MSC secretomes in musculoskeletal and wound‑healing settings. Normoxic and hypoxic CCM conditions are both already included in Figure 3, and we have clarified in the Results text and have now included the raw data for all conditions as Supplemental Data Angiogenesis Analysis.

Comment 6:

Fig 4. Please include normopoxic CCM and both from ES-MSC and IPFP-MSC CCM.

Response 6:

We appreciate the reviewer’s suggestion to include the normoxic CCM conditions from both ES‑MSC and IPFP‑MSC in Fig. 4. In the main figure, we chose to present only those conditions that showed statistically significant differences relative to controls (day 7) in order to maintain clarity and focus on the most biologically relevant effects. To ensure full transparency and allow detailed comparison across all culture conditions, we have now included the complete raw dataset (including normoxic ES‑MSC and IPFP‑MSC CCM) as an Excel file/pdf file as a supplemental data set (Supplemental Data Angiogenesis Analysis). We have also clarified in the figure legend and Results that all experimental conditions are reported in the supplemental dataset, while Fig. 4 is restricted to conditions with significant changes.

The New Figure 4 legend:

Comment 7:

The authors used two types of MSC: one from human embryonic mesenchymal stem cells (ES-MSC) and the other from fat pad mesenchymal stem cells (IPFP-MSC). What kind of ES-MSC? Why at all the IPFP-MSC were selected for this work? Some paper demonstrate that EVs from that cells are toxic to nucleus pulposus tissues [Zhou Z et al. Osteoarthritic infrapatellar fat pad aggravates cartilage degradation via activation of p38MAPK and ERK1/2 pathways. Inflamm Res. 2021 Dec;70(10-12):1129-1139. doi: 10.1007/s00011-021-01503-9].

Response 7:

We thank the reviewer for these questions. The ES-MSC are a cell line that we have derived from ESC (Tannenbaum et al. 2012 PMID: 22745653; Grogan et al; 2023 PMID:36458467) that have multi-differentiation qualities and are clinically relevant.

While there are mixed reports of IPFP-MSC on biological activity and responses as the reviewer points out, we have studied human IPFP-MSC cell lines for years and have shown their promise for regeneration of cartilage and meniscus tissues (van Schaik et al. 2021 PMID: 30373381; Dorthé et al. 2022 PMID: 35186903, Grogan et al. 2020 PMID: 31134817). These cells also represent an alternative and clinically relevant adult human derived cell line.

Comment 8:

Fig 5 describes the protocol with cell incubation, marks are not clear, please replace with the table.

Response 8:

We thank the reviewer for this suggestion. We have improved the clarity of Fig. 5 by refining the labels and layout to more intuitively convey the temporal sequence and branching of experimental conditions.

Comment 9:

Fig 6 shows GAG release by cartilage disks under CCM and EVsm what about IPFP-MSC CCM and EVs?

Response 9:

We thank the reviewer for this important point. In Fig. 6, we focused on ES‑MSC CCM and EVs because our primary objective in this study was to evaluate the modulation of OA cartilage by ES‑MSC–derived secretome fractions. We chose to analyze ES-MSC secretomes because of the greater reproducibility and efficacy.

Comment 10:

Fig 7 shows GAG release and gene expression under EVs treatment. Which EVs and doses?

Response 10:

We thank the reviewer for this helpful comment. In Fig. 7, the data correspond to EVs derived from ES‑MSCs, applied at a dose of 1 × 10¹⁰ (10B) particles per well. We have now clarified the EV source and particle dose in the Methods and in the Fig. 7 legend.

Comment 11:

Methods

The authors prepared CCM by concentrating super on 100 kDa cartridges loosing small molecules and enriching by EVs. Most papers use unconcentrated super [Kay AG et al. Mesenchymal Stem Cell-Conditioned Medium Reduces Disease Severity and Immune Responses in Inflammatory Arthritis. Sci Rep. 2017;7: 18019 10.1038/s41598-017-18144-w; Suteja RC et al. In Vitro and In Vivo Potential of Human Stem Cell-Derived Conditioned Medium (Secretome) and Exosomes as a Novel Treatment for Osteoarthritis: A Systematic Review of Experimental Studies. Clin Orthop Surg. 2025 Oct;17(5):797-814. doi: 10.4055/cios25023; Chen W et al. Conditioned medium of mesenchymal stem cells delays osteoarthritis progression in a rat model by protecting subchondral bone, maintaining matrix homeostasis, and enhancing autophagy. J Tissue Eng Regen Med. 2019 Sep;13(9):1618-1628. doi: 10.1002/term.2916]. Why the authors decided to use CCM?

Response 11:

We thank the reviewer for this thoughtful comment and for highlighting these important studies on unconcentrated conditioned medium. In our work, we elected to use concentrated conditioned medium (CCM) rather than unconcentrated supernatant for two main reasons. First, one of the major aims of this study was to isolate exosome‑enriched fractions by size‑exclusion chromatography, for which a prior concentration step is standard to obtain sufficient particle yield and enable reproducible SEC fractionation. Second, concentrating the medium allowed us to achieve EV/exosome doses in the range of billions of particles per treatment, which in our preliminary experiments produced more robust and consistent biological responses at volumes that were practical for our culture format. We fully agree that unconcentrated conditioned medium represents a valuable and widely used approach. We have now clarified this distinction and our rationale for using CCM in the Methods and Discussion.

Comment 12:

Funny that all the preparations ES-MCK-CCM, IPFP-MSC-CCM, ES-MSC-EVs (called by SEC), and IPFP-MSC-EVs gave the same amount of EVs (49 billions per mL). Probably the authors adjusted to that amount, but stated as it.

Response 12:

We thank the reviewer for noting the apparent similarity in reported EV concentrations. Our original text used the overall mean value (49.2±12.7×10⁹ particles/mL) across multiple ES‑MSC‑CCM, IPFP‑MSC‑CCM, and corresponding SEC preparations, which may have given the impression that each individual preparation yielded the same concentration. We have now clarified in the Abstract, Results, and Supplemental Table 1 that 50 Billion particles/ml represents an average yield across independent batches, and we explicitly report the specific data from each source to avoid any misunderstanding.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

N/A

Author Response

We thank the reviewer for this careful review.

Reviewer 2 Report

Comments and Suggestions for Authors

I no have further questions

Author Response

We thank the reviewer for this careful review.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors did minor revision. All major remarks were not met. See pdf file.

Comments for author File: Comments.pdf

Author Response

General:

SEC is the method not the vesicles

We thank the reviewer for this comment. We now define the samples derived from SEC fractions as “EVs” to clearly separate the method from the product.

Why are there two figure legends?

The instruction from the IJMS journal was to provide a brief figure title. We have deleted the brief title and have kept only one legend for all figures.

Figure 1: Figure legend usually includes the description of the figures WHITHOUT the result discussion. All the discussions should be given in the text. Revise, please.

We have removed the discussion of the results from the figure legends.

What is negative stained?

Negative stain TEM is a commonly used rapid, high-contrast sample preparation technique for visualizing biological specimens (viruses, proteins, nanoparticles). The typo in “negative stained TEM” was fixed.

In proliferation of which cells?

We have clarified that it was proliferation of IPFP-MSC.

Figure 2: I still do not understand why the authors provide repeated results? A, B, C are the same as D, E, F. Besides the results do not correlate. Second besides: the abscissa marked particles per billions, why the data are labeled 10B, 500M? Please correct everywhere like 10, 5, 1, 0.5.

We thank the reviewer for this comment. As recommended, we now only present the data generated from the CCM and EV exposures. We have also corrected the title for the abscissa.

Keep only one legend. The authors can give the first vertion but edited: Figure 2. Cell proliferation after exposure to the ES-MSC concentrated conditioned medium (CCM) and EVs (SEC). ES-MSC (A, D), IPEP-MSC (B, E) and HUVEC (C, F) were incubated with CCM or SEC for 24 h (?).

We have kept only one legend for all figures. We now only present the data generated from the CCM and EV exposures (see previous response).

Figure 3: Still I cannot see the difference between normoxia and hypoxia in both ES-MSC and IPFP-MSC.

We agree with the reviewer that there do not seem to be visible differences in between normoxia and hypoxia conditions. We conducted histomorphometry of the microvascular networks to measure: total network length, total segment length, total branch length, and mesh index. We compared measurements between each CCM condition and the Control condition (no CCM treatment). Normoxia and Hypoxia CCM from ES-MSC significantly increased these measurements (relative to control). Compared to the Control condition on day 7, Hypoxia CCM from ES-MSC maintained greater network integrity as measured by total network length (41%), total segment length (67%), total branch length (51%), and mesh index (48%). We did not find significant differences between Normoxia and Hypoxia CCM from IPFP-MSC.

We have clarified in the Results:

“At the 3-day timepoint (Figure 3), control gels (HUVEC without CCM) exhibited a network with high numbers of nodes, junctions, and meshes. However, by day 7, the networks in control gels disintegrated with substantial regression of network indices, marked decreases in node count, junctions, mesh area, and total segment length (p<0.05 day 7 vs. day 3). On day 7, CCM harvested from hypoxic ES-MSC maintained greater network integrity as measured by total network length, total segment length, total branch length, and mesh index, relative to day 7 control gels (p<0.05, Figure 3, Figure 4, and Supplemental Data Angiogenesis Analysis). Whereas the other CCM treatments did not result in a statistically significant difference relative to the day 7 control condition.”

Figure 5: All that should be presented as a Table, the figure is not easy to understand.

We appreciate the reviewer’s suggestion to convert the figure to a table. We believe that a figure is more descriptive and have modified the figure to make it easier to understand.

Figure 4: I cannot see with or without, control must be without CCM

We have specified in the figure legend that Control is without any CCM.

Figure 6: not only secretome but SEC, and add what is in A-C.

Figure 6 has been updated. We have clarified the treatments and moved the results to the main text and revised the Figure 6 legend:

Figure 6. Glycosaminoglycan (GAG) release profiles from ex vivo human osteoarthritic cartilage tissue treated with ES-MSC secretomes. Human cartilage explants (N=3 donors; 70M, 71M and 76F) were pre-treated with IL-1b for three days before treatment with either ES-MSC derived CCM or EVs (10 billion particles). Control (CNT) explants were not pre-treated with IL-1b. A. GAG release profiles three days after CCM treatment. B. GAG release profiles three days after EV treatment. (Key: CNT = Control; IL-1b = IL-1b treatment only; IL-1b→CCM = 3-day IL-1b treatment followed by CCM treatment; IL-1b→EV = 3-day IL-1b treatment followed by EV treatment).

Figure 7: Secretome or SEC? And EV, not SEC, not EVs. Does CNT mean control? All abbreaviations must be explained in the legend. In the text CCM was used not EV, why is EV written in the abscissa?

As recommended, we have modified Figure 7, corrected the abscissa, clarified all abbreviations, and revised Figure 7 legend:

Figure 7. Concentrated secretome (CCM) treatment modulates glycosaminoglycan (GAG) release and catabolic gene expression. A-B. Explants pretreated with IL-1b continued to release more GAG. GAG release from controls (without IL-1b pretreatment) remained unchanged. CCM treatment (10 billion particles) reduced GAG release in IL-1b pretreated and control explants (*p<0.05). C-F. IL-1b pre-treatment significantly increased gene expression of catabolic genes in explants (fold change relative to untreated explants).

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The authors at least corrected the nomenclature (EV instead of many others) and figure 2. Figure 3 is not convincing but let it be.