Pneumatospinning and Electrospinning Scaffolds for Meniscus Regeneration Using Human Embryonic-Derived Mesenchymal Stem Cells
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors must mention clearly the main aim of the current manuscript.
I believe that the authors could make more to put in evidence the neotissue formation not just the laminate scaffold viability. This aspect is very important and deserves a special section and figure.
Also, I suggest presenting separately the results for mechanical testing of implanted scaffolds.
The text from line 517 to 522 is not necessary.
I expect to find some comments related to the comparation between these laminate constructs and other composite designs.
Even the authors say in conclusion that “The advancement to laminate scaffolds offers a promising solution to the challenges of meeting mechanical and bioactivity requirements for partial meniscus replacement grafts.”, I didn’t find anything about bioactivity. How was the bioactivity quantified? Maybe the authors could use another term or provide some explanations.
Some other problems are related to the figures (see below):
-In figure 2, the authors show also some SEM images. I believe that it is not adequate to be used at this part related to the fabrication methods used.
- SEM images from figure 4 must be used at original size. Also, some explanation about the red mark on figure 4-E must be provided in text.
-Figure 5 is not clearly, and the scale is not visible.
- Also, for figure 6 the scale is not clear.
Author Response
Reviewer 1:
Reviewer 1 comment 1. The authors must mention clearly the main aim of the current manuscript.
Author response to Comment 1:
In the last paragraph of the Introduction, we have more clearly stated the principal aim and secondary aims of the study: “The principal aim was to combine a pneumatospun collagen scaffold with clinically relevant embryonic-derived MSCs (ES-MSCs) and to enhance bioactivity by immobilizing growth factors using heparin-conjugation. We evaluated neo-tissue formation and mechanical integration to analyze the effect of growth factor immobilization in a human OA meniscus explant model. A secondary aim was to test proof of concept of fabricating a laminate scaffold that would enhance mechanical properties while satisfying biocompatibility, cell colonization, and neo tissue formation with potential for use in partial meniscus replacements.”
Reviewer 1 comment 2. I believe that the authors could make more to put in evidence the neotissue formation not just the laminate scaffold viability. This aspect is very important and deserves a special section and figure.
Author response to comment 2:
We agree that neotissue formation is important. The evidence of neotissue formation in the laminate scaffolds is displayed in specific histological and immunohistochemical staining in Figure 9 (New Figure 10) and Figure 10 (New Figure 11), for comparison with old Figure 6 (New Figure 7). In the new Figure 10, we used Safranin O, picrosirius red, and collagen immunostaining to show the deposition of extracellular matrix components. In the new Figure 11, we show data that the laminate scaffolds supported neo-meniscus like gene expression. After implantation in human ex vivo tissues for 6 weeks, Safranin O staining showed neo tissue formation and integration with the native host meniscus tissue. Finally, we noted enhanced mechanical pushout qualities (shear modulus) with growth factor conjugation which is supported by the evidence of histologic integration.
Reviewer 1 comment 3. Also, I suggest presenting separately the results for mechanical testing of implanted scaffolds.
Author response to comment 3:
As recommended, we have separately presented the results for mechanical testing of implanted scaffolds under the following section: “TGF-β enhances mechanical integration of implanted scaffolds”
Reviewer 1 comment 4. The text from line 517 to 522 is not necessary.
Author response to comment 4:
We have now revised the paragraph: “Advanced in vitro and ex vivo systems have become increasingly important for preclinical evaluation (see the official NIH policy statement (https://www.nih.gov/news-events/news-releases/nih-prioritize-human-based-research-technologies). We had previously characterized an ex vivo model to reproducibly assess cartilage-scaffold integration and quantify tissue bonding strength [53]. We used a similar ex vivo model for meniscus tissue integration to provide a translationally relevant approach that supports biomechanical assessment of scaffold integration aligned with evolving research policies.”
Reviewer 1 comment 5. expect to find some comments related to the comparation between these laminate constructs and other composite designs.
Author response to comment 5:
As recommended, we have extended the Discussion to include comparisons between our laminate construct and other composite designs:
The revised discussion:
A range of laminate and composite scaffolds have been tested for meniscus regeneration using diverse material combinations and cell sources. Reported configurations include silk fibroin and wool keratin blends [54], meniscus ECM with methacrylated gelatin [55], PLA with natural hydrogels [24], cellulose and chitosan composites [56] , polycaprolactone (PCL) with silk fibroin [36], as well as multicomponent scaffolds containing PCL, silk fibroin, gelatin, and ascorbic acid seeded with adipose-derived MSCs [57]. Additionally, bioprinted composite hydrogels with aligned synthetic microfibers in methacrylated gelatin using meniscus fibroblasts or BM-MSCs have demonstrated promising results [58]. Collectively, these works highlight incremental improvements in combining mechanical strength with cell-compatible environments. However, many of these composite constructs rely on either dense fiber meshes that limit cellular infiltration or hydrogel-rich matrices with limited capacity for load bearing.
We and others have laminated electrospun scaffolds to address these limitations [24,59,60]. Fisher et al showed increased stiffness of layered electrospun PCL scaffolds seeded with bovine MSC [59,60]. We seeded human avascular meniscus cells in a hydrogel composed of collagen type II, chondroitin sulfate, and hyaluronan on layers of electrospun PLA. While these approaches did meet biomechanical requirements, the composite thickness was only a few hundred micrometers [24]. Building on these prior efforts, the present laminate scaffold advances the field by integrating a mechanically robust coaxial electrospun PLA/collagen layer with bioactive, porous pneumatospun collagen exterior layers. Unlike conventional electrospun scaffolds that offer mechanical integrity at the expense of cell infiltration and unlike purely hydrogel-based or single-fiber composite systems that compromise structural robustness, this laminate design leverages outer pneumatospun layers to facilitate cell seeding, viability, and matrix formation, while the electrospun core significantly increased the overall elastic modulus (from 0.1 to 2.4 MPa). The cell migration and matrix elaboration across internal interfaces further support its clinical relevance [14–16]. Taken together, these features position this scaffold at least as effective and in some respects superior to published prototypes in enabling both mechanical performance and robust cell-mediated integration, aligning with the paradigm of functional biomimicry for complex tissue engineering [15,16].
Reviewer 1 comment 6. Even the authors say in conclusion that “The advancement to laminate scaffolds offers a promising solution to the challenges of meeting mechanical and bioactivity requirements for partial meniscus replacement grafts.”, I didn’t find anything about bioactivity. How was the bioactivity quantified? Maybe the authors could use another term or provide some explanations.
Author response to comment 6:
We agree that the term ‘bioactivity’ should be better defined. In the revised text, we now specify that bioactivity in this context refers to growth factor–mediated enhancement of meniscogenesis, as evidenced by increased Safranin O staining, collagen II deposition, collagen fiber polarization, and upregulation of genes such as ACAN and COL2A1 in growth factor–immobilized scaffolds. We have revised the Abstract and Conclusion to directly link ‘bioactivity’ to these measured histologic, molecular, and mechanical readouts.
Reviewer 1 comment 7. Some other problems are related to the figures (see below):
-In figure 2, the authors show also some SEM images. I believe that it is not adequate to be used at this part related to the fabrication methods used.
Author response to comment 7:
We appreciate the recommendations for improving the Figures and have moved the SEM images from Figure 2 to the new Figure 3.
Reviewer 1 comment 8.- SEM images from figure 4 must be used at original size. Also, some explanation about the red mark on figure 4-E must be provided in text.
Author response to comment 8:
SEM images in Figure 4 (new Figure 5) are at the original size. We have clarified the figure legend to note that the red rectangle in panel E denotes the region shown in panel F.
Reviewer 1 comment 9.-Figure 5 is not clearly, and the scale is not visible.
Author response to comment 9:
We have updated scale bars in Figure 5 (new Figure 6).
Reviewer 1 comment 10 - Also, for figure 6 the scale is not clear.
Author response to comment 7:
As recommended, we have updated scale bars in Figure 6 (new Figure 7).
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study evaluated the ES-MSCs cultured on pneumatospun collagen scaffolds in forming meniscus-like regenerated tissue and repairing human OA meniscal defects in vitro. Overall, the data are quite comprehensive, but more in-depth and detailed discussion is still needed, and the image quality needs to be improved. The following are some suggestions:
- All the pictures in Figures 1 and 2 are in low quality, they should also be supplemented in the supplementary data.
- All Figure captions should be carefully checked and revised. The current captions are too brief and simple to get sufficient information, each panel should be explained in the caption.
- Figure 2 A-D showed the laminate scaffold fabrication process. It is recommended to add explanatory text indicating the meaning of different colors and layers.
- Figure 4 showed the SEM images of pneumatospun fibers, revealing bead-like structures and fiber architecture. Is this uneven distribution a typical characteristic of pneumatospinning, and can it be optimized by adjusting the spinning parameters?
- The author should use arrows or apply colors to point out the cells and ECM In Figure 4F.
- How many days were the scaffold and cells cultured in Figure 5? And what’s the size of the scaffold?
- In Figure 6, was a cell-seeded or cell-free scaffold implanted into the human OA meniscus model? What’s the purpose to use the fresh tissue? Is it to simulate the in vivo repair environment? Did the authors observe cell migration? And what culture medium was used? This part should be discussed in more detailed and logical way.
- In Figure 6A, compared to the surrounded meniscus tissue, all growth factor-treated scaffolds site showed deeper color in Col-II staining other than Col-I staining, does this indicate the formation of more hyaline cartilage than fibrocartilage? What does this mean for meniscus repair?
- Has the author attempted the in vivo implantation? In the absence of in vivo data, how can the reliability of these in vitro data be assessed?
- Has the author tested the mechanical strength of the scaffold itself? How is the degradability of the scaffold?
- Please check the manuscript carefully, including units, special characters, all abbreviations must be displayed in full upon their first appearance.
Author Response
Reviewer 2:
Reviewer 2 comment 1. This study evaluated the ES-MSCs cultured on pneumatospun collagen scaffolds in forming meniscus-like regenerated tissue and repairing human OA meniscal defects in vitro. Overall, the data are quite comprehensive, but more in-depth and detailed discussion is still needed, and the image quality needs to be improved. The following are some suggestions:
Author response to comment 1:
We thank the reviewer for this complimentary overview and constructive review.
Reviewer 2 comment 2. All the pictures in Figures 1 and 2 are in low quality, they should also be supplemented in the supplementary data.
Author response to comment 2:
We followed journal instructions to insert figures in our MS Word manuscript. The journal then converted our Word document to PDF which appears to have reduced the resolution. We have improved the quality of Figures 1 and 2 but the conversion to pdf may still affect the image resolution. As recommended, we have moved panels of Figure 1 to supplemental data.
Reviewer 2 comment 3. All Figure captions should be carefully checked and revised. The current captions are too brief and simple to get sufficient information, each panel should be explained in the caption.
Author response to comment 3:
We agree that the current figure captions are too brief. The journal’s instructions were to place the Figures in the text with only “a short title”. The Figure legends were located at the end of the original manuscript. We have placed the full Figure legends below each Figure.
Reviewer 2 comment 4. Figure 2 A-D showed the laminate scaffold fabrication process. It is recommended to add explanatory text indicating the meaning of different colors and layers.
Author response to comment 4:
As recommended, we have modified Figure 2 A-D and added explanatory text indicating the meaning of different colors and layers.
Reviewer 2 comment 5. Figure 4 showed the SEM images of pneumatospun fibers, revealing bead-like structures and fiber architecture. Is this uneven distribution a typical characteristic of pneumatospinning, and can it be optimized by adjusting the spinning parameters?
Author response to comment 5:
The reviewer is correct. Beads tend to form when pneumatospinning collagen into scaffolds as we and others have reported (Polk S et al, Biofabrication, 2018 PMID: 30109859; Dorthé et al, Front Bioeng Biotechnol, 2022, PMID: 35186903]. Bead formation has been linked to surface‑tension effects and is mitigated by optimizing solution formulation and spinning parameters. Others have intentionally pneumatospun polymers with beads for drug delivery (e.g. Anaya-Mancipe JM, ACS Appl Mater Interfaces, 2025, PMID: 40208007). To reduce bead density, we adjusted polymer concentration and tuned airflow to avoid excessive turbulence.
Reviewer 2 comment 6. The author should use arrows or apply colors to point out the cells and ECM In Figure 4F.
Author response to comment 6:
We appreciate this advice. We have colored the cells green and added arrows to indicate the deposition of ECM in Figure 4 (new Figure 5).
Reviewer 2 comment 7. How many days were the scaffold and cells cultured in Figure 5? And what’s the size of the scaffold?
Author response to comment 7:
The cells were cultured upon the 3.5 mm by 2-3mm thick scaffolds for a total of five weeks. We have now added this information to the Results section and in the legend for Figure 5 (new Figure 6).
Reviewer 2 comment 8. In Figure 6, was a cell-seeded or cell-free scaffold implanted into the human OA meniscus model? What’s the purpose to use the fresh tissue? Is it to simulate the in vivo repair environment? Did the authors observe cell migration? And what culture medium was used? This part should be discussed in more detailed and logical way.
Author response to Comment 8:
We have clarified that cell‑seeded scaffolds (containing ES‑MSCs) were implanted into fresh human OA meniscus explants and that the use of freshly harvested OA tissue with living cells is intended to approximate a more physiologic repair environment. We also specify that constructs were cultured in serum‑free chondrogenic differentiation medium supplemented with TGFb3, with details cross‑referenced to the Methods. We have reorganized the corresponding Results paragraph to present the experimental design, culture conditions, histology, histomorphometry, and mechanical outcomes in a more logical sequence.
We did not analyze cell migration in this study. In a previous publication we had characterized out ex vivo explant repair model and reported that PDGF significantly enhanced cell migration from host meniscus into implanted decellularized tissues (Lee KI, Olmer M, Baek J, D'Lima DD, Lotz MK. Platelet-derived growth factor-coated decellularized meniscus scaffold for integrative healing of meniscus tears. Acta Biomater. 2018 PMID: 29908335).
Reviewer 2 comment 9. In Figure 6A, compared to the surrounded meniscus tissue, all growth factor-treated scaffolds site showed deeper color in Col-II staining other than Col-I staining, does this indicate the formation of more hyaline cartilage than fibrocartilage? What does this mean for meniscus repair?
Author response to Comment 9:
We agree with the reviewer that Col-II stain intensity is visibly higher than the intensity of Col-I stain. We could not quantify the relative intensity of the IHC stain between the two collagen types because of the differences in antibodies and IHC processes, for example differences in the exposure times based on negative and positive controls. Hyaline cartilage only contains Col-II. The avascular meniscus is fibrocartilaginous and contains a mixture of collagen types I and II and glycosaminoglycans.
Reviewer 2 comment 10. Has the author attempted the in vivo implantation? In the absence of in vivo data, how can the reliability of these in vitro data be assessed?
Author response to Comment 10:
We agree that in vivo validation is an important next step. We performed in vivo subcutaneous implantation in nude mice and have included the preliminary results in Figures 12 and 13. In the Limitations section, we now explicitly acknowledge that the present work is restricted to in vitro, ex vivo human OA meniscus model, and in vivo mouse subcutaneous models. The performance under physiological joint loading and remodeling in vivo remains to be tested. The combined use of histology, gene expression, polarized light analysis, and biomechanical push‑out testing in human tissue explants provides a comprehensive laboratory test platform for initial proof of concept. Future in vivo studies of meniscus repair are needed to confirm long‑term integration, durability, and functional restoration.
Reviewer 2 comment 11. Has the author tested the mechanical strength of the scaffold itself? How is the degradability of the scaffold?
Author response to Comment 11:
We have reported the tensile modulus of laminate scaffolds and corresponding yield and peak stress values in the Results. Regarding degradability, we did not conduct a systematic degradation study of the laminate scaffold. We did perform preliminary experiments of biodegradation in vivo and have added this data. We had previously reported that electrospun collagen scaffolds were almost completed bioresorbed by 6 weeks (Baek J et al, Nanomedicine, 2022, PMID: 34991339) In this study, our pneumatospun scaffolds remained relatively intact up to 6 weeks after implantation. We have included these results in Figure 12.
Reviewer 2 comment 12. Please check the manuscript carefully, including units, special characters, all abbreviations must be displayed in full upon their first appearance.
Author response to Comment 12:
We thank the reviewer for this careful review. The entire manuscript has been thoroughly proofread to correct units, special characters, and symbols, to ensure that all abbreviations (e.g., ES‑MSC, DoP, PLA, OA) are defined in full at their first appearance in the text and figure legends.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript entitled "Pneumatospinning and electrospinning scaffolds for meniscus regeneration using human embryonic derived mesenchymal stem cells" is well designed and well written. It is evident that the authors have invested a lot of time and effort into this comprehesive study. The study aims to offer an alternative for meniscus pathology in order to further protect against knee degeneration. Despite the direct clinical application is still limited, it offers new insights of cell therapies in meniscal pathology. I recommend for publication after minor revision. I would add to the limitations of the study the fact that the defect they have caused is unlikely to appear in clinical setting, therefore the injuries found in clinica settings, are a lot harder to be treated with a similar techniques as the authors described.
Author Response
Reviewer 3:
Reviewer 3 comment 1.
The manuscript entitled "Pneumatospinning and electrospinning scaffolds for meniscus regeneration using human embryonic derived mesenchymal stem cells" is well designed and well written. It is evident that the authors have invested a lot of time and effort into this comprehesive study. The study aims to offer an alternative for meniscus pathology in order to further protect against knee degeneration. Despite the direct clinical application is still limited, it offers new insights of cell therapies in meniscal pathology. I recommend for publication after minor revision. I would add to the limitations of the study the fact that the defect they have caused is unlikely to appear in clinical setting, therefore the injuries found in clinica settings, are a lot harder to be treated with a similar techniques as the authors described.
Author response to comment 1:
We are grateful for the reviewer’s positive evaluation and this thoughtful suggestion. In the limitations section, we now state that “Our ex vivo model does not replicate the complex tear patterns and the loading conditions observed in clinical meniscus injuries. Future studies will therefore need to evaluate this system in more clinically representative tears and under physiological loading.”
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors perform all suggested improvements and the manuscript could be published in the current form.
Reviewer 2 Report
Comments and Suggestions for AuthorsI don’t have further questions on this revised manuscript, thanks.
