Three‐dimensional printed biomimetic multilayer scaffolds coordinated with sleep‐related small extracellular vesicles: A strategy for extracellular matrix homeostasis and macrophage polarization to enhance osteochondral regeneration

Cartilage defects resulting from injury or degeneration are a common clinical problem, and due to its avascular nature, articular cartilage has poor self‐healing capacity. Three‐dimensional (3D) bioprinting has attracted great attention in tissue engineering. Melatonin (MT), a hormone mainly secreted at night, plays an important role in tissue repair. Small extracellular vesicles (sEV) are considered ideal drug delivery vehicles and MT‐sEV (sleep‐related sEV) have the potential ability to promote cartilage regeneration. Here, biomimetic multilayer scaffolds were fabricated using 3D bioprinting. A double network hydrogel, composed of methacrylated hyaluronic acid and gelatin methacryloyl (HG), was prepared. MT‐sEV and HG hydrogel were used to create a cartilage layer. A bone layer was formed using poly(ε‐caprolactone) and hydroxyapatite ultralong nanowires. Additionally, two bioinks were alternately printed at the interface layer. The results of RNA sequencing revealed the potential regulatory mechanisms. MT‐sEV showed promotional effects on cell migration, proliferation, chondrogenic differentiation, and extracellular matrix (ECM) deposition. Moreover, MT‐sEV altered macrophage polarization and regulated the expression of inflammatory cytokines. In vivo experiments demonstrated that the biomimetic multilayer scaffolds promoted cartilage regeneration. These experiments demonstrated the ability of MT‐sEV to regulate the immune microenvironment and promote the secretion of ECM, providing a promising strategy for cartilage regeneration.


INTRODUCTION
Cartilage and subchondral bone injuries are prevalent clinical issues that can arise from various causes including trauma, osteoarthritis, tumors, and other diseases.These injuries severely impact the quality of life for patients. 14][5] Macrophages, crucial cells of the inflammatory response, are mainly divided into two types: the pro-inflammatory M1 type and the antiinflammatory M2 type. 6M1 macrophage infiltration aggravates the inflammatory response and suppresses ECM synthesis by reducing the expression of collagen and aggrecan (ACAN). 7Inflammatory cytokines induce matrix-degrading proteases in articular cartilage, breaking the synthetic-catabolic balance of ECM.M2 macrophages secrete anti-inflammatory cytokines that suppress the inflammatory response and enhance catabolic activity of chondrocytes, thereby promoting cartilage regeneration. 8oreover, anti-inflammatory cytokines increase chondrocyte viability and promote the secretion of ECM by chondrocytes, expediting cartilage formation.Therefore, promoting M2 polarization represents a viable strategy for facilitating cartilage repair.
0][11] Cartilage and subchondral bone have complex structures and different tissue compositions.The simultaneous repair of cartilage, subchondral bone, and bone-cartilage interface is a challenge in the field of tissue engineering, attracting widespread attention. 12The extrusion-based 3D printing technology allows for the fabrication of scaffolds that closely mimic the physiological structure of native osteochondral tissue, thus enhancing the reparative outcomes. 13oly(ε-caprolactone) (PCL) is commonly used in bone repair due to its excellent biocompatibility and low immunogenicity. 14Hydroxyapatite (HAP) is an inorganic component of the bone matrix that is capable of inducing bone degeneration, and HAP ultralong nanowire (HAPUW) has high flexibility and excellent mechanical properties.Incorporating HAPUW into polymers is a well-established technique with desirable effects on bone repair. 15Cartilage repair remains a challenging problem to date.Hydrogel scaffolds printed by 3D bioprinting are proven to facilitate cartilage regeneration, providing a promising therapeutic strategy for cartilage injury. 16,17Methacrylated hyaluronic acid (HAMA) has attracted widespread attention for its superior mechanical properties and excellent biocompatibility. 18evertheless, pure HAMA hydrogel lacks temperature sensitivity. 19As a temperature-sensitive hydrogel, gelatin methacryloyl (GelMA) possesses excellent cell compatibility and bioactivity due to its specific amino acid sequence that contributes to cell adhesion. 20,21However, the poor mechanical properties of GelMA restrict its application in cartilage regeneration.][24] Three-dimensional printed scaffolds combined with bioactive factors that possess immunomodulatory property is a common strategy to promote osteochondral repair.6][27] MT has been proven to shift macrophage polarization toward M2 phenotype by regulating oxidative stress homeostasis. 28In recent years, it has been discovered that MT has a pro-regeneration effect on cartilage due to its multifaceted actions, including reduction of inflammation, modulation of the circadian rhythm, and synthesis of ECM. 29 In our previous study, we identified sleep-related circular RNA by inducing cell sleep with MT, and demonstrated that combining sleep-related circular RNA with extracellular vesicles (sEV) is a promising therapeutic approach for the treatment of osteoarthritis. 30owever, the therapeutic potential of incorporating sleeprelated sEV secreted by MT-pretreated cells with scaffolds for osteochondral defects and the underlying molecular mechanisms remain elusive.
In this study, biomimetic multilayer scaffolds were fabricated using 3D bioprinting technology.The HG double network hydrogel loaded with MT-sEV was used to generate the cartilage layer and PCL/HAPUW (PCLUW) was used for the bone layer.Two bioinks were printed alternately in the interface layer.The potential molecular mechanisms of the whole MT-sEV on cartilage repair were interrogated by RNA sequencing.The overall effects of MT-sEV on cell migration, chondrogenic differentiation, cell proliferation, and macrophage polarization were explored in in vitro experiments.The effect of biomimetic scaffolds on cartilage repair was explored in vivo.

Characteristics of hydrogels
HG hydrogel was obtained based on GelMA and HAMA hydrogels.The compressive modulus was analyzed to characterize the mechanical properties of three hydrogels.
The results showed that 10% GelMA hydrogel presented a compressive modulus of 14.92 ± 2.91 kPa, while the compressive modulus of 2% HAMA hydrogel was 24.74 ± 1.17 kPa (Figure 1A).In comparison, the modulus of HG hydrogel was 189.10 ± 6.94 kPa, which corresponds to approximately 7.6 and 12.6 times that of HAMA and GelMA hydrogels, respectively.The compressive modulus of HG hydrogel was higher than that of HAMA and GelMA hydrogels, which was beneficial for the repair of cartilage tissue. 31The compressive stress-strain curve of HG hydrogel is shown in Figure S1.Rheological experiments were carried out to investigate the change in the storage modulus (G′) and loss modulus (G″) of GelMA (Figure 1B), HAMA (Figure S2), and HG hydrogels (Figure 1C) as the temperature increased from 5 • C to 35 • C. The results showed that when the temperature was below 33 • C, the G′ of GelMA hydrogel was higher than G″, indicating that GelMA was in the gel phase.However, when the test temperature exceeded 33 • C, the G′ of GelMA hydrogel was smaller than G″, which suggested that GelMA was in the sol phase.When the G′ was equal to G″, GelMA hydrogel was in a semi-solid state.In addition, HG hydrogel showed a gel-sol transition with temperature variations, existing in a gel phase below 32 • C and a fluid phase above 32 • C. In contract, the G′ of HAMA hydrogel was approximately equal to G″, and both G′ and G″ were <3 Pa throughout the measurements, which showed that HAMA hydrogel was in the sol or biphasic phase and lacked a gel-to-sol transition.The results demonstrated that like GelMA hydrogel, HG hydrogel was thermosensitive and suitable for extrusion-based 3D bioprinting.

Preparation of biomimetic scaffolds
HG hydrogel was created to print the cartilage layer, and PCL was mixed with HAPUW to obtain PCLUW for the bone layer (Figure 1D).The results of lap-shear test demonstrated the adhesion between PCLUW and HG hydrogel (Figure S3).The compressive modulus of PCLUW was 52.30 ± 4.82 MPa, which was lower than that of natural bone. 324][35] The compressive stress-strain curve of PCLUW is shown in Figure S4.We utilized a dual-channel 3D printer to generate biomimetic scaffolds consisting of an HG cartilage layer, a PCLUW bone layer and a HG/PCLUW interface layer.A schematic representation of 3D bioprinting is shown in Figure 1E and the biomimetic scaffolds are displayed in Figure 1F.Scanning electron microscopy (SEM) was used to characterize the morphologies of biomimetic scaffolds.HG hydrogel had a porous structure that facilitated the transmission of nutrients.HG and PCLUW were alternately printed at the interface layer to limit the ingrowth of blood vessels (Figure 1F).

Isolation and identification of sEV
The method of isolating sEV has been described in our previous research. 36We identified the isolated sEV by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM).NTA of the particle sizedistribution showed that the particle size of sEV was in the range of 30-200 nm, similar to the findings of a previous study (Figure S5). 37The results of TEM showed that sEV exhibited a characteristic globular morphology similar to reports in previous literature (Figure S6). 38

Biocompatibility of the scaffolds
Cells of the chondrocyte cell line C28/I2 were seeded onto biomimetic scaffolds loaded with MT-sEV and SEM was used to observe the morphology of the cells within the scaffolds.A schematic illustration of C28/I2 cells cultured on the scaffolds is shown in Figure 2A.Representative SEM images revealed that C28/I2 cells with normal morphology adhered on scaffolds after 3 days of culture (Figure 2B).After 5 days of culture, the C28/I2 cells exhibited excellent spreading behavior on biomimetic scaffolds, which suggested that cells seeded onto scaffolds maintain proliferation capacity.Furthermore, the addition of MT-sEV did not affect cell activity (Figure 2C).A live/dead assay was performed to evaluate cell viability.C28/I2 cells were cultured on scaffolds and stained with calcein-AM (green) and propidium iodide (red).The results showed that cells attached well to the surface of scaffolds and fewer dead cells were present after 3 days of culture (Figure 2D).Confocal microscopy revealed an apparent increase in cell number and a good distribution of C28/I2 cells after 5 days of culture (Figure 2E).Our experiments demonstrated excellent biocompatibility of both the scaffolds and MT-sEV.

RNA sequencing and analysis
Bioinformatics analysis was conducted to investigate the underlying regulatory mechanisms of MT-sEV on cellular behavior.A volcano plot showed that there were 736 differentially expressed genes (DEGs), comprising 349 up-regulated and 387 down-regulated genes in the experimental groups (Figure 3A).Further analysis of the DEGs showed that (Figure 3B): (1) versican (VCAN), bone morphogenetic protein-6 (BMP6), and cartilage oligomeric matrix protein (COMP) were highly expressed in the MT-sEV group, which suggested that MT-sEV might promote ECM synthesis [39][40][41] ; (2) a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5), matrix metalloproteinase-1 (MMP1), and MMP3 were associated with ECM degradation and their expression was low in the MT-sEV group 42 ; (3) paired-like homeodomain transcription factor-2 exhibited a high level of expression, which was associated with cell proliferation 43 ; (4) insulinlike growth factor binding protein-3 was implicated in cell migration, 44 which was highly expressed in the MT-sEV group; (5) interleukin-1β (IL-1β) and IL-6 receptor (IL-6R) were linked to chronic inflammation and their expression was low in the MT-sEV group. 45Some pathways were identified via gene ontology enrichment analysis (Figure 3C): positive regulation of cell migration and cell adhesion, suggesting that MT-sEV might play a role in cell migration and adhesion, and negative regulation of inflammatory response, suggesting that MT-sEV had a modifying effect on inflammation.The results of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) showed that MT-sEV treatment decreased the expression of pro-inflammatory cytokines (tumor necrosis factor-α [TNF-α], IL-1β, and IL-18).In contrast, the expression level of anti-inflammatory cytokines (IL-4 and IL-10) was increased in the MT-sEV group (Figure 3D).Reactome enrichment analysis was performed to further evaluate the signaling pathways affected by MT-sEV.The results of reactome enrichment are shown as a bubble chart in Figure 4A.The enrichment signaling pathways included ECM proteoglycans, collagen formation, ECM organization, elastic fiber formation, collagen biosynthesis, and modifying enzymes, which were correlated with the synthesis and degradation of cartilage ECM.Gene set enrichment analysis was further performed and the results are shown in Figure 4B.Multiple pathways related to ECM synthesis were significantly enriched in the MT-sEV group, including ECM organization, collagen biosynthesis and modifying enzymes, and ECM proteoglycans.Moreover, the nucleotide-bindingoligomerization-domain-containing proteins-1/2 signaling pathway was downregulated in the MT-sEV group, which suggested that MT-sEV might play a role in the immune response.
The results of qRT-PCR (Figure 4C) indicated that MT-sEV reduced the gene expression related to ECM degradation (MMP1, MMP3, and ADAMTS5) and induced the expression of ECM formation-related genes (BMP6, COMP, and VCAN).In addition, MT-sEV upregulated the expression level of type II collagen (COL II), which is associated with chondrogenic differentiation, as well as markers associated with osteogenic differentiation, including osteocalcin (OCN), type I collagen (COL I), and BMP2 (Figure S7).

The effects of MT-sEV on cell migration, proliferation
Transwell assay was performed to assess the effect of MT-sEV on cell migration.Bone marrow-derived mesenchymal stem cells (BMSCs) and C28/I2 cells were seeded into upper chamber, and the migrated cells were stained using crystal violet.As seen in Figure 5A,B, the sEV group had more migrated BMSCs and C28/I2 cells that migrated into the lower chamber compared to the control group.The results also showed that there were more migrated BMSCs and C28/I2 cells in the MT-sEV group compared to the sEV group.In addition, the migrated BMSCs and C28/I2 cells of the MT-sEV group exhibited a uniformly spread morphology.The results of migration assay suggested that MT-sEV significantly increased BMSCs and C28/I2 migration.Figure 5C shows a schematic diagram of transwell experiments.
The effect of MT-sEV on cell proliferation was investigated by EdU proliferation experiments.As shown in Figure 5D,E, a small increase in EdU-positive BMSCs and C28/I2 cells was determined in the sEV group compared with the control group.Furthermore, there were more EdU-positive BMSCs and C28/I2 cells in the MT-sEV group than in the control and sEV groups.The results demonstrated that MT-sEV had a greater ability to promote cell proliferation than sEV.
Chondrocyte pellets were stained with alcian blue and safranin O to observe the capacity of MT-sEV to induce chondrogenic differentiation and ECM deposition (Figure 5F,G).The staining results of the control group demonstrated successful differentiation of BMSCs into chondrocytes and secretion of ECM.The results also showed that sEV bring about a limited improvement in ECM deposition compared to the control group.In contrast, the MT-sEV group showed intense positive staining with Alcian blue and Safranin O, while the control and sEV groups showed only slight positive staining with both.Correspondingly, MT-sEV promoted both chondrogenic differentiation of BMSCs and ECM deposition by chondrocytes.

Regulation of macrophage activity by MT-sEV
When cartilage is damaged, macrophages are recruited to the joint cavity and participate in the process of cartilage regeneration. 8Lipopolysaccharide was utilized for inducing macrophage polarization toward M1 phenotype, while IL-4 and IL-13 were used to induce macrophage polarization toward M2 phenotype.Immunofluorescence experiments were performed to evaluate the immunomodulatory effects of MT-sEV (Figure 6A,B).The results showed that the sEV group had decreased iNOS expression and increased CD206 expression compared to the control group.Furthermore, MT-sEV displayed a superior capacity to enhance the M2/M1 ratio compared to sEV.These observations were verified by flow cytometry (Figure 6C,D).The results of flow cytometry showed that sEV decreased the proportion of the M1 (F4/80+/CD86+) macrophage type from 46.31% to 39.37% and increased the M2 (F4/80+/CD206+) macrophage type from 33.41% to 63.22%.The F4/80+/CD86+ ratio was 31.50% and the F4/80+/CD206+ ratio reached 79.98% in the MT-sEV group, suggesting that MT-sEV have a stronger capacity to facilitate macrophage polarization toward the M2 phenotype than sEV.

Effects of biomimetic scaffolds loaded with MT-sEV
BMSCs were co-cultured with scaffolds and underwent osteogenic and chondrogenic differentiation, respectively.Immunofluorescence results showed that BMSCs cocultured with scaffolds exhibited higher expression levels of OCN and COL II compared to control group (Figure S8).The therapeutic effects of biomimetic scaffolds loaded with MT-sEV for cartilage regeneration were evaluated by staining with hematoxylin and eosin (H&E), toluidine blue, and safranin O/fast green staining (Figure 7).The results of H&E and toluidine blue staining showed that the regenerated tissue in the control and HG + PCLUW groups contained fibrous tissue and cartilaginous tissues that differed from normal cartilage tissue.In addition, the regenerated tissue did not fill the entirety of the defect region.In contrast, the HG@MT-sEV + PCLUW group exhibited a typical cartilage tissue structure and the regenerated cartilage integrated well with surrounding normal cartilage tissue.Furthermore, a large amount of deposited collagen was observed in the HG@MT-sEV + PCLUW group, as opposed to limited collage deposition in the control and HG + PCLUW groups.Safranin O/Fast green staining demonstrated that there was more regenerated cartilage in the HG@MT-sEV + PCLUW group compared to the control and HG + PCLUW groups.Moreover, the thickness of regenerated cartilage in the HG@MT-sEV + PCLUW group was similar to that of the surrounding native tissue.Immunohistochemical staining of COL II revealed mass collagen deposited in HG@MT-sEV + PCLUW group.However, control and HG + PCLUW groups had limited collage deposition.
In summary, biomimetic multilayer scaffolds loaded with MT-sEV promote cartilage regeneration by stimulating cell migration, promoting cell proliferation and chondrogenic differentiation, facilitating ECM secretion, and regulating the inflammatory microenvironment (Figure 8).

DISCUSSION
Articular cartilage is influenced by the microenvironment and has a poor self-healing capacity due to the lack of vascularity and the small number of chondrocytes.The repair of cartilage defects remains a challenge in tissue engineering.Three-dimensional bioprinting scaffolds with biological functions form a current focus in the study of cartilage regeneration. 46ydrogels have received great attention as promising materials for tissue engineering due to their excellent hydrophilicity and similarity to ECM. 47 Hydrogels have different mechanical and biological characteristics depending on their distinct composition, and they can be mixed with water-soluble drugs and retain their biological activity.48,49 The biomechanical property of hydrogels is considered to be a key factor for cartilage repair.50 Cartilaginous repair tissue can be integrated into the adjacent tissue when the mechanical property of the hydrogel is similar to that of native cartilage tissue.The addition of a polymer and the construction of double-network hydrogels are effective strategies to enhance the mechanical properties of hydrogels.51,52 In this study, an HG doublenetwork hydrogel with superior thermosensitivity was fabricated by mixing GelMA and HAMA hydrogels. Th resulting HG hydrogel retained the advantages of each component but also showed a significant improvement in mechanical properties compared with those of the pure hydrogels, making it a better match to the biomechanical demands of cartilage repair.
Currently, 3D bioprinting is attracting extensive interest in the cartilage regeneration field due to its advantages of precision, complexity, flexibility, and personalized customization. 53The osteochondral interface needs to be considered for the design of osteochondral biomimetic scaffolds due to the different physiological structures and mechanical properties of cartilage and bone. 54Besides, neovascularization during bone defect regeneration might invade the nascent cartilage, which may lead to microenvironmental aberrance, and the development of calcified cartilage and fibrocartilage. 55Therefore, it is necessary to avoid blood vessel invasion during cartilage regeneration. 56Here, a biomimetic multilayer scaffold was fabricated for cartilage regeneration by 3D bioprinting with an alternate printing structure at the osteochondral interface.The cartilage layer and bone layer were made of HG hydrogel and PCLUW, respectively.HG hydrogel and PCLUW were printed alternately at the cartilage-bone interface to prevent blood vessel invasion.
It is well known that sEV are lipid bilayer membrane vesicles secreted by most cell types.Furthermore, sEV carry a variety of cargo, including lipids, proteins, and nucleic acids, which play a role in intracellular communication. 57The membrane and contents of sEV can be modified to efficiently deliver a variety of drugs, which has been a key focus of cell-free therapy in recent years with a broad range of clinical applications. 58Liposomes are also one of the delivery vehicles for drugs, with a spherical structure and lipid molecular layers.In contrast to liposomes, sEV have low immunogenicity and excellent biocompatibility because the components of sEV are similar to the cellular composition.In addition, sEV are well stabilized in tissues and are easily phagocytosed by cells.Modified sEV loaded with specific microRNAs promote chondrogenic differentiation of BMSCs to enhance cartilage repair. 59Previous study showed that sEV loaded with miR-92a-3p inhibited the expression of WNT5A, which enhanced chondrogenesis and suppressed the chondrocyte phenotype. 60In addition, sEV derived from kartogeninpretreated MSCs enhanced chondrocyte anabolism and cartilage regeneration. 61Cartilage regeneration is currently considered to be a complex process that is regulated by multifaceted factors.Multifunctional sEV that regulate diverse cell activities provide a microenvironment suitable for chondrocytes and form one of the hotspots in tissue regeneration.
Sleep is involved in the self-repair mechanism of the human body through maintaining tissue homeostasis, cellular metabolic balance, and regulating inflammation regulation. 62As an important hormone that regulates the circadian rhythm, MT has been demonstrated to directly modulate the expression of diverse circadian clock genes and restore circadian rhythm, which can regulate diverse cellular functions and has potential therapeutic effects on multiple diseases. 63MT may inhibit inflammation and oxidative stress in cartilage to retard osteoarthritis progression and stimulate cartilage regeneration.In addition, MT preserves the proliferation and differentiation properties of BMSCs, enhancing their therapeutic effect. 64In our previous study, we found that MT increased the expression of circRNA3503 and isolated circRNA3503-loaded sEV, which alleviated apoptosis and maintained the balance between synthesis and degradation of ECM. 30 Herein, we obtained sleep-related sEV by combining MT with sEV, and demonstrated that they simulate endogenous tissue repair mechanisms.Considering that RNA and proteins of sEV may play a synergistic role, we explored the role of MT-sEV as a whole complex.Our results confirmed that MT-sEV play a role in promoting cartilage regeneration.
Cell proliferation and migration are factors affecting tissue regeneration.Successful cartilage repair requires stem cell proliferation and migration toward the defect region, which are regulated by multiple factors.Previous studies have found that MT exerts therapeutic effects by enhancing viability and migration of stem cells. 65Our experiments demonstrated that MT-sEV have the potential to promote cartilage regeneration through a synergistic combination of enhanced migration and increased proliferation.
Articular cartilage consists of chondrocytes and ECM secreted by chondrocytes.Chondrocytes, the only cell type present within cartilage, are the key to cartilage defect repair.MSCs migrate to the damaged site and differentiate into chondrocytes after cartilage injury, which is beneficial for cartilage regeneration.MSCs are capable of differentiation into osteoblasts, adipocytes, and chondrocytes and chondrogenic differentiation is regulated by various factors. 66Previous study has demonstrated that sEV isolated from MSCs increase the expression of cartilage ECM components and promote cartilage repair. 67ur experiments have demonstrated that MT-sEV have a stronger ability to promote chondrogenic differentiation of BMSCs and to enhance matrix synthesis of chondrocytes compared with sEV.
Cartilage injury initiates an immune response with activated macrophages secreting cytokines.Pro-inflammatory cytokines secreted by activated M1 macrophages, such as IL-1β, TNF-α, IL-6, disturb ECM metabolic homeostasis by reducing the expression of ECM anabolic markers, including COL II and ACAN, and promoting the expression of proteins associated with ECM catabolism degradation. 68urthermore, these cytokines inhibit chondrocyte migration and enhance chondrocyte apoptosis. 69The secretion of matrix metalloproteinases by M1 macrophages also contributes to ECM degradation. 70On the other hand, M2 macrophages secrete anti-inflammatory cytokines, including IL-10 and transforming growth factor-β, which suppress the expression of pro-inflammatory factors.This alleviates the negative effects of the inflammatory microenvironment on ECM remodeling. 71The anti-inflammatory cytokines induce chondrocytes to increase the expression of COL II and sex determining region Y-box 9, which promotes ECM synthesis and inhibits ECM degradation.As a result, the imbalance in ECM homeostasis is reversed.Additionally, these cytokines play a critical role in inhibiting chondrocyte apoptosis. 72herefore, modulating the immune microenvironment has been considered as a potential approach for cartilage regeneration.Our experiments have demonstrated that MT-sEV inhibit macrophage polarization to M1 but promote macrophage polarization to M2, which enables chondrocytes to maintain the capacity of ECM synthesis and thus promote cartilage regeneration.
In summary, HG double network hydrogel was prepared with excellent mechanical properties.We printed biomimetic multilayer scaffolds by employing 3D bioprinting technology, and show that they had superior biocompatibility.MT-sEV were isolated and we explored the potential molecular mechanisms by which MT-sEV promoted cartilage regeneration.In vitro experiments demonstrated the effects of MT-sEV on promoting cell migration, chondrogenic differentiation and ECM deposition.Furthermore, MT-sEV had the capacity to polarize the macrophage population toward the M2 type.Biomimetic scaffolds containing MT-sEV had the capacity to promote cartilage regeneration, which is a promising therapeutic strategy for the treatment of cartilage defects.

EXPERIMENTAL
All animal experiments were approved by the Animal Care Committee of Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, and followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.All methods can be found in the Supporting Information.

F I G U R E 2
Biocompatibility of the HG@MT-sEV cartilage layer.(A) Schematic illustration of the co-culture experiments.Created with BioRender.com.(B) Scanning electron microscopy (SEM) images of co-culture experiments.The images showed that C28/I2 cells were seeded on a methacrylated hyaluronic acid and gelatin methacryloyl (HG) cartilage layer or an HG@MT-sEV cartilage layer and cultured for 3 days.Scale bar: 200 µm.(C) SEM images of C28/I2 cells cultured on an HG cartilage layer or an HG@MT-sEV cartilage layer for 5 days.Scale bar: 200 µm.(D) The fluorescence images of live/dead assay with calcein-AM (green) and propidium iodide (PI, red).C28/I2 cells were cultured on an HG cartilage layer or an HG@MT-sEV cartilage layer for 3 days.Scale bar: 500 µm.(E) C28/I2 cells were cultured for 5 days and live/dead assay was performed.Scale bar: 500 µm.MT-sEV, small extracellular vesicles secreted by melatonin-pretreated cells.

F I G U R E 3
Identification of differentially expressed genes (DEGs) and analysis of effects on inflammation.(A) Volcano plot of DEGs.(B) Heatmap of clustering analysis.(C) Gene ontology enrichment analysis.(D) The relative mRNA expression of pro-inflammatory and anti-inflammatory cytokines.*p < .05,**p < .01,***p < .001.

F I G U R E 4
Analysis of the effect on cartilage regeneration.(A) Reactome pathway enrichment analysis.(B) Gene set enrichment analysis.(C) The relative mRNA expression of cytokines related to extracellular matrix (ECM) synthesis and degradation.*p < .05,***p < .001,****p < .0001.F I G U R E 5 The effects of small extracellular vesicles secreted by melatonin-pretreated cells (MT-sEV) on cell proliferation, cell migration, and chondrogenic differentiation.(A) Transwell experiment of bone marrow-derived mesenchymal stem cells (BMSCs) was performed to evaluate the effect of MT-sEV on cell migration.Scale bar: 500 µm.(B) Transwell experiment of C28/I2 cells.Scale bar: 500 µm.(C) Schematic representation of the transwell assay.Created with BioRender.com.(D) EdU staining of BMSCs to determine the effect of MT-sEV on cell proliferation.Scale bar: 200 µm.(E) EdU cell proliferation staining of C28/I2 cells.Scale bar: 200 µm.(F) Alcian blue staining of chondrocyte spheres.Scale bar: 200 µm.(G) Safranin O staining of chondrocyte spheres.Scale bar: 200 µm.

F I G U R E 6
The capacity of small extracellular vesicles secreted by melatonin-pretreated cells (MT-sEV) to influence macrophage polarization.(A) Immunofluorescence staining of iNOS was used to identify M1 macrophages.Scale bar: 100 µm.(B) Immunofluorescent staining of CD206 to evaluate the effect of MT-sEV on M2 polarization.Scale bar: 100 µm.(C) Representative flow cytometry contour plots of CD86-positive macrophages.(D) Flow cytometry contour plots of CD206-positive macrophages.F I G U R E 7 Histological analysis of cartilage defect repair in a rabbit model.Hematoxylin & eosin, toluidine blue, safranin O/fast green, and type II collagen (COL II) immunohistochemical staining at 12 weeks.FI G U R E 8 Schematic illustration of biomimetic multilayer scaffolds enhancing osteochondral regeneration.Illustration credit: Lina Cao.

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O N F L I C T O F I N T E R E S T S TAT E M E N TThe authors declare no conflict of interest.D ATA AVA I L A B I L I T Y S TAT E M E N TAll related information in this study is available upon reasonable request.O R C I DShi-Cong Tao https://orcid.org/0000-0002-8706-6138RE F E R E N C E S Conceptualization: Shi-Cong Tao and Shang-Chun Guo.Data curation: Xu-Ran Li, Qing-Song Deng, and Po-Lin Liu.Formal analysis: Xu-Ran Li, Shu-Hang He, and Yuan Gao.Funding acquisition: Shi-Cong Tao and Shang-Chun Guo.Investigation: Xu-Ran Li, Qing-Song Deng, and Po-Lin Liu.Methodology: Xu-Ran Li, Qing-Song Deng, and Po-Lin Liu.Project administration: Shi-Cong Tao and Shang-Chun Guo.Resources: Fei Wang, Chang-Ru Zhang, Zhan-Ying Wei, and Xiao-Qiu Dou.Supervision: Shi-Cong Tao and Shang-Chun Guo.Validation: Xu-Ran Li and Qing-Song Deng.Visualization: Xu-Ran Li.Writingoriginal draft: Xu-Ran Li.Writing-review and editing: Shi-Cong Tao and Helen Dawes.We thank the Institute of Translational Medicine Shanghai Jiao Tong University, National Research Center forTranslational Medicine (Shanghai), Shanghai Institute for Biological Sciences, and the Animal Experimental Center of Shanghai Sixth People's Hospital for assistance.We wish to acknowledge current and previous members of our laboratory.In addition, we thank OE Biotech, Solarbio, Novoprotein, RiboBio, SUNP Biotech, Cyagen Biosciences, and Shiyanjia Lab for providing technical support.We sincerely thank Lina Cao for the design of the schematic diagram.Some illustrations were created with BioRender.com.The present study was supported by the National Natural Science Foundation of China (grant numbers 81802226, 81871834, 82072530, and 82372566), the Shanghai Pujiang Program (grant number 2019PJD038), the Shanghai "Rising Stars of Medical Talent" Youth Development Program (Youth Medical Talents-Specialist Program), the Shanghai Jiao Tong University School of Medicine "Two-Hundred Talent" Program (2022-017), and the Shanghai Sixth People's Hospital Excellent Young Scientist Development Program (ynyq202101).
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