Injectable Photo-cross-linkable Porous Composite Hydrogels with Exosomes for M2 Macrophage Polarization and Cartilage Defect regeneration

Injectable Photo-cross-linkable Porous Composite Hydrogels with Exosomes for M2 Macrophage Polarization and Cartilage Defect regeneration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Injectable Photo-cross-linkable Porous Composite Hydrogels with Exosomes for M2 Macrophage Polarization and Cartilage Defect regeneration Yanchi Bi, Xiao Xiao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5954768/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In the initial stages of osteoarthritis(OA), the deterioration of cartilage in the superficial region results in the formation of superficial cartilage defects. It is anticipated that a therapy based on human umbilical cord mesenchymal stem cell exosomes(hUCMSCs-Exos) will make a notable contribution to the promotion of M2 macrophage polarization and the facilitation of cartilage repair. This study demonstrates that injectable photo-cross-linkable porous gelatin methacryloyl(GelMA)/silk fibroin glycidyl methacrylate(SilMA) hydrogels encapsulating human umbilical cord mesenchymal stem cell exosomes(hUCMSCs-Exos)represent a straightforward and effective approach for cartilage regeneration. The encapsulation of exosomes in hydrogels has been demonstrated to have no significant impact on the physical properties, as evidenced by physical studies. Furthermore, studies based on in vitro cell models and in vivo models in rats have demonstrated that exosomes released from hydrogels can promote M2 polarization, inhibit M1 polarization, and facilitate cartilage regeneration by regulating the PI3K/AKT signaling pathway. This process facilitates the repair of cartilage defects. The findings of our research have led to the development of an injectable photo-cross-linkable hydrogel for superficial cartilage regeneration, which represents a promising minimally invasive treatment for cartilage defects with the aid of arthroscopy. Silk fibroin Exosomes Cartilage defect Immunomodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. INTRODUCTION The prevalence of cartilage defects resulting from injury, osteoarthritis (OA), and other pathological conditions is a significant and growing burden on the healthcare system [ 1 ]. In the initial stages, cartilage defects are classified as partial-thickness cartilage defects (PCD), which are superficial cartilage defects characterised by loss of extracellular matrix and alterations in the metabolism and viability of chondrocytes. This can lead to a worsening of OA and, in severe cases, complete joint destruction [ 2 ]. However, cartilage is unable to regenerate due to the absence of blood vessels and the limited proliferation capacity of chondrocytes[ 1 ]. Despite recent advances in microfracture techniques and cell-based cartilage regeneration therapies, there is still no effective treatment for early-stage cartilage defects[ 3 ], [ 4 ]. Consequently, there is an urgent need to develop advanced tissue-engineered biomaterials for the regeneration of superficial cartilage defects. In a clinical context, mesenchymal stem cells (MSCs) have demonstrated potential for the regeneration of cartilage defects due to their capacity to rejuvenate and to reduce inflammation[ 5 ]. Among the various types of MSCs, human umbilical cord-derived MSCs (hUCMSCs) are of particular interest due to their non-invasive availability and robust regenerative capacity[ 6 ]. Despite the favourable safety and efficacy profile of hUCMSCs in cartilage defect regeneration, the lack of control and regulation of their in vivo growth and differentiation presents a significant challenge to the standardisation of clinical translation[ 7 ]. Moreover, there is mounting evidence that the therapeutic effects of hUCMSCs are predominantly attributable to paracrine effects. Additionally, it has been demonstrated that hUCMSC-derived exosomes (hUCMSC-EXO) can facilitate cartilage regeneration, upregulate the expression of cartilage-specific genes and glycosaminoglycan synthesis, stimulate the secretion of anti-inflammatory factors, and promote cell mitosis and migration [ 8 ]. The current administration of hUCMSC-EXO is via injection. However, electrostatic repulsion induces a strong tendency for exosomes to distribute in synovial fluid, resulting in low cartilage retention and insufficient therapeutic effects[ 9 ]. Moreover, the recovery of cartilage lesions usually takes a considerable length of time, but the half-life of exosomes in the body is relatively short, and they often cannot continuously promote the repair of cartilage defects[ 10 ]. Therefore, it is necessary to develop methods for fixation and sustained-release systems to ensure that exosomes exert the maximum therapeutic effect. The recent advancements in regenerative medicine and tissue engineering have paved the way for the utilisation of sophisticated biomaterials in the treatment of cartilage defects[ 11 ]. Hydrogels are biocompatible and possess loose and porous structural properties, which render them suitable for use as exosome carriers, thereby prolonging the retention time in specific areas and regulating the release of exosomes[ 12 ]. Concurrently, hydrogel implantation and injection represent the primary strategies for contemporary cartilage repair, offering a foundation for the utilisation of exosomes in situ for cartilage and osteochondral interface reconstruction[ 13 ]. Recent studies have demonstrated that the encapsulation of MSC-EXOs in hydrogels effectively results in efficient and sustained delivery for tissue engineering applications[ 14 ]. Furthermore, Zhang et al. demonstrated that injectable mussel-inspired high-adhesion hydrogels loaded with exosomes can promote in situ cartilage and ECM regeneration in rats[ 15 ]. Furthermore, thermosensitive hydrogels loaded with exosomes derived from M2 macrophages have been demonstrated to facilitate cartilage repair by stimulating synovial lymphangiogenesis[ 16 ]. This suggests that hydrogels can be employed as a fixed and sustained-release vector for exosomes in cartilage repair. Silk fibroin (SF)-based hydrogels have garnered considerable interest due to their straightforward processing, multifunctional functionalisation, high water content and three-dimensional porous structure that closely resembles that of natural ECM[ 17 ]. Furthermore, functionalised SF hydrogels have been demonstrated to regulate the migration, proliferation and differentiation of stem cells[ 18 ]. Nevertheless, SF-glycidyl methacrylate (SilMA) is not an appropriate choice for direct use in cartilage regeneration due to its lack of larger pores at the micro or macro scale. Previous studies have demonstrated that gelatin methacrylate (GelMA), which exhibits a highly porous structure, can be readily fabricated and that porous GelMA/SilMA hydrogels can facilitate epithelial sealing[ 19 ]and ocular surface reconstruction[ 20 ]. Despite the high biocompatibility and capacity for cell growth promotion observed in porous GelMA/SilMA hydrogels, their potential as a fixed and sustained-release carrier for exosomes and as a promoter of cartilage defect repair remains to be confirmed. The objective of this study was to develop an injectable photocurable porous GelMA/SilMA hydrogel encapsulating exosomes (PSE) for the promotion of cartilage defect regeneration and M2 polarization of macrophages. The characteristics of the PSE hydrogel were evaluated to ascertain its suitability for cartilage regeneration. The biological effects of the PSE hydrogel in promoting the regeneration of damaged cartilage cells and the polarization of M2 macrophages were confirmed in vitro cell experiments and in vivo rat cartilage defect models. Furthermore, the chemokine signalling pathway of the PSE hydrogel in promoting the repair of damaged cartilage cells was also investigated. 2. MATERIALS AND METHODS 2.1 Materials GelMA (EFL-GM-PR-001), SilMA (EFL-SilMA-001) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP) were purchased from EFL (Suzhou, China). Rat Chondrocyte Cells, hUCMSCs, RAW 264.7, and other cell culture-related reagents were purchased from Procell(Wuhan,China). The primary antibodies and live/dead cell double staining kit were purchased from Abcam (Cambridge, USA).The secondary antibodies were purchased from Invitrogen (Carlsbad, USA). A CCK-8 assay kit was obtained from GLPBIO(Montclair, CA,USA). All other solvents and agents were of analytical grade and used without further purification. 2.2 Isolation and identification of exosomes When hUCMSCs reached 80–90% confluence, they were washed and incubated for another 48h in serum-free medium. The medium was collected and centrifuged at 300×g for 15 min at 4°C and then at 2500×g for 15 min. After centrifugation, the supernatant was filtered using a 0.22 µm filter to remove the remaining cells and debris. Next, a 15-mL Amicon Ultra-15 Centrifugal Filter Unit (Merck Millipore) was used to transfer the filtered solution. The solution was centrifuged at 4000×g until the volume in the upper compartment was concentrated to approximately 200 uL. The ultrafiltered solution was washed with phosphate-buffered saline (PBS) and centrifugation was repeated three times. The washed ultrafiltration liquid containing exosomes was placed on a 30% sucrose/D2O cushion in an Ultra-Clear™ tube (Beckman Coulter, Brea, CA) and ultra-centrifuged for 1 h at 100,000×g. The pellets were resuspended in PBS and centrifuged at 4000×g to concentrate the volume to approximately 200 µL. Transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA) with a Zetaview (Particle Metrix, Munich, Germany) was performed for the exosome size distribution measurement. Exosome size was calculated using NTA software (Malvern, Shanghai, China). The proteins were subsequently used for Western blot analysis for TSG101, Alix, CD9 and Calnexin markers to detect exosome components after protein extraction. In order to analyze the effect of these radicals from H2O2 on the exosomes structural integrity, 1 mL exosomes (200 µg/mL) were treated with 20 µl HRP (5 mg/mL) and 20 µl H2O2 (0.5% w/v) in 26°C for 24 h, followed by the NTA experiments. 2.3 Preparation of P/S6 hydrogels and PSE hydrogels According to the manufacturer’s instructions, 0.6 g of freezedried porous GelMA was dissolved in 10 mL of PBS to generate a homogeneous 6% w/v homogeneous solution with continuous stirring in a water bath in the dark at 37°C for 1 h. Similarly, freezedried SilMA was dissolved in PBS with 0.25% LAP at room temperature for 1 h at a concentration of 20% (w/v).Finally, the 6% w/v porous GelMA solution was uniformly mixed with the 20% w/v SilMA solution at feed ratios of 6:1 to give the samples P/S6. For the hydrogel used in the cell and animal experiments, a hydrogel with exosomes (200 µg/mL) was prepared, where the exosomes were mixed with P/S6 solution to form the PSE hydrogel. All steps were performed under sterile conditions, and the prepared solutions were sterilized using a 0.22 µm filter. The hydrogel precursors were exposed to visible light (405 nm, 30 mW/cm2 ) from a light source for 30 s to form the hydrogel. For transparency, swelling ratio (SR) and degradation tests, the P/S6 hydrogel precursors(120ul) and the PSE hydrogel precursors(120µL) were injected into cylindrical molds (10 mm diameter; 2 mm height) to form disc-shaped hydrogels. 2.4 Characterization of the P/S6 hydrogel and the PSE hydrogel For morphological analysis, a Hitachi SU 8010 (Hitachi, Tokyo, Japan) was used for cryo-field emission scanning electron microscopy (FE-SEM). Dynamic structural changes were identified using Fourier transform infrared spectroscopy (FTIR) on a Nicolet FTIR 6700 spectrometer (Thermo Fisher, Massachusetts, USA). The compressive strength of the hydrogels was determined using DMA (DMA, Q800, TA Instruments, USA) to calculate the stress-strain curve of the hydrogels. In addition, the storage modulus (G') and loss modulus (G'') of the hydrogels were measured using rheological tests (Physica MCR301, Anton Paar) at a fixed strain (5%) and dynamic oscillation frequency (10 − 0.1 Hz). In order to determine swelling ratio (SR), 200 µL hydrogels were exposed to blue light for 20 s and then incubated in PBS at 37°C. The swollen weight (Ws) and the dry weight (Wd) was measured at each indicated time point (0,1, 2, 4, 6, 8, and 10 h). The SR was calculated according to the following equation: $$\:SR=\frac{Ws-Wd}{Wd}$$ In the context of in vitro degradation tests, the initial dry weight (Wo) of the hydrogels was ascertained by weighing them prior to immersion. This was followed by the immersion of these hydrogels into a solution of PBS containing lysozyme (1mg/mL, GLPBIO) at 37 degrees Celsius for 21 days. At the designated time points (7, 14 and 21 days), the samples were lyophilised and weighed (Wt). The degradation ratio was subsequently calculated using the following formula: $$\:Degradation（\%）＝\frac{Wt}{Wo}\times\:100\%$$ The cumulative and daily release profiles were evaluated using a bicinchoninic acid (BCA) reagent test kit (Beyotime). The PSE hydrogels were incubated at 37°C in PBS. The supernatant was collected on days 1,3,5, 7,10, 14and 21 for free exosome (Exo) detection using BCA to assess the release profile of Exo from the PSE. 2.5 In vitro studies 2.5.1 In vitro biocompatibility assessment of P/S6 and PSE hydrogels In vitro biocompatibility was evaluated through the implementation of cell viability and proliferation experiments. To conduct the Live/Dead staining test, chondrocytes at a density of 1×10⁶ were seeded into 12-well culture plates and co-incubated with P/S6 or PSE hydrogel extracts for 24 hours. The Live/Dead solution was then prepared in the ratio of 1 ml: 3 µl: 5 µl (PBS: calcein-AM(Invitrogen): PI(Invitrogen)) and added in each group and incubation was done for 30 min at 37 ℃. After 1×106 chondrocytes were co-cultured with P/S6 or PSE hydrogel extracts for 1, 3, and 5 days, 100 µl/ml CCK-8 solution (GLPBIO) was added to each well and incubated for 2 h. Subsequently, 100 µl of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 450 nm with a microplate analyser (BioTech). 2.5.2 Co-culture of chondrocytes and RAW264.7 cells with hydrogel The DMEM basal medium was soaked with a 20% volume fraction of P/S6 and PSE hydrogel for a period of 24 hours in order to prepare hydrogel extracts. Chondrocytes (P1 generation) were seeded into 6-well plates and treated with IL-1β (10 ng/ml) for 24 hours, thereby creating an in vitro model of damaged chondrocytes. The damaged chondrocytes were then cultured with P/S6 and PSE hydrogel extracts for three days. Similarly, RAW264.7 cells were seeded onto 6-well plates and cultured with P/S6 and PSE hydrogel extract for three days. The cell culture medium was prepared as follows: the hydrogel extract was extracted with DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum. 2.5.3 Immunofluorescence The samples were fixed with 4% paraformaldehyde in PBS for a period of 30 minutes. Subsequently, the cells or tissues were treated with 0.2% Triton X-100 (Beyotime) for 10 minutes and blocked with 3% bovine serum albumin (BSA, Beyotime) for one hour at room temperature. The samples were incubated at 4°C overnight with the primary antibodies. Subsequently, the sample was washed three times with PBS and incubated with the secondary antibody for a period of 2 hours at room temperature. Subsequently, the sample was subjected to Hoechst (Beyotime) staining. Subsequently, the stained samples were imaged using a confocal microscope (Leica). The results were analysed using the ImageJ software. 2.5.4 Gene expression Total cellular RNA was extracted using an RNA extraction kit (Vazyme) and subsequently reverse-transcribed into cDNA using an EVO-MLV RT kit (Vazyme). qRT-PCR analysis was conducted using LightCycler 480 SYBR Green Master Mix (Vazyme). The endogenous control employed was the reference gene GAPDH. The relative gene expression was quantified using the 2-ΔΔCt method. The experiment was conducted in triplicate. 2.5.5 Western Blot The cells were homogenised in RIPA buffer (CWBIO) containing protease and phosphatase inhibitors (Thermo Fisher) in order to create a cell homogenate. The supernatants were then lysed on ice for 30 minutes and subsequently collected by centrifuging at 12,000 rpm at 4°C for a further 30 minutes. The total protein concentration was determined using the BCA kit (Beyotime). The supernatants were mixed with loading buffer (Beyotime), and equal amounts (40 µg) of protein were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in accordance with the manufacturer's instructions. Subsequently, the separated protein was transferred to a polyvinylidene difluoride (PVDF, Thermo Fisher) membrane, which was then blocked with 5% skim milk for one hour. Thereafter, the membrane was incubated with primary antibodies overnight. Subsequently, the PVDF membrane was incubated with the secondary antibody for one hour prior to the application of the enhanced chemiluminescence (ECL) kit (Thermo Fisher) for visualisation. Prior to each subsequent step, the membranes were washed thrice with Tris-Buffered Saline with Tween® 20 (TBST). The results were analysed using the ImageJ software. 2.6 In vivo studies 2.6.1 Ethics statement The research involving animal subjects was approved by the Animal Experiment Ethics Committee of Qingdao Municipal Hospital. All experimental procedures on animals were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. 2.6.2 Osteochondral defect models As previously described in the literature, 3-month-old Sprague Dawley rats (n = 20) were randomly divided into three groups: control, P/S6 and PSE groups, with each group containing rats weighing 200–250 g. Following anaesthesia, the rats were shaved and disinfected, and then their right knee joints were exposed. Subsequently, a 2 mm diameter and 1 mm height hole was created in the centre of the trochlear groove using a drill. In order to compare the effects of the different experimental groups on cartilage defect regeneration, the control group was treated with PBS, while the other two experimental groups were treated with P/S6 hydrogel and PSE hydrogel, respectively. In the P/S6 and PSE groups, the hydrogel was introduced into the defect via syringe injection. At the 6th and 10th weeks post-surgery, the rats were euthanised and subjected to histological analysis. 2.6.3 Histological analysis and immunohistochemistry At 6 and 10 weeks, the rats were sacrificed and whole femurs were collected. Samples were fixed in 4% paraformaldehyde (pH 7.5) for 1 day and decalcified in decalcifying solution for 21 days, the samples were then embedded in paraffin and cut into 5 µm sections. Three specimens were randomly selected from each group at 6 and 10 weeks for histological analysis. The specimens were soaked in 10% formalin overnight, decalcified, embedded in paraffin, and sectioned for staining. The specimens were stained with hematoxylin and eosin (H&E) and Safranin O/Fast Green. Three specimens were randomly selected from each group at 6 weeks for immunohistochemistry. The rabbit anti-COL-2 (Abcam), MMP-13 (Abcam), Arg-1 (Gentex), and CD206 (Gentex) primary antibodies were used for immunohistochemistry. The results were analysed using the ImageJ software. 2.7 Transcriptome Analysis The in vitro function of the PSE hydrogel was analysed by transcriptome analysis of IL-1β-treated chondrocytes co-cultured with the hydrogel, conducted by Novogene (Beijing, China). Total RNA was extracted using TRIzol reagent. RNA purity and quantification were assessed using a NanoDrop 2000 spectrophotometer, and RNA integrity was assessed using an Agilent 2100 bioanalyzer. The sequencing libraries were then constructed using the VAHTS Universal V6 RNA-seq Library Preparation kit according to the manufacturer's instructions. Transcriptome sequencing and analysis were performed by Novogene (Beijing, China). The libraries were sequenced on the Illumina Novaseq 6000 platform and 150 bp paired-end reads were generated. Raw readings were generated for each sample. Raw readings in fastq format were first processed using fastp1, and low-quality data were removed to obtain clean readings. Clean readings from each sample were retained for subsequent analysis. Clean readings were mapped to the reference genome using HISAT22. The FPKM3 of each gene was calculated, and the read counts of each gene were obtained by HTSeq-count4. PCA analysis was performed using R (v 3.2.0) to assess the bio-reproducibility of the samples, and DESeq25 was used for differential expression analysis. A q-value 2.0 was used as the threshold for significantly differentially expressed genes (DEGs). 2.8 Statistical analysis The results presented are representative of at least three independent experiments and are expressed as mean ± standard error of the mean (SEM). The student's t-test and paired ANOVA with a post-hoc test (GraphPad Prism 9.0) were used to determine the statistical significance between the intervention and control groups. A one-way analysis of variance model was used to compare multiple groups. The significance level was set at 0.05. 3. RESULTS AND DISCUSSION 3.1 Characterization of hUCMSC-EXOs To confirm the isolation of hUCMSCs-derived exosomes, transmission electron microscopy (TEM) was employed to obtain micrographs, which revealed a round or elliptical morphology with a complete cell membrane structure (Fig. 1 A, B, C). Furthermore, the qNano® system was employed for quantification and size profiling. The particle size distribution was found to be predominantly around 100 nm, consistent with that of exosomal dimensions (Fig. 1 D). Additionally, Western blotting analysis demonstrated that the nanoparticles expressed the exosomal-specific markers CD9 and TSG101 (Fig. 1 E). Therefore, the aforementioned results confirm that the exosomes were successfully isolated from hUCMSCs. 3.2 Synthesis and characterization of the porous hydrogels with and without exosomes A mixture of porous GelMA/SilMA hydrogel and exosomes was prepared under ultraviolet irradiation with a photoinitiator. A scanning electron microscope (SEM) was employed to examine the structural morphology of the porous GelMA/SilMA hydrogel following the freeze-drying process. The SEM images revealed the presence of a highly porous structure and an interconnected network throughout the sample. Scanning electron microscopy (SEM) imaging demonstrated that the hydrogel exhibited a porous network structure (Fig. 2 A), and that the exosomes were attached to the internal surface of the porous hydrogel (Fig. 2 B). It has been demonstrated that the highly porous structure can provide a favourable microenvironment for cell adhesion, proliferation and migration . The strain-stress curves of the hydrogels demonstrate that when the strain reaches 75%, the compressive strength of the PSE hydrogel is 0.41 MPa, and the compressive strength of the P/S6 hydrogel is 0.46 MPa. This indicates that both hydrogels meet the compressive strength of human cartilage, as previously reported[ 21 ]. The addition of exosomes has been observed to have a minimal effect on the mechanical properties of the hydrogels. (Fig. 2 C) The results of the rheological tests demonstrated that the storage modulus (G') of P/S6 and PSE was significantly higher than the loss modulus (G''), indicating that they exhibited the characteristics of stable viscoelastic solids[ 22 ]. Furthermore, the discrepancy between the mean storage modulus of PSE and P/S6 was not substantial, suggesting that the incorporation of exosomes did not alter the mechanical characteristics of the hydrogel (Fig. 2 D). The secondary structural changes of the hydrogels were analysed using FTIR. Following the addition of exosomes, the FTIR spectra of the PSE and P/S6 hydrogels exhibited no shift at 1650 cm-1, indicating that the incorporation of exosomes did not result in significant structural alterations in the hydrogels (Fig. 2 E). Furthermore, the swelling ratio and biodegradation rate are of significant importance with regard to the practical application of the material. Following a 60-minute immersion in PBS, the swelling ratio of the PSE hydrogel and P/S6 hydrogel exhibited a marked increase, reaching values exceeding 6 (Fig. 2 F). Subsequently, a 21-day enzymatic degradation study was conducted using lysozyme-containing PBS to evaluate the applicability of the PSE hydrogel and P/S6 hydrogel. Both hydrogel samples exhibited relatively rapid degradation during the initial seven-day period, followed by a gradual deceleration over time (Fig. 2 G). Concurrently, BCA detection demonstrated that the PSE hydrogel could sustain continuous exosome release, with approximately 85% of the exosomes released into PBS for at least 14 days (Fig. 2 H), aligning with the biodegradation curve of the hydrogel and achieving the desired slow release of exosomes. 3.3 Biocompatibility of PSE and P/S6 hydrogels The results of the live/dead staining demonstrated that following a period of one day in culture within the P/S6 and PSE hydrogel extracts, the majority of rat chondrocytes exhibited green fluorescence, with only a minimal number displaying red fluorescence. This suggests that the hydrogels are biocompatible (Fig. 5 A). A semi-quantitative analysis utilising a live/dead assay demonstrated that the survival rate of cells in the P/S6 and PSE hydrogel extracts exceeded 90%, with the cells exhibiting high vitality (Fig. 5 B). Furthermore, a CCK-8 assay was conducted to assess the proliferative capacity of chondrocytes in P/S6 and PSE hydrogel extracts. The CCK-8 results demonstrated that the cell proliferation rate increased with the incubation time, indicating that the cells were undergoing both growth and proliferation (Fig. 5 C). Previous studies have also yielded analogous findings, indicating that the incorporation of an optimal amount of silk fibroin can enhance the proliferative effect[ 23 ]. 3.4 PSE hydrogel promotes the repair of IL-1β-damaged chondrocytes In order to evaluate the efficacy of PSE hydrogel in repairing cartilage, cartilage cells that had been pretreated with IL-1β were cultured in hydrogel extract for a period of three days. Following this, the cells were analysed using immunofluorescence, qPCR and western blot. Additionally, a control group was constituted by untreated healthy cartilage cells cultured in hydrogel extract for three days. The extracellular matrix (ECM) of cartilage is primarily composed of proteoglycans and collagen II. Immunofluorescence images demonstrate that the PSE group exhibited elevated levels of immunofluorescence for aggregated proteoglycans and collagen II. The expression of the cartilage-forming gene SOX-9 was markedly elevated in comparison to the control group, thereby indicating that the exosomes present within the hydrogel enhanced the efficacy of cartilage differentiation (Fig. 4 A). Furthermore, the immunofluorescence images demonstrated that the expression of MMP-13 was diminished in the PSE group. MMP-13 is not only a key matrix-degrading enzyme in chondrocytes, but its expression level is also frequently employed as an indicator of the severity of cartilage damage. This not only corroborates the therapeutic efficacy of PSE on damaged chondrocytes, but also its prospective advantages for preserving the structure of articular cartilage in patients with damaged cartilage (Fig. 4 A). The aforementioned conclusion was corroborated by the semi-quantitative analysis of the immunofluorescence images (Fig. 4 B). Furthermore, qPCR analysis revealed that the expression levels of ACAN, SOX-9 and COL-II genes were significantly elevated in the PSE group compared to the P/S6 group. Conversely, the expression level of the MMP13 gene was significantly reduced. These findings suggest that Exos can mitigate the detrimental effects of IL-1β on chondrocytes, and that porous hydrogels combined with Exos can enhance the repair of damaged chondrocytes (Fig. 4 C). Western blot analysis also demonstrated that PSE hydrogels exhibited a markedly superior repair efficacy for damaged cartilage, exceeding that of P/S6 hydrogels. This finding aligns with the qPCR results, which indicated that the combined use of Exos can further enhance the repair of damaged chondrocytes (Fig. 4 C). Western blot results also demonstrated that the repair effect of PSE hydrogel on damaged cartilage was significant and markedly superior to that of P/S6 hydrogel, which was consistent with the qPCR results (Fig. 4 D, E). These findings collectively suggest that the PSE hydrogel can directly exert a robust repair effect on damaged chondrocytes. 3.5 PSE hydrogel effectively promotes the polarization of M2 macrophages in vitro Following damage to cartilage, the inflammatory response at the injury site disrupts the anabolic and catabolic balance of chondrocytes, which in turn results in the degradation of the ECM via matrix-degrading enzymes[ 24 ]. Given that macrophages are the primary mediators of inflammation following injury, we sought to examine the polarising impact of PSE hydrogel on RAW264.7 cells in an in vitro setting. The expression of relevant inflammatory molecules was then detected by immunofluorescence and qPCR in RAW264.7 cells cultured in P/S6 and PSE hydrogel extracts, with the aim of verifying the immunomodulatory properties of PSE hydrogel. On day 3, the immunofluorescence results demonstrated that the fluorescence intensity of the M2 phenotype markers Arg-1 and CD206 in the PSE group was markedly higher than that in the control group and the P/S6 group. This suggests that the hUCMSC-EXOs loaded in the hydrogel facilitated the M2 polarization of macrophages (Fig. 5 A, B). Furthermore, the qPCR results on day 3 demonstrated that the PSE hydrogel markedly elevated the expression levels of Arg-1 and CD206, whereas the expression level of iNOS was diminished. Exosome vesicles are nano-scale vesicles containing nucleic acids, proteins and other components. They primarily exert their biological effects by releasing active ingredients extracellularly for cell communication and transmission[ 25 ]. A substantial body of evidence has demonstrated that MSC-derived exosomes are a rich source of molecules that regulate immunity, including mRNA, miRNA, cytokines and chemokines, which play a pivotal role in modulating the phenotype and function of immune cells[ 26 ]. The results demonstrated that hUCMSC-Exos exert a robust immunomodulatory impact by facilitating the M2 polarization of macrophages, which may prove advantageous for the repair of damaged cartilage. 3.6 PSE promotes cartilage repair in the knee joints of rats and regulates the polarization of macrophages A rat patellar cartilage defect was created for the purpose of evaluating the efficacy of cartilage regeneration and the inflammatory response following the injection of PSE hydrogel. At 6 and 12 weeks post-surgery, the distal femurs were collected for gross observations and histological staining analysis (Fig. 6 A). The gross analysis conducted at 6 and 12 weeks revealed that the cartilage surface in the PSE hydrogel group exhibited a smoother and more continuous appearance than that observed in the control and P/S6 groups (Fig. 6 B). Histological changes to the cartilage were analysed using H&E and Safranin O/Fast Green staining. In contrast to the control group, in which the patellar groove defect was filled with new fibrous tissue and minimal cartilage formation was observed, the PSE hydrogel treatment group demonstrated notable cartilage formation in the defect area at six weeks postoperatively and a smooth surface at 10 weeks. It is noteworthy that the boundary between the regenerated cartilage and the native tissue was rarely discernible in the PSE group, suggesting the formation of hyaline cartilage in situ (Fig. 6 C,D). The immunochemical analysis of ECM expression in defective cartilage demonstrated that the PSE hydrogel is capable of promoting the regeneration of superficial cartilage defects in the knee joint. The PSE group exhibited elevated collagen II expression and diminished MMP-13 expression relative to the control and P/S6 groups (Fig. 7 A). These findings indicate that PSE hydrogel not only inhibits the expression of MMP-13 in chondrocytes, but also significantly promotes the expression of collagen type II in chondrocytes. This suggests that PSE hydrogel can promote anabolic rather than catabolic metabolism in chondrocytes by directly supplementing ECM. Furthermore, in comparison to the P/S6 hydrogel treatment group, the PSE hydrogel demonstrated a more pronounced promotion of COL II expression, thereby substantiating the chondroprotective effects and cartilage regeneration potential of hUCMC-derived Exos. To confirm that PSE hydrogel can modulate the inflammatory response following growth plate injury in vivo, the macrophage phenotype polarization was detected using the unique markers CD206 and Arg-1 in the injured area. As observed in the in vitro studies, the expression of CD206 and Arg-1 at the injury site was higher in the PSE group than in the control and P/S6 groups (Fig. 7 B,C). This suggests that PSE is capable of inducing M2 polarization of macrophages at the injury site. The role of immunoreactivity in the context of tissue injury and repair, particularly in chondrocytes, is of paramount importance. Pro-inflammatory immune cells produce inflammatory cytokines, including IL-1β, iNOS and TNF-α, which are rapidly upregulated, leading to overexpression of matrix metalloproteinases, such as MMP-1 and MMP-13. This, in turn, results in chondrocyte apoptosis and matrix degradation following cartilage injury[ 27 ]. M2 macrophages, also referred to as wound-healing macrophages, are capable of creating an anti-inflammatory environment that is conducive to tissue repair and remodeling[ 28 ]. It has been demonstrated in previous studies that the injection of an M2 macrophage activator into the joint cavity of animals with osteoarthritis can prevent the formation of osteophytes and slow the progression of cartilage damage[ 29 ]. The injection of PSE hydrogel at the injury site resulted in the induction of a higher expression of anti-inflammatory factors, thereby regulating the immune microenvironment and promoting the survival of chondrocytes and cartilage regeneration. 3.7 Transcriptome analysis reveals the mechanism by which PSE hydrogel promotes cartilage cell repair The cells were divided into three groups: a control group comprising healthy chondrocytes (WT), an IL-1β + P/S6 group (KO), and an IL-1β + PSE group (OE). Transcriptomics was employed to elucidate the potential mechanism through which the PSE hydrogel affects the regeneration of damaged cartilage. To assess the stability of the three groups, Pearson correlation and principal component analysis were conducted (Fig. 8 A and B). The results demonstrated that the correlation coefficients of the specimens in each group were within an acceptable range (R2 > 0.95, n = 3), indicating good biological reproducibility and that the transcriptome data could be utilised for further analysis. Subsequently, a Venn diagram and heat map analysis were conducted between the three groups (Fig. 8 C, D), which demonstrated the overlap of differentially expressed genes and cluster analysis between different comparison groups. This allowed for the subsequent analysis of differentially expressed genes to be carried out. Furthermore, a volcano plot analysis of differentially expressed genes (DEGs) was conducted (Fig. 8 E). In comparison to the P/S6 group, the PSE group exhibited upregulation of 946 genes and downregulation of 1,394 genes. Among the differentially expressed genes, the MMP13 gene exhibited decreased expression in the PSE group, whereas the ACAN and COL II genes demonstrated significant upregulation. These findings align with the results of previous cell and animal experiments. This provides further evidence that the PSE hydrogel has a protective effect on chondrocytes and is capable of promoting cartilage regeneration. To gain further insight into the mechanism by which PSE hydrogel exerts its chondroprotective effect, we conducted the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed genes (Fig. 8 F). The top enriched up-KEGG pathways associated with the PSE and P/S6 groups were analysed to ascertain the mechanisms by which the PSE hydrogel exerts its chondroprotective effects. The results demonstrated that the PI3K/AKT signalling pathway and ECM-receptor interaction are activated by the PSE hydrogel, which participates in the production or synthesis of ECM. This includes the positive regulation of extracellular matrix containing collagen, collagen binding and collagen biosynthesis processes, and the promotion of ECM regeneration. In conclusion, it can be posited that PSE hydrogel has the capacity to regulate cell signalling and facilitate the regeneration of cartilage defects. However, further research is required to elucidate the underlying mechanisms in greater detail. 4. CONCLUSIONS In this study, a porous GelMA/SilMA hydrogel loaded with hUCMSC-Exos (PSE hydrogel) was prepared, which exhibited excellent physical properties and biocompatibility. The PSE hydrogel was observed to modulate the immune microenvironment, promote M2 macrophage polarization, and protect damaged cartilage by activating cell communication signal pathways, thereby facilitating the repair of cartilage defects in the rat patellar groove. In light of these findings, this study has resulted in the creation of a promising biomaterial for patients with superficial cartilage defects, which can be injected under arthroscopic surgery. Declarations Author Contributions Yanchi Bi contributed to the conception and design of the study. Liang Zhu wrote the manuscript. Haibo Zhao collected data for the work. XX, YTB supervised the manuscript. All authors read and approved the final manuscript.. Funding This work was supported by the Shandong Provincial Key R&D program [Science and Technology Demonstration Project] in 2021 [grant no. 2021SFGC0502]; the Qingdao Science and Technology Plan Project Science and Technology Benefiting the People Demonstration and Guidance Project in 2022 [grant no. 22-3-7-smjk-5-nsh]; Qingdao Shinan District Science and Technology Bureau public domain science and technology support plan project [grant no.2023-2-011-YY]. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Acknowledgments We are grateful for all the research applied to this article. Conflicts of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Y. 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[Online]. Available: https://pubmed.ncbi.nlm.nih.gov/31231454/ H. Zhang et al. , ‘Synovial macrophage M1 polarisation exacerbates experimental osteoarthritis partially through R-spondin-2’, Ann Rheum Dis , vol. 77, no. 10, pp. 1524–1534, Oct. 2018, doi: 10.1136/annrheumdis-2018-213450. A. J. Nixon et al. , ‘Disease-Modifying Osteoarthritis Treatment With Interleukin-1 Receptor Antagonist Gene Therapy in Small and Large Animal Models’, Arthritis Rheumatol , vol. 70, no. 11, pp. 1757–1768, Nov. 2018, doi: 10.1002/art.40668. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.jpeg Abstract. Injectable Photo-cross-linkable Porous Composite Hydrogels with Exosomes for M2 Macrophage Polarization and Cartilage Defect regeneration Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5954768","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":411484638,"identity":"e9c67975-2c50-4b93-a18e-3094629c8978","order_by":0,"name":"Yanchi Bi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYBACA2YwdYCHn3Qtkg1Ea4FQBxgMDhCrxZydx/BzQc0dGePjyRsYflRsI6zFspnHWHrGsWc8ZmeeFTD2nLlNhMMO8xhI8zYc5jG7kWPAzNhGnBbj3yAtxjNI0GIGtsVAglgtls1sZdY8xw7zSAD9cpAov5jzH958m6fmsD1/e/LGBz8qiNDCwMABjRqGBKKjhv0BXAuROkbBKBgFo2CkAQDEVTk0SA+oGwAAAABJRU5ErkJggg==","orcid":"","institution":"Qingdao University","correspondingAuthor":true,"prefix":"","firstName":"Yanchi","middleName":"","lastName":"Bi","suffix":""},{"id":411484640,"identity":"6a4ed6ea-69e3-4dd8-b545-b3123450fc37","order_by":1,"name":"Xiao Xiao","email":"","orcid":"","institution":"Central Laboratories, University of Health and Rehabilitation Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2025-02-04 04:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5954768/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5954768/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75694975,"identity":"e5557692-65b3-4b59-bed7-adeb3e73ccd9","added_by":"auto","created_at":"2025-02-07 08:07:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":251099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation and identification of hUCMSC-EXOs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B, C) Representative TEM images of hUCMSC-EXOs. (D) NTA results of hUCMSC-EXOs. (E) Western blot analysis of CD9 and TSG101 in hUCMSC-EXOs.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5954768/v1/df4d0af88c519a0b2e2f6227.png"},{"id":75694974,"identity":"c1f03c03-5a65-405e-ab4e-8f82ff70b783","added_by":"auto","created_at":"2025-02-07 08:07:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":286774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of PSE and P/S6 hydrogels.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) SEM image of the PSE and P/S6 hydrogels. (C) Strain-stress curves of PSE and P/S6 hydrogels. (D) Rheological analysis of PSE and P/S6 hydrogels. (E) FTIR spectra of PSE and P/S6 hydrogels. (F) Swelling ratio of PSE and P/S6 hydrogels. (G) Degradation curves of PSE and P/S6 hydrogels. (H) Cumulative release curve of exosomes from PSE hydrogels. (n=3).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5954768/v1/266eb5c123ee6bd5115c4be0.png"},{"id":75695726,"identity":"92520ad6-1d7c-46a1-bd3a-6fab06e99b48","added_by":"auto","created_at":"2025-02-07 08:15:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":353325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChondrocyte viability and proliferation in P/S6 and PSE hydrogel extracts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Live/dead assay showing chondrocyte viability in P/S6 and PSE hydrogels. Scale bar = 100 μm. (B) Semi-quantitative analysis of chondrocyte viability by live/dead assay. (C) CCK-8 assay measured after 1, 3 and 5 days of cell culture in each group (n = 3). Data are expressed as mean ± SD, n = 3, analysed using ANOVA and Tukey's multiple comparison test).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5954768/v1/b944671220331f0e2286531c.png"},{"id":75694769,"identity":"b26bbfb7-2f48-4400-bc3d-d4735a6f1a2b","added_by":"auto","created_at":"2025-02-07 07:59:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":553187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePSE hydrogel promotes the repair of damaged cartilage cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The immunofluorescence staining intensity of ACAN, SOX9, MMP13 and COLⅡ in each group. Scale bar = 500 μm. (B) Quantitative analysis of the fluorescence intensity of ACAN, SOX9, MMP13 and COLⅡ (n = 3). (C) qPCR determination of the mRNA levels of ACAN, SOX9, MMP13 and COLⅡ (n = 3). (D) Western Blot analysis of ACAN, SOX9, MMP13 and COLⅡ protein bands in each group. (E) Quantitative analysis of ACAN, SOX9, MMP13 and COLⅡ protein expression in each group (n = 3). ANOVA followed by Tukey's test was used for statistical analysis (*p \u0026lt; 0.05, **p \u0026lt; 0.01 and ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5954768/v1/e5eb10f04065556169c36bec.png"},{"id":75694771,"identity":"fd7b77fe-8e62-45f4-b42e-9fcfbd2ea30f","added_by":"auto","created_at":"2025-02-07 07:59:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":485857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePSE hydrogel effectively promotes the polarization of M2 macrophages in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunofluorescence of RAW264.7 cells in each group for Arg-1, CD206 and iNOS. Scale bar = 200 μm. (B) Quantitative analysis of the fluorescence intensity of Arg-1, CD206 and iNOS in each group (n = 3). (C) mRNA expression of anti-inflammatory cytokines (Arg-1, CD206) and pro-inflammatory cytokines (iNOS) in each group (n = 3).ANOVA followed by Tukey's test was used for statistical analysis (*p \u0026lt; 0.05, **p \u0026lt; 0.01 and ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5954768/v1/272434dcb9e06a8d97dbacd3.png"},{"id":75694978,"identity":"0d38431f-77b7-469b-87c3-7b527ab639ea","added_by":"auto","created_at":"2025-02-07 08:07:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":749975,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepair effect of PSE hydrogel on superficial cartilage defects in rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Surgical procedure of injecting PSE hydrogel into the superficial cartilage defect in the patella groove of the distal femur. (B) A cursory observation of the integrity of the knee cartilage 6 and 10 weeks after hydrogel injection. (C) H\u0026amp;E staining images of the rat knee 6 and 12 weeks after hydrogel injection. (F) Safranin O/Fast Green staining images of rat knees 6 and 12 weeks after hydrogel injection.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5954768/v1/4f99d9b89dd8708a8c9d6551.png"},{"id":75694985,"identity":"f048573f-c348-43fc-b055-8f2f0549e452","added_by":"auto","created_at":"2025-02-07 08:07:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":522387,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of proteins at the site of cartilage defect after hyaluronic acid injection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunohistochemical staining of COL-Ⅱ and MMP13 protein at the defect site. (B)Immunohistochemical staining of ARG-1 and CD206 protein at the defect site. (C) Quantitative analysis of immunohistochemical staining of defective areas (n = 3). ANOVA followed by Tukey's test was used for statistical analysis (*p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5954768/v1/2163b6fd69b1f245019514cf.png"},{"id":75694782,"identity":"1c3717f8-be0b-4891-918f-26561c4dbed2","added_by":"auto","created_at":"2025-02-07 07:59:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":430079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of PSE hydrogel-promoted chondrocyte repair.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Analysis of biological replicates between the three groups. (B) Principal component analysis (PCA) between the three groups. (C) Co-expression Venn diagram between the three groups. (D) Heatmap of the differential gene set between the three groups. (E) Volcano plot of differential expression mRNA transcriptomics analysis between the PSE group and the P/S6 group. (E) Enriched up-KEGG terms in the PSE group and the P/S6 group. (n=3)\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5954768/v1/f31149a2dec32212a6d4c7b4.png"},{"id":105903655,"identity":"6b6cd280-086c-4303-a4f9-858efe7f360e","added_by":"auto","created_at":"2026-04-01 09:45:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4838577,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5954768/v1/ec531d6b-c621-41e8-9597-c2db7ce90258.pdf"},{"id":75694764,"identity":"bb13a9b7-3b65-4e61-ac60-ae3ae5bdc7a7","added_by":"auto","created_at":"2025-02-07 07:59:48","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":382326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbstract. Injectable Photo-cross-linkable Porous Composite Hydrogels with Exosomes for M2 Macrophage Polarization and Cartilage Defect regeneration\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GraphicalAbstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5954768/v1/c828e590d66e087c4ead4095.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Injectable Photo-cross-linkable Porous Composite Hydrogels with Exosomes for M2 Macrophage Polarization and Cartilage Defect regeneration","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe prevalence of cartilage defects resulting from injury, osteoarthritis (OA), and other pathological conditions is a significant and growing burden on the healthcare system [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In the initial stages, cartilage defects are classified as partial-thickness cartilage defects (PCD), which are superficial cartilage defects characterised by loss of extracellular matrix and alterations in the metabolism and viability of chondrocytes. This can lead to a worsening of OA and, in severe cases, complete joint destruction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, cartilage is unable to regenerate due to the absence of blood vessels and the limited proliferation capacity of chondrocytes[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite recent advances in microfracture techniques and cell-based cartilage regeneration therapies, there is still no effective treatment for early-stage cartilage defects[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Consequently, there is an urgent need to develop advanced tissue-engineered biomaterials for the regeneration of superficial cartilage defects.\u003c/p\u003e \u003cp\u003eIn a clinical context, mesenchymal stem cells (MSCs) have demonstrated potential for the regeneration of cartilage defects due to their capacity to rejuvenate and to reduce inflammation[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among the various types of MSCs, human umbilical cord-derived MSCs (hUCMSCs) are of particular interest due to their non-invasive availability and robust regenerative capacity[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite the favourable safety and efficacy profile of hUCMSCs in cartilage defect regeneration, the lack of control and regulation of their in vivo growth and differentiation presents a significant challenge to the standardisation of clinical translation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, there is mounting evidence that the therapeutic effects of hUCMSCs are predominantly attributable to paracrine effects. Additionally, it has been demonstrated that hUCMSC-derived exosomes (hUCMSC-EXO) can facilitate cartilage regeneration, upregulate the expression of cartilage-specific genes and glycosaminoglycan synthesis, stimulate the secretion of anti-inflammatory factors, and promote cell mitosis and migration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The current administration of hUCMSC-EXO is via injection. However, electrostatic repulsion induces a strong tendency for exosomes to distribute in synovial fluid, resulting in low cartilage retention and insufficient therapeutic effects[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, the recovery of cartilage lesions usually takes a considerable length of time, but the half-life of exosomes in the body is relatively short, and they often cannot continuously promote the repair of cartilage defects[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, it is necessary to develop methods for fixation and sustained-release systems to ensure that exosomes exert the maximum therapeutic effect.\u003c/p\u003e \u003cp\u003eThe recent advancements in regenerative medicine and tissue engineering have paved the way for the utilisation of sophisticated biomaterials in the treatment of cartilage defects[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Hydrogels are biocompatible and possess loose and porous structural properties, which render them suitable for use as exosome carriers, thereby prolonging the retention time in specific areas and regulating the release of exosomes[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Concurrently, hydrogel implantation and injection represent the primary strategies for contemporary cartilage repair, offering a foundation for the utilisation of exosomes in situ for cartilage and osteochondral interface reconstruction[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Recent studies have demonstrated that the encapsulation of MSC-EXOs in hydrogels effectively results in efficient and sustained delivery for tissue engineering applications[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, Zhang et al. demonstrated that injectable mussel-inspired high-adhesion hydrogels loaded with exosomes can promote in situ cartilage and ECM regeneration in rats[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, thermosensitive hydrogels loaded with exosomes derived from M2 macrophages have been demonstrated to facilitate cartilage repair by stimulating synovial lymphangiogenesis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This suggests that hydrogels can be employed as a fixed and sustained-release vector for exosomes in cartilage repair.\u003c/p\u003e \u003cp\u003eSilk fibroin (SF)-based hydrogels have garnered considerable interest due to their straightforward processing, multifunctional functionalisation, high water content and three-dimensional porous structure that closely resembles that of natural ECM[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, functionalised SF hydrogels have been demonstrated to regulate the migration, proliferation and differentiation of stem cells[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nevertheless, SF-glycidyl methacrylate (SilMA) is not an appropriate choice for direct use in cartilage regeneration due to its lack of larger pores at the micro or macro scale. Previous studies have demonstrated that gelatin methacrylate (GelMA), which exhibits a highly porous structure, can be readily fabricated and that porous GelMA/SilMA hydrogels can facilitate epithelial sealing[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]and ocular surface reconstruction[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Despite the high biocompatibility and capacity for cell growth promotion observed in porous GelMA/SilMA hydrogels, their potential as a fixed and sustained-release carrier for exosomes and as a promoter of cartilage defect repair remains to be confirmed.\u003c/p\u003e \u003cp\u003eThe objective of this study was to develop an injectable photocurable porous GelMA/SilMA hydrogel encapsulating exosomes (PSE) for the promotion of cartilage defect regeneration and M2 polarization of macrophages. The characteristics of the PSE hydrogel were evaluated to ascertain its suitability for cartilage regeneration. The biological effects of the PSE hydrogel in promoting the regeneration of damaged cartilage cells and the polarization of M2 macrophages were confirmed in vitro cell experiments and in vivo rat cartilage defect models. Furthermore, the chemokine signalling pathway of the PSE hydrogel in promoting the repair of damaged cartilage cells was also investigated.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eGelMA (EFL-GM-PR-001), SilMA (EFL-SilMA-001) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP) were purchased from EFL (Suzhou, China). Rat Chondrocyte Cells, hUCMSCs, RAW 264.7, and other cell culture-related reagents were purchased from Procell(Wuhan,China).\u003c/p\u003e \u003cp\u003eThe primary antibodies and live/dead cell double staining kit were purchased from Abcam (Cambridge, USA).The secondary antibodies were purchased from Invitrogen (Carlsbad, USA).\u003c/p\u003e \u003cp\u003eA CCK-8 assay kit was obtained from GLPBIO(Montclair, CA,USA). All other solvents and agents were of analytical grade and used without further purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Isolation and identification of exosomes\u003c/h2\u003e \u003cp\u003eWhen hUCMSCs reached 80\u0026ndash;90% confluence, they were washed and incubated for another 48h in serum-free medium. The medium was collected and centrifuged at 300\u0026times;g for 15 min at 4\u0026deg;C and then at 2500\u0026times;g for 15 min. After centrifugation, the supernatant was filtered using a 0.22 \u0026micro;m filter to remove the remaining cells and debris. Next, a 15-mL Amicon Ultra-15 Centrifugal Filter Unit (Merck Millipore) was used to transfer the filtered solution. The solution was centrifuged at 4000\u0026times;g until the volume in the upper compartment was concentrated to approximately 200 uL. The ultrafiltered solution was washed with phosphate-buffered saline (PBS) and centrifugation was repeated three times. The washed ultrafiltration liquid containing exosomes was placed on a 30% sucrose/D2O cushion in an Ultra-Clear\u0026trade; tube (Beckman Coulter, Brea, CA) and ultra-centrifuged for 1 h at 100,000\u0026times;g. The pellets were resuspended in PBS and centrifuged at 4000\u0026times;g to concentrate the volume to approximately 200 \u0026micro;L.\u003c/p\u003e \u003cp\u003eTransmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA) with a Zetaview (Particle Metrix, Munich, Germany) was performed for the exosome size distribution measurement. Exosome size was calculated using NTA software (Malvern, Shanghai, China). The proteins were subsequently used for Western blot analysis for TSG101, Alix, CD9 and Calnexin markers to detect exosome components after protein extraction. In order to analyze the effect of these radicals from H2O2 on the exosomes structural integrity, 1 mL exosomes (200 \u0026micro;g/mL) were treated with 20 \u0026micro;l HRP (5 mg/mL) and 20 \u0026micro;l H2O2 (0.5% w/v) in 26\u0026deg;C for 24 h, followed by the NTA experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of P/S6 hydrogels and PSE hydrogels\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer\u0026rsquo;s instructions, 0.6 g of freezedried porous GelMA was dissolved in 10 mL of PBS to generate a homogeneous 6% w/v homogeneous solution with continuous stirring in a water bath in the dark at 37\u0026deg;C for 1 h. Similarly, freezedried SilMA was dissolved in PBS with 0.25% LAP at room temperature for 1 h at a concentration of 20% (w/v).Finally, the 6% w/v porous GelMA solution was uniformly mixed with the 20% w/v SilMA solution at feed ratios of 6:1 to give the samples P/S6. For the hydrogel used in the cell and animal experiments, a hydrogel with exosomes (200 \u0026micro;g/mL) was prepared, where the exosomes were mixed with P/S6 solution to form the PSE hydrogel. All steps were performed under sterile conditions, and the prepared solutions were sterilized using a 0.22 \u0026micro;m filter. The hydrogel precursors were exposed to visible light (405 nm, 30 mW/cm2 ) from a light source for 30 s to form the hydrogel.\u003c/p\u003e \u003cp\u003eFor transparency, swelling ratio (SR) and degradation tests, the P/S6 hydrogel precursors(120ul) and the PSE hydrogel precursors(120\u0026micro;L) were injected into cylindrical molds (10 mm diameter; 2 mm height) to form disc-shaped hydrogels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Characterization of the P/S6 hydrogel and the PSE hydrogel\u003c/h2\u003e \u003cp\u003eFor morphological analysis, a Hitachi SU 8010 (Hitachi, Tokyo, Japan) was used for cryo-field emission scanning electron microscopy (FE-SEM).\u003c/p\u003e \u003cp\u003eDynamic structural changes were identified using Fourier transform infrared spectroscopy (FTIR) on a Nicolet FTIR 6700 spectrometer (Thermo Fisher, Massachusetts, USA).\u003c/p\u003e \u003cp\u003eThe compressive strength of the hydrogels was determined using DMA (DMA, Q800, TA Instruments, USA) to calculate the stress-strain curve of the hydrogels.\u003c/p\u003e \u003cp\u003eIn addition, the storage modulus (G') and loss modulus (G'') of the hydrogels were measured using rheological tests (Physica MCR301, Anton Paar) at a fixed strain (5%) and dynamic oscillation frequency (10\u0026thinsp;\u0026minus;\u0026thinsp;0.1 Hz).\u003c/p\u003e \u003cp\u003eIn order to determine swelling ratio (SR), 200 \u0026micro;L hydrogels were exposed to blue light for 20 s and then incubated in PBS at 37\u0026deg;C. The swollen weight (Ws) and the dry weight (Wd) was measured at each indicated time point (0,1, 2, 4, 6, 8, and 10 h). The SR was calculated according to the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:SR=\\frac{Ws-Wd}{Wd}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn the context of in vitro degradation tests, the initial dry weight (Wo) of the hydrogels was ascertained by weighing them prior to immersion. This was followed by the immersion of these hydrogels into a solution of PBS containing lysozyme (1mg/mL, GLPBIO) at 37 degrees Celsius for 21 days. At the designated time points (7, 14 and 21 days), the samples were lyophilised and weighed (Wt). The degradation ratio was subsequently calculated using the following formula:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Degradation（\\%）＝\\frac{Wt}{Wo}\\times\\:100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe cumulative and daily release profiles were evaluated using a bicinchoninic acid (BCA) reagent test kit (Beyotime). The PSE hydrogels were incubated at 37\u0026deg;C in PBS. The supernatant was collected on days 1,3,5, 7,10, 14and 21 for free exosome (Exo) detection using BCA to assess the release profile of Exo from the PSE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 In vitro studies\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 In vitro biocompatibility assessment of P/S6 and PSE hydrogels\u003c/h2\u003e \u003cp\u003eIn vitro biocompatibility was evaluated through the implementation of cell viability and proliferation experiments. To conduct the Live/Dead staining test, chondrocytes at a density of 1\u0026times;10⁶ were seeded into 12-well culture plates and co-incubated with P/S6 or PSE hydrogel extracts for 24 hours. The Live/Dead solution was then prepared in the ratio of 1 ml: 3 \u0026micro;l: 5 \u0026micro;l (PBS: calcein-AM(Invitrogen): PI(Invitrogen)) and added in each group and incubation was done for 30 min at 37 ℃. After 1\u0026times;106 chondrocytes were co-cultured with P/S6 or PSE hydrogel extracts for 1, 3, and 5 days, 100 \u0026micro;l/ml CCK-8 solution (GLPBIO) was added to each well and incubated for 2 h. Subsequently, 100 \u0026micro;l of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 450 nm with a microplate analyser (BioTech).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Co-culture of chondrocytes and RAW264.7 cells with hydrogel\u003c/h2\u003e \u003cp\u003eThe DMEM basal medium was soaked with a 20% volume fraction of P/S6 and PSE hydrogel for a period of 24 hours in order to prepare hydrogel extracts. Chondrocytes (P1 generation) were seeded into 6-well plates and treated with IL-1β (10 ng/ml) for 24 hours, thereby creating an in vitro model of damaged chondrocytes. The damaged chondrocytes were then cultured with P/S6 and PSE hydrogel extracts for three days. Similarly, RAW264.7 cells were seeded onto 6-well plates and cultured with P/S6 and PSE hydrogel extract for three days. The cell culture medium was prepared as follows: the hydrogel extract was extracted with DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Immunofluorescence\u003c/h2\u003e \u003cp\u003eThe samples were fixed with 4% paraformaldehyde in PBS for a period of 30 minutes. Subsequently, the cells or tissues were treated with 0.2% Triton X-100 (Beyotime) for 10 minutes and blocked with 3% bovine serum albumin (BSA, Beyotime) for one hour at room temperature. The samples were incubated at 4\u0026deg;C overnight with the primary antibodies. Subsequently, the sample was washed three times with PBS and incubated with the secondary antibody for a period of 2 hours at room temperature. Subsequently, the sample was subjected to Hoechst (Beyotime) staining. Subsequently, the stained samples were imaged using a confocal microscope (Leica). The results were analysed using the ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4 Gene expression\u003c/h2\u003e \u003cp\u003eTotal cellular RNA was extracted using an RNA extraction kit (Vazyme) and subsequently reverse-transcribed into cDNA using an EVO-MLV RT kit (Vazyme). qRT-PCR analysis was conducted using LightCycler 480 SYBR Green Master Mix (Vazyme). The endogenous control employed was the reference gene GAPDH. The relative gene expression was quantified using the 2-ΔΔCt method. The experiment was conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.5 Western Blot\u003c/h2\u003e \u003cp\u003eThe cells were homogenised in RIPA buffer (CWBIO) containing protease and phosphatase inhibitors (Thermo Fisher) in order to create a cell homogenate. The supernatants were then lysed on ice for 30 minutes and subsequently collected by centrifuging at 12,000 rpm at 4\u0026deg;C for a further 30 minutes. The total protein concentration was determined using the BCA kit (Beyotime). The supernatants were mixed with loading buffer (Beyotime), and equal amounts (40 \u0026micro;g) of protein were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in accordance with the manufacturer's instructions. Subsequently, the separated protein was transferred to a polyvinylidene difluoride (PVDF, Thermo Fisher) membrane, which was then blocked with 5% skim milk for one hour. Thereafter, the membrane was incubated with primary antibodies overnight. Subsequently, the PVDF membrane was incubated with the secondary antibody for one hour prior to the application of the enhanced chemiluminescence (ECL) kit (Thermo Fisher) for visualisation. Prior to each subsequent step, the membranes were washed thrice with Tris-Buffered Saline with Tween\u0026reg; 20 (TBST). The results were analysed using the ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.6 In vivo studies\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Ethics statement\u003c/h2\u003e \u003cp\u003e The research involving animal subjects was approved by the Animal Experiment Ethics Committee of Qingdao Municipal Hospital. All experimental procedures on animals were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Osteochondral defect models\u003c/h2\u003e \u003cp\u003eAs previously described in the literature, 3-month-old Sprague Dawley rats (n\u0026thinsp;=\u0026thinsp;20) were randomly divided into three groups: control, P/S6 and PSE groups, with each group containing rats weighing 200\u0026ndash;250 g. Following anaesthesia, the rats were shaved and disinfected, and then their right knee joints were exposed. Subsequently, a 2 mm diameter and 1 mm height hole was created in the centre of the trochlear groove using a drill. In order to compare the effects of the different experimental groups on cartilage defect regeneration, the control group was treated with PBS, while the other two experimental groups were treated with P/S6 hydrogel and PSE hydrogel, respectively. In the P/S6 and PSE groups, the hydrogel was introduced into the defect via syringe injection. At the 6th and 10th weeks post-surgery, the rats were euthanised and subjected to histological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3 Histological analysis and immunohistochemistry\u003c/h2\u003e \u003cp\u003eAt 6 and 10 weeks, the rats were sacrificed and whole femurs were collected. Samples were fixed in 4% paraformaldehyde (pH 7.5) for 1 day and decalcified in decalcifying solution for 21 days, the samples were then embedded in paraffin and cut into 5 \u0026micro;m sections. Three specimens were randomly selected from each group at 6 and 10 weeks for histological analysis. The specimens were soaked in 10% formalin overnight, decalcified, embedded in paraffin, and sectioned for staining. The specimens were stained with hematoxylin and eosin (H\u0026amp;E) and Safranin O/Fast Green.\u003c/p\u003e \u003cp\u003eThree specimens were randomly selected from each group at 6 weeks for immunohistochemistry. The rabbit anti-COL-2 (Abcam), MMP-13 (Abcam), Arg-1 (Gentex), and CD206 (Gentex) primary antibodies were used for immunohistochemistry. The results were analysed using the ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Transcriptome Analysis\u003c/h2\u003e \u003cp\u003eThe in vitro function of the PSE hydrogel was analysed by transcriptome analysis of IL-1β-treated chondrocytes co-cultured with the hydrogel, conducted by Novogene (Beijing, China). Total RNA was extracted using TRIzol reagent. RNA purity and quantification were assessed using a NanoDrop 2000 spectrophotometer, and RNA integrity was assessed using an Agilent 2100 bioanalyzer. The sequencing libraries were then constructed using the VAHTS Universal V6 RNA-seq Library Preparation kit according to the manufacturer's instructions. Transcriptome sequencing and analysis were performed by Novogene (Beijing, China). The libraries were sequenced on the Illumina Novaseq 6000 platform and 150 bp paired-end reads were generated. Raw readings were generated for each sample. Raw readings in fastq format were first processed using fastp1, and low-quality data were removed to obtain clean readings. Clean readings from each sample were retained for subsequent analysis. Clean readings were mapped to the reference genome using HISAT22. The FPKM3 of each gene was calculated, and the read counts of each gene were obtained by HTSeq-count4. PCA analysis was performed using R (v 3.2.0) to assess the bio-reproducibility of the samples, and DESeq25 was used for differential expression analysis. A q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and Fold Change\u0026thinsp;\u0026gt;\u0026thinsp;2.0 was used as the threshold for significantly differentially expressed genes (DEGs).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe results presented are representative of at least three independent experiments and are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The student's t-test and paired ANOVA with a post-hoc test (GraphPad Prism 9.0) were used to determine the statistical significance between the intervention and control groups. A one-way analysis of variance model was used to compare multiple groups. The significance level was set at 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of hUCMSC-EXOs\u003c/h2\u003e \u003cp\u003eTo confirm the isolation of hUCMSCs-derived exosomes, transmission electron microscopy (TEM) was employed to obtain micrographs, which revealed a round or elliptical morphology with a complete cell membrane structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, C). Furthermore, the qNano\u0026reg; system was employed for quantification and size profiling. The particle size distribution was found to be predominantly around 100 nm, consistent with that of exosomal dimensions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Additionally, Western blotting analysis demonstrated that the nanoparticles expressed the exosomal-specific markers CD9 and TSG101 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Therefore, the aforementioned results confirm that the exosomes were successfully isolated from hUCMSCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Synthesis and characterization of the porous hydrogels with and without exosomes\u003c/h2\u003e \u003cp\u003eA mixture of porous GelMA/SilMA hydrogel and exosomes was prepared under ultraviolet irradiation with a photoinitiator. A scanning electron microscope (SEM) was employed to examine the structural morphology of the porous GelMA/SilMA hydrogel following the freeze-drying process. The SEM images revealed the presence of a highly porous structure and an interconnected network throughout the sample. Scanning electron microscopy (SEM) imaging demonstrated that the hydrogel exhibited a porous network structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and that the exosomes were attached to the internal surface of the porous hydrogel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). It has been demonstrated that the highly porous structure can provide a favourable microenvironment for cell adhesion, proliferation and migration .\u003c/p\u003e \u003cp\u003eThe strain-stress curves of the hydrogels demonstrate that when the strain reaches 75%, the compressive strength of the PSE hydrogel is 0.41 MPa, and the compressive strength of the P/S6 hydrogel is 0.46 MPa. This indicates that both hydrogels meet the compressive strength of human cartilage, as previously reported[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The addition of exosomes has been observed to have a minimal effect on the mechanical properties of the hydrogels. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC)\u003c/p\u003e \u003cp\u003eThe results of the rheological tests demonstrated that the storage modulus (G') of P/S6 and PSE was significantly higher than the loss modulus (G''), indicating that they exhibited the characteristics of stable viscoelastic solids[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, the discrepancy between the mean storage modulus of PSE and P/S6 was not substantial, suggesting that the incorporation of exosomes did not alter the mechanical characteristics of the hydrogel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe secondary structural changes of the hydrogels were analysed using FTIR. Following the addition of exosomes, the FTIR spectra of the PSE and P/S6 hydrogels exhibited no shift at 1650 cm-1, indicating that the incorporation of exosomes did not result in significant structural alterations in the hydrogels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eFurthermore, the swelling ratio and biodegradation rate are of significant importance with regard to the practical application of the material. Following a 60-minute immersion in PBS, the swelling ratio of the PSE hydrogel and P/S6 hydrogel exhibited a marked increase, reaching values exceeding 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Subsequently, a 21-day enzymatic degradation study was conducted using lysozyme-containing PBS to evaluate the applicability of the PSE hydrogel and P/S6 hydrogel. Both hydrogel samples exhibited relatively rapid degradation during the initial seven-day period, followed by a gradual deceleration over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Concurrently, BCA detection demonstrated that the PSE hydrogel could sustain continuous exosome release, with approximately 85% of the exosomes released into PBS for at least 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), aligning with the biodegradation curve of the hydrogel and achieving the desired slow release of exosomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Biocompatibility of PSE and P/S6 hydrogels\u003c/h2\u003e \u003cp\u003eThe results of the live/dead staining demonstrated that following a period of one day in culture within the P/S6 and PSE hydrogel extracts, the majority of rat chondrocytes exhibited green fluorescence, with only a minimal number displaying red fluorescence. This suggests that the hydrogels are biocompatible (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). A semi-quantitative analysis utilising a live/dead assay demonstrated that the survival rate of cells in the P/S6 and PSE hydrogel extracts exceeded 90%, with the cells exhibiting high vitality (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Furthermore, a CCK-8 assay was conducted to assess the proliferative capacity of chondrocytes in P/S6 and PSE hydrogel extracts. The CCK-8 results demonstrated that the cell proliferation rate increased with the incubation time, indicating that the cells were undergoing both growth and proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Previous studies have also yielded analogous findings, indicating that the incorporation of an optimal amount of silk fibroin can enhance the proliferative effect[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4 PSE hydrogel promotes the repair of IL-1β-damaged chondrocytes\u003c/h2\u003e \u003cp\u003eIn order to evaluate the efficacy of PSE hydrogel in repairing cartilage, cartilage cells that had been pretreated with IL-1β were cultured in hydrogel extract for a period of three days. Following this, the cells were analysed using immunofluorescence, qPCR and western blot. Additionally, a control group was constituted by untreated healthy cartilage cells cultured in hydrogel extract for three days.\u003c/p\u003e \u003cp\u003eThe extracellular matrix (ECM) of cartilage is primarily composed of proteoglycans and collagen II. Immunofluorescence images demonstrate that the PSE group exhibited elevated levels of immunofluorescence for aggregated proteoglycans and collagen II. The expression of the cartilage-forming gene SOX-9 was markedly elevated in comparison to the control group, thereby indicating that the exosomes present within the hydrogel enhanced the efficacy of cartilage differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Furthermore, the immunofluorescence images demonstrated that the expression of MMP-13 was diminished in the PSE group. MMP-13 is not only a key matrix-degrading enzyme in chondrocytes, but its expression level is also frequently employed as an indicator of the severity of cartilage damage. This not only corroborates the therapeutic efficacy of PSE on damaged chondrocytes, but also its prospective advantages for preserving the structure of articular cartilage in patients with damaged cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The aforementioned conclusion was corroborated by the semi-quantitative analysis of the immunofluorescence images (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFurthermore, qPCR analysis revealed that the expression levels of ACAN, SOX-9 and COL-II genes were significantly elevated in the PSE group compared to the P/S6 group. Conversely, the expression level of the MMP13 gene was significantly reduced. These findings suggest that Exos can mitigate the detrimental effects of IL-1β on chondrocytes, and that porous hydrogels combined with Exos can enhance the repair of damaged chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Western blot analysis also demonstrated that PSE hydrogels exhibited a markedly superior repair efficacy for damaged cartilage, exceeding that of P/S6 hydrogels. This finding aligns with the qPCR results, which indicated that the combined use of Exos can further enhance the repair of damaged chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Western blot results also demonstrated that the repair effect of PSE hydrogel on damaged cartilage was significant and markedly superior to that of P/S6 hydrogel, which was consistent with the qPCR results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). These findings collectively suggest that the PSE hydrogel can directly exert a robust repair effect on damaged chondrocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5 PSE hydrogel effectively promotes the polarization of M2 macrophages in vitro\u003c/h2\u003e \u003cp\u003eFollowing damage to cartilage, the inflammatory response at the injury site disrupts the anabolic and catabolic balance of chondrocytes, which in turn results in the degradation of the ECM via matrix-degrading enzymes[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Given that macrophages are the primary mediators of inflammation following injury, we sought to examine the polarising impact of PSE hydrogel on RAW264.7 cells in an in vitro setting. The expression of relevant inflammatory molecules was then detected by immunofluorescence and qPCR in RAW264.7 cells cultured in P/S6 and PSE hydrogel extracts, with the aim of verifying the immunomodulatory properties of PSE hydrogel.\u003c/p\u003e \u003cp\u003eOn day 3, the immunofluorescence results demonstrated that the fluorescence intensity of the M2 phenotype markers Arg-1 and CD206 in the PSE group was markedly higher than that in the control group and the P/S6 group. This suggests that the hUCMSC-EXOs loaded in the hydrogel facilitated the M2 polarization of macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Furthermore, the qPCR results on day 3 demonstrated that the PSE hydrogel markedly elevated the expression levels of Arg-1 and CD206, whereas the expression level of iNOS was diminished.\u003c/p\u003e \u003cp\u003eExosome vesicles are nano-scale vesicles containing nucleic acids, proteins and other components. They primarily exert their biological effects by releasing active ingredients extracellularly for cell communication and transmission[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A substantial body of evidence has demonstrated that MSC-derived exosomes are a rich source of molecules that regulate immunity, including mRNA, miRNA, cytokines and chemokines, which play a pivotal role in modulating the phenotype and function of immune cells[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The results demonstrated that hUCMSC-Exos exert a robust immunomodulatory impact by facilitating the M2 polarization of macrophages, which may prove advantageous for the repair of damaged cartilage.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6 PSE promotes cartilage repair in the knee joints of rats and regulates the polarization of macrophages\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA rat patellar cartilage defect was created for the purpose of evaluating the efficacy of cartilage regeneration and the inflammatory response following the injection of PSE hydrogel. At 6 and 12 weeks post-surgery, the distal femurs were collected for gross observations and histological staining analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The gross analysis conducted at 6 and 12 weeks revealed that the cartilage surface in the PSE hydrogel group exhibited a smoother and more continuous appearance than that observed in the control and P/S6 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Histological changes to the cartilage were analysed using H\u0026amp;E and Safranin O/Fast Green staining. In contrast to the control group, in which the patellar groove defect was filled with new fibrous tissue and minimal cartilage formation was observed, the PSE hydrogel treatment group demonstrated notable cartilage formation in the defect area at six weeks postoperatively and a smooth surface at 10 weeks. It is noteworthy that the boundary between the regenerated cartilage and the native tissue was rarely discernible in the PSE group, suggesting the formation of hyaline cartilage in situ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC,D).\u003c/p\u003e \u003cp\u003eThe immunochemical analysis of ECM expression in defective cartilage demonstrated that the PSE hydrogel is capable of promoting the regeneration of superficial cartilage defects in the knee joint. The PSE group exhibited elevated collagen II expression and diminished MMP-13 expression relative to the control and P/S6 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). These findings indicate that PSE hydrogel not only inhibits the expression of MMP-13 in chondrocytes, but also significantly promotes the expression of collagen type II in chondrocytes. This suggests that PSE hydrogel can promote anabolic rather than catabolic metabolism in chondrocytes by directly supplementing ECM. Furthermore, in comparison to the P/S6 hydrogel treatment group, the PSE hydrogel demonstrated a more pronounced promotion of COL II expression, thereby substantiating the chondroprotective effects and cartilage regeneration potential of hUCMC-derived Exos.\u003c/p\u003e \u003cp\u003eTo confirm that PSE hydrogel can modulate the inflammatory response following growth plate injury in vivo, the macrophage phenotype polarization was detected using the unique markers CD206 and Arg-1 in the injured area. As observed in the in vitro studies, the expression of CD206 and Arg-1 at the injury site was higher in the PSE group than in the control and P/S6 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB,C). This suggests that PSE is capable of inducing M2 polarization of macrophages at the injury site. The role of immunoreactivity in the context of tissue injury and repair, particularly in chondrocytes, is of paramount importance. Pro-inflammatory immune cells produce inflammatory cytokines, including IL-1β, iNOS and TNF-α, which are rapidly upregulated, leading to overexpression of matrix metalloproteinases, such as MMP-1 and MMP-13. This, in turn, results in chondrocyte apoptosis and matrix degradation following cartilage injury[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. M2 macrophages, also referred to as wound-healing macrophages, are capable of creating an anti-inflammatory environment that is conducive to tissue repair and remodeling[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It has been demonstrated in previous studies that the injection of an M2 macrophage activator into the joint cavity of animals with osteoarthritis can prevent the formation of osteophytes and slow the progression of cartilage damage[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The injection of PSE hydrogel at the injury site resulted in the induction of a higher expression of anti-inflammatory factors, thereby regulating the immune microenvironment and promoting the survival of chondrocytes and cartilage regeneration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Transcriptome analysis reveals the mechanism by which PSE hydrogel promotes cartilage cell repair\u003c/h2\u003e \u003cp\u003eThe cells were divided into three groups: a control group comprising healthy chondrocytes (WT), an IL-1β\u0026thinsp;+\u0026thinsp;P/S6 group (KO), and an IL-1β\u0026thinsp;+\u0026thinsp;PSE group (OE). Transcriptomics was employed to elucidate the potential mechanism through which the PSE hydrogel affects the regeneration of damaged cartilage. To assess the stability of the three groups, Pearson correlation and principal component analysis were conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and B). The results demonstrated that the correlation coefficients of the specimens in each group were within an acceptable range (R2\u0026thinsp;\u0026gt;\u0026thinsp;0.95, n\u0026thinsp;=\u0026thinsp;3), indicating good biological reproducibility and that the transcriptome data could be utilised for further analysis. Subsequently, a Venn diagram and heat map analysis were conducted between the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, D), which demonstrated the overlap of differentially expressed genes and cluster analysis between different comparison groups. This allowed for the subsequent analysis of differentially expressed genes to be carried out. Furthermore, a volcano plot analysis of differentially expressed genes (DEGs) was conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). In comparison to the P/S6 group, the PSE group exhibited upregulation of 946 genes and downregulation of 1,394 genes. Among the differentially expressed genes, the MMP13 gene exhibited decreased expression in the PSE group, whereas the ACAN and COL II genes demonstrated significant upregulation. These findings align with the results of previous cell and animal experiments. This provides further evidence that the PSE hydrogel has a protective effect on chondrocytes and is capable of promoting cartilage regeneration.\u003c/p\u003e \u003cp\u003eTo gain further insight into the mechanism by which PSE hydrogel exerts its chondroprotective effect, we conducted the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). The top enriched up-KEGG pathways associated with the PSE and P/S6 groups were analysed to ascertain the mechanisms by which the PSE hydrogel exerts its chondroprotective effects. The results demonstrated that the PI3K/AKT signalling pathway and ECM-receptor interaction are activated by the PSE hydrogel, which participates in the production or synthesis of ECM. This includes the positive regulation of extracellular matrix containing collagen, collagen binding and collagen biosynthesis processes, and the promotion of ECM regeneration. In conclusion, it can be posited that PSE hydrogel has the capacity to regulate cell signalling and facilitate the regeneration of cartilage defects. However, further research is required to elucidate the underlying mechanisms in greater detail.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eIn this study, a porous GelMA/SilMA hydrogel loaded with hUCMSC-Exos (PSE hydrogel) was prepared, which exhibited excellent physical properties and biocompatibility. The PSE hydrogel was observed to modulate the immune microenvironment, promote M2 macrophage polarization, and protect damaged cartilage by activating cell communication signal pathways, thereby facilitating the repair of cartilage defects in the rat patellar groove. In light of these findings, this study has resulted in the creation of a promising biomaterial for patients with superficial cartilage defects, which can be injected under arthroscopic surgery.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYanchi Bi contributed to the conception and design of the study. Liang Zhu wrote the manuscript. Haibo Zhao collected data for the work. XX, YTB supervised the manuscript. All authors read and approved the final manuscript..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Shandong Provincial Key R\u0026amp;D program [Science and Technology Demonstration Project] in 2021 [grant no. 2021SFGC0502]; the Qingdao Science and Technology Plan Project Science and Technology Benefiting the People Demonstration and Guidance Project in 2022 [grant no. 22-3-7-smjk-5-nsh]; Qingdao Shinan District Science and Technology Bureau public domain science and technology support plan project [grant no.2023-2-011-YY].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for all the research applied to this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eY. 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Nixon \u003cem\u003eet al.\u003c/em\u003e, \u0026lsquo;Disease-Modifying Osteoarthritis Treatment With Interleukin-1 Receptor Antagonist Gene Therapy in Small and Large Animal Models\u0026rsquo;, \u003cem\u003eArthritis Rheumatol\u003c/em\u003e, vol. 70, no. 11, pp. 1757\u0026ndash;1768, Nov. 2018, doi: 10.1002/art.40668.\u003c/li\u003e\n\u003c/ol\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Silk fibroin, Exosomes, Cartilage defect, Immunomodulation","lastPublishedDoi":"10.21203/rs.3.rs-5954768/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5954768/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the initial stages of osteoarthritis(OA), the deterioration of cartilage in the superficial region results in the formation of superficial cartilage defects. It is anticipated that a therapy based on human umbilical cord mesenchymal stem cell exosomes(hUCMSCs-Exos) will make a notable contribution to the promotion of M2 macrophage polarization and the facilitation of cartilage repair. This study demonstrates that injectable photo-cross-linkable porous gelatin methacryloyl(GelMA)/silk fibroin glycidyl methacrylate(SilMA) hydrogels encapsulating human umbilical cord mesenchymal stem cell exosomes(hUCMSCs-Exos)represent a straightforward and effective approach for cartilage regeneration. The encapsulation of exosomes in hydrogels has been demonstrated to have no significant impact on the physical properties, as evidenced by physical studies. Furthermore, studies based on in vitro cell models and in vivo models in rats have demonstrated that exosomes released from hydrogels can promote M2 polarization, inhibit M1 polarization, and facilitate cartilage regeneration by regulating the PI3K/AKT signaling pathway. This process facilitates the repair of cartilage defects. The findings of our research have led to the development of an injectable photo-cross-linkable hydrogel for superficial cartilage regeneration, which represents a promising minimally invasive treatment for cartilage defects with the aid of arthroscopy.\u003c/p\u003e","manuscriptTitle":"Injectable Photo-cross-linkable Porous Composite Hydrogels with Exosomes for M2 Macrophage Polarization and Cartilage Defect regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 07:59:42","doi":"10.21203/rs.3.rs-5954768/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd54ba1c-e082-43be-bee1-3213a6bdda8d","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T09:44:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-07 07:59:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5954768","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5954768","identity":"rs-5954768","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}