Bone marrow mesenchymal stem cell-derived exosomes prevent osteoarthritis by regulating synovial macrophage polarization

Identification of BMSCs

In the present study, we isolated BMSCs that were of irregular shape, spindle shape, and polygon in the bright field images (Figure 1A). The results of alizarin red, oil red O and Alimin Blue staining confirmed that BMSCs had multi-directional differentiation potentials, including differentiation into osteoblasts, adipocytes and chondrocytes (Figure 1B). BMSCs were identified by flow cytometry, and were positive for the mesenchymal markers CD44 and CD90, while they were negative for the hematopoietic cell surface marker, CD45 (Figure 1C).

Figure 1. Characterization of bone marrow mesenchymal stem cells (BMSCs). (A) BMSCs exhibited a representative spindle-like morphology. Scale bar: 100 μm. (B) BMSCs showed osteogenic pluripotent differentiation abilities, adipogenesis, and cartilage formation. Scale bar: 100 μm. (C) Flow cytometry analysis of characteristic cell surface markers of BMSCs. The mesenchymal markers, CD44 and CD90, were positive, while the hematopoietic cell surface marker, CD45, was negative.

Characterization of BMSC-derived exosomes

Exosomes were isolated from the supernatant of BMSCs by ultrafiltration and centrifugation. Purified BMSC-derived exosomes were identified by NTA, transmission electron microscopy and western blotting. Transmission electron microscopy showed that BMSC-derived exosomes showed a typical cup-like morphology (Figure 2A). NTA detection revealed that the average vesicle diameter was about 140 nm (Figure 2B). Western blot analysis of exosome lysates revealed positive expression of the exosomal surface markers CD63, CD81 and CD9, while no cerulein was detected (Figure 2C). Collectively, these results confirm the successful separation of exosomes from the supernatant of BMSCs.

Figure 2. Characterization and uptake of BMSC exosomes. (A) Transmission electron microscopy (TEM) showed that BMSC-derived exosomes had a typical cup-like morphology. Scale bar: 50 nm. (B) Nanosight Tracking Analysis (NTA) detection revealed that the average vesicle diameter was about 140 nm. (C) Western blot analysis of exosome lysates revealed positive expression of the exosome surface markers, CD63, CD81 and CD9. No cerulein was detected in BMSC-derived exosomes. (D) Detection of exosome uptake by BMSCs. Dil : red, DAPI: blue. Scale bar: 20 μm.

To investigate the feasibility of using BMSC-derived exosomes to treat OA, we examined the cellular uptake of Dil-labeled exosomes (Dil-exosomes). After RAW264.7 cells were incubated with Dil-exosomes for 24 h, Dil-exosomes were observed in the cytoplasm by fluorescence microscopy to confirm uptake (Figure 2C). In vivo experiments further demonstrated that Dil-exosomes could accumulate in the knee joint and be taken up by synovial cells following injection (Figure 3C).

Figure 3. BMSC exosomes prevented cartilage destruction in a rat model. (A) Representative images of HE staining in knee sections. Scale bar: 50 μm. (B) Representative images of Safranin O / Fast Green staining in knee sections. Scale bar: 50 μm. (C) Representative adoptive Dil-labelled BMSC-Exos (green fluorescence) in a knee joint. Scale bar: 50 μm (D) Evaluation of cartilage destruction using the OARSI scoring system. *p < 0.05 compared to the Sham group; # p < 0.05 compared to the PBS group.

BMSC-derived exosomes relieve osteoarthritis

MSC-derived exosomes isolated from various tissue sources have potential therapeutic benefits in many diseases [22–24], including cartilage damage and repair. We hypothesized that BMSC-derived exosomes can alleviate OA. In this study, in order to evaluate the therapeutic effect of BMSC-derived exosomes in OA, intra-articular injections were performed with PBS or BMSC-derived exosomes at 4 weeks postoperatively. At the 8^th week after operation, the PBS group showed severe cartilage degradation, markedly thinned articular cartilage, increased surface fibrosis area, abnormal distribution of chondrocytes (Figure 3A and 3B), decreased expression of type II collagen, which is the main component of hyaline cartilage, and increased expression of type X collagen, which is the main component of fibrocartilage (Figure 4A–4D). Micro CT analysis and three-dimensional (3D) modeling of the knee showed a significant increase in the number and surface area of the osteophyte around the joint (Figure 4E and 4F). In contrast, in the exosomes group, cartilage was less degraded, chondrocytes maintained near-normal morphology and distribution, and the number and surface area of osteophytes around the joints were less than those in the PBS group. The OARSI score was significantly lower in the exosomes group (Figure 3D). The western blot results of cartilage tissue showed that compared with the exosomes group, the expression of the hypertrophic gene, collagen X, and runx2 was decreased. The expression of the chondrogenic genes, collagen II and sox9, was also increased (Figure 4G–4K). These findings indicated that BMSC-derived exosomes inhibit cartilage degradation and osteophyte formation during OA progression in rats.

Figure 4. BMSC exosomes inhibited chondrocyte hypertrophy and reduced osteophyte formation in the rat model. (A) and (B) Immunohistochemistry and analysis of collagen II in knee sections. Scale bar: 50 μm. (C) and (D) Immunohistochemistry and analysis of collagen X in knee sections. Scale bar: 50 μm. (E) Micro-CT scan and three-dimensional reconstruction of the knee joint in each experimental group. (F) Osteophyte score and quantitative analysis of the volume of the region of interest (ROI). (G–K) Western blot results and analyses of cartilage tissue. *p < 0.05 compared to the Sham group; # p < 0.05 compared to the PBS group.

BMSC-derived exosomes change the M1/M2 subsets of synovial macrophages

As mentioned previously, M1 macrophages increase the inflammatory response during OA. In this study, we found that BMSC-derived exosomes can alleviate the progression of OA. However, whether exosomes promote the phenotypic transformation of synovial macrophages from M1 to M2 is unknown. To address this question, knee joint sample sections were used for immunohistochemistry and immunofluorescence to detect changes in the synovial M1 / M2 subsets. Compared with the sham group, synovial hyperplasia and cell infiltration were significantly increased in the PBS group, and the synovitis score was significantly increased (Figure 5A and 5B). F4/80+ (macrophage marker) -positive cells in synovial tissue were significantly increased, and the ratio of F4/80+iNOS+ (M1-like macrophage markers) positive cells also increased significantly (Figure 5E and 5F). However, Arg1+ (M2-like macrophage markers) increased slightly (Figure 5C and 5D). Compared with the PBS group, synovial hyperplasia and cell infiltration were reduced in the Exo group, the ratio of M1-positive cells was significantly decreased, and the ratio of M2-positive cells was increased. ELISA detection of synovial fluid showed that the levels of the pro-inflammatory cytokines IL-1β, TNF-α were significantly decreased in the Exo group, while the level of the anti-inflammatory cytokine IL-10 was increased (Figure 5G–5I). These findings indicate that BMSC-derived exosomes change the M1/M2 subsets of synovial macrophages during OA progression in rats and decrease the level of inflammation in synovial fluid.

Figure 5. BMSC exosomes reduced synovial macrophage infiltration, induced changes in M1/M2 subsets, and reduced inflammation in the rat model. (A) Representative images of HE staining in synovial tissue. Scale bar: 50 μm. (B) Quantification of synovitis scores in synovial tissue. (C) and (D) Immunohistochemistry and analysis of Arg1 in synovial tissue, respectively. Scale bar: 50 μm. (E) and (F) Immunofluorescence and analysis of F4/80 and iNOS, respectively, in synovial tissue. Scale bar: 50 μm. (G–J) Determination of the cytokines, IL-1β, IL-6, TNF-α, and IL-10 in synovial fluid from each test group. * p < 0.05 compared to the Sham group; # p < 0.05 compared to the PBS group.

BMSC-derived exosomes promote the phenotypic transformation of macrophages from M1 to M2 in vitro

We further verified the regulation of BMSC-derived exosomes on macrophage polarization in vitro. LPS was used to induce RAW264.7 polarization to the M1 phenotype. Cells were incubated with LPS (50 ng/mL) or LPS (50 ng / ml) + Exo (1 μg/mL) for 24 h, and then the cells and supernatant were collected for assaying. Flow Cytometry and immunofluorescence were used to detect macrophage typing, and ELISA was used to determine the cytokine content in the supernatant. Immunofluorescence results showed that the proportion of M1 macrophages (F4/80+iNOS+) was significantly up-regulated after LPS treatment, while the proportion of M2 macrophages (F4/80+Arg1+) was not noticeably different. Compared with the LPS group, the M1 ratio of the exosomes-treatment group was significantly decreased, while the M2 ratio was increased (Figure 6C–6F). The results of flow cytometry experiments showed that the proportion of CD11c+CD11b+(M1) in the Exo-treatment group was significantly decreased, while the proportion of CD206+CD11b+(M2) was increased (Figure 6B). The ELISA results in the supernatant showed that the levels of the pro-inflammatory cytokines IL-1β, IL-6, TNF-α were significantly decreased in the exosomes-treatment group, while the levels of the anti-inflammatory cytokine IL-10 was increased (Figure 6G–6J). Those data indicate that BMSC-derived exosomes promote the phenotypic transformation of macrophages from M1 to M2 in vitro and reduce inflammation.

Figure 6. BMSC exosomes promote the phenotypic transformation of macrophages from M1 to M2 and reduce inflammation. (A) The protocol of the co-culture system. (B) Detection of CD11c (M1 marker) and CD206 (M2 marker) expression in RAW264.7 cells by flow cytometry. (C) and (D) Detection of INOS (M1 marker) by immunofluorescence in RAW264.7 cells and statistical results. Scale bar: 100 μm. (E) and (F) Detection and analysis of Arg-1 (M2 marker) in RAW264.7 cells by immunofluorescence and statistical results. Scale bar: 100 μm. (G–J) Determination of the cytokines, IL-1β, IL-6, TNF-α and IL-10 in supernatants. *p < 0.05 compared to the Control group; # p < 0.05 compared to the LPS group.

Transformation of macrophage polarization maintains chondrogenic characteristics and inhibits hypertrophy of chondrocytes

In order to investigate whether changes in macrophage polarization have an effect on chondrocytes, we co-cultured macrophages with chondrocytes for 72 h, and then analyzed the chondrocytes (Figure 6A). The results of immunofluorescence assays showed that compared with the control group, the collagen X gene of the cartilage hypertrophy in the LPS-treatment group was significantly up-regulated, while the proportion of the collagen II gene was decreased (Figure 7A–7D). Compared with the LPS group, the expression of collagen X in the Exo-treatment group was significantly decreased, while that of collagen II was significantly increased. Western blot results showed that compared with the LPS group, the expression of collagen X and runx2 were decreased, while the expressions of collagen II and sox9 were increased (Figure 7E–7I). The results of toluidine blue staining showed the markedly increased expression of GAG in chondrocytes cultured with Raw264.7 cells induced by LPS and exosomes (Figure 7J). These data indicate that the phenotypic conversion of macrophages from M1 to M2 maintain chondrogenic characteristics and inhibit hypertrophy of chondrocytes.

Figure 7. Macrophages treated with BMSC exosomes maintain chondrogenic characteristics and inhibit hypertrophy of chondrocytes. (A) and (B) Detection of the expression of collagen II by immunofluorescence in chondrocytes and statistical results. Scale bar: 100 μm. (C) and (D) Detection of the expression of collagen X by immunofluorescence in chondrocytes and statistical results. Scale bar: 100 μm. (E–I) Detection of the expression of collagen II, collagen X, SOX9 and Runx2 in chondrocytes by western blotting and statistical results. (J) Image of toluidine blue staining of chondrocytes. Scale bar: 200 μm. *p < 0.05 compared to the Control group; # p < 0.05 compared to the LPS group.

Isolation and culture of cells

All animal experiments were performed in accordance with the Animal Research Committee of Nanjing Medical University. Rats were euthanized by CO₂ inhalation, the knee joint was separated, the soft tissue around the joint was removed, and the cartilage tissue was cut into pieces for use. The femur and sacral cavity were rinsed repeatedly, the rinsing solution was collected, the pellet was resuspended after centrifugation and inoculated in Dulbecco's Modified Eagle’s Medium/Nutrient Mixture F-12 medium (DMEM/F-12, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% cyan-streptomycin (P/S, Sigma, USA) at 37° C in humid air with 5% CO₂. Cartilaginous tissue was washed three times with phosphate buffered saline (PBS, Gibco, USA) and digested with 0.2% type II collagenase (Sigma-Aldrich, USA) at 37° C for 6-8 h. The pellet was resuspended after centrifugation and inoculated in high-glucose Dulbecco's Modified Eagle’s Medium (HG-DMEM, Gibco, USA) containing 10% FBS and 1% P/S at 37° C in humid air with 5% CO2. All medium was replaced every 3 days and passaging was performed when 80%~90 of cells is merged.

Identification of BMSCs

For surface markers, five-passage BMSCs were used with flow cytometry to identify the mesenchymal markers, CD44 and CD90, and the hematopoietic cell surface marker, CD45. For multidirectional differentiation potential, osteogenic induction medium, adipogenic induction medium and chondrogenic induction medium (all from Cyagen, China) were used to replace the complete medium to induce differentiation. The process of inducing differentiation was performed as described in the manufacturer's manual.

Preparation and identification of exosomes

When BMSCs fused to 70%-80%, they were washed twice with PBS and then incubated in serum-free stem cell medium for 48 h (Gibco, USA). The medium was collected and centrifuged at 300 × g for 10 min and then centrifuged at 2,000 × g for 10 min at 4° C. The supernatant was collected and filtered using a 0.22 μm filter to remove cell debris. The supernatant was then transferred to an Amicon Ultra-15 centrifugal filter (Burlington, USA) and centrifuged at 4,000 × g at 4° C until the volume was reduced to 200 μL. Then, the sample was washed twice by ultrafiltration PBS and ultrafiltered again to 200 μL.

For exosome purification, the medium was applied to a 30% sucrose/D₂O pad in an Ultra-Clear^TM tube (Beckman Coulter, USA) and centrifuged at 100,000 × g for 60 min at 4° C using an optima L-100 XP Ultracentrifuge (Beckman Coulter, USA). Partially purified exosomes were recovered using an 18 g needle, diluted with PBS, and centrifuged through the filter unit at 4,000 ×g at 4° C until the final volume reached 200 μL. The solution was stored at -80° C or used immediately for the experiment. The concentration and size distribution of exosomes were measured using Nanosight Tracking Analysis (NTA). Transmission electron microscopy (TEM) was used to identify the morphology of exosomes. Exosome surface marker proteins, including CD81, CD9 and CD63 were analyzed by western blot.

Exosomes uptake

To fluorescently label exosomes, Dil solution (Eugene, USA) was added to PBS and incubated with exosomes according to the manufacturer's instructions. Excess Dil dye was removed by ultracentrifugation at 100,000 × g for 1 h at 4° C. The pellet was washed three times with PBS and resuspended. The Dil-labeled exosomes (Dil-exosomes) were incubated with RAW264.7 cells for 24 h. Then, cells were washed with PBS and fixed in 4% paraformaldehyde, and the uptake of Dil-exosomes was observed using laser confocal microscopy. Dil-exosomes were also injected into the joint cavity of model rats (described below). After 2 h, the rats were anesthetized and the knee joint was removed for the preparation of sections. Sections were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescence microscope.

Incubation of exosomes and RAW264.7 cells

The murine macrophage RAW264.7 cell line was purchased from the American Type Culture Collection (ATCC) and cultured in HG-DMEM medium containing 10% FBS and 1% P/S. The cells cultured in that medium were defined as M0 macrophages. M0 macrophages were incubated with 50 ng / ml LPS (Beyotime, China) or 50 ng/mL LPS + 1 μg/mL BMSC-derived exosomes for 24 h. Cells were harvested after centrifugation and macrophage polarization was identified using flow cytometry and immunofluorescence. The supernatant was collected and tested for cytokine expression levels.

Co-culture of RAW264.7 cells and chondrocytes

RAW264.7 cells were co-cultured with chondrocytes utilizing Transwell cell culture inserts (Corning, USA) with a 0.4 μm pore sized membrane. Briefly, RAW264.7 cells were seeded on the upper chamber of the co-culture system with 50 ng/mL LPS or 50 ng / ml LPS + 1 μg / ml BMSC-derived exosomes for 24 h. Then, the RAW264.7 cells were washed three times with PBS and chondrocytes were seeded into the lower chamber of the co-culture system. RAW264.7 cells were then co-cultured with chondrocytes for 72 h, after which chondrocytes were collected and the phenotype was identified by western blotting and immunofluorescence.

Western blot analysis

Total protein was extracted from cells and tissues, and the protein concentration was determined using a BCA assay kit (Beyotime, China). SDS loading buffer (5X) was added to the protein sample, boiled for 10 min to denature the proteins, and stored at -20° C or used immediately for testing. The protein sample was loaded onto an SDS-PAGE gel and transferred to a 0.22 um PVDF membrane by electrophoresis. After membrane transfer, the PVDF membrane was incubated with blocking solution (Beyotime, China) for 2 h at room temperature and then incubated with the following primary antibodies overnight at 4° C (Abcam, USA): anti-Collagen X antibody, anti-Collagen II antibody, anti-Runx2 antibody, anti-SOX9 antibody, and anti-GAPDH antibody. After washing three times with TBST for 10 min each time, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA) for 1 h at room temperature. Blots were detected using an enhanced chemiluminescence kit (Beyotime, China).

Cell fluorescence analysis

Cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 20 min, and blocked with PBS containing 3% bovine serum albumin (BSA, Yeasen, China) for 1 h. Then, cells were incubated overnight with the following primary antibodies: anti-F4/80 antibody (eBioscience, USA), anti-iNOS antibody (Abcam, USA), anti-ARG1 antibody (Proteintech, China), anti-Collagen X antibody (Abcam), USA), and anti-Collagen II antibody (Abcam, USA). Alexa 488 or Alexa 594 dye-labeled secondary antibodies (Jackson, USA) were added and incubated for 1 h at room temperature. Nuclear staining was performed for 5 min by the addition of 4',6-diamidino-2-phenylindole (Beyotime, China) solution. Immunoreactivity was visualized using a fluorescence microscope (Carl Zeiss, Germany).

Flow cytometry analysis

Cells were washed with PBS and incubated with 3% BSA for 1 h to block non-specific antigen binding. Cells were resuspended after centrifugation and incubated with the following fluorescein-conjugated antibodies for 30 minutes at 37° C in the dark: FITC anti-CD44 antibody (BD Biosciences, USA), PE anti-CD90 antibody (BD Biosciences, USA), PE anti-CD45 antibody (BD Biosciences, USA), PE anti-CD11b antibody (BD Biosciences, USA), APC anti-CD206 antibody (eBioscience, USA), and FITC anti- CD11c antibody (BD Biosciences, USA). Cells were then collected and washed with PBS after centrifugation. Fluorescence was evaluated by BD Cyan flow cytometry (BD Bioscience, USA) and further analysis was performed using FlowJo software (Tree Star Inc., USA).

Toluidine blue staining

To observe secreted GAG in the cartilage matrix, chondrocytes cultured in the co-culture system were washed with PBS and fixed in 4% paraformaldehyde for 15 min, then stained with toluidine blue (Solarbio, China), according to the manufacturer's instructions. The results with an optical microscope (Leica, Germany).

OA model and experimental group

Healthy adult male Sprague–Dawley rats (weighing 200−250 g) were purchased from the Animal Center of Nanjing Medical University (Nanjing, Jiangsu, China). In the animal facility, all animals were maintained under specific pathogen-free (SPF) conditions. The experimental procedure was approved by the Animal Care and Use Committee of the Institutional Laboratory of Nanjing Medical University, and maximized attention to limiting the number of animals used in this study. The OA model was induced using the modified Hulth technique. Chloral hydrate (10%; 0.1 mL/100 g) was intraperitoneally injected into the anesthetized rats, the right knee joint was taken longitudinally 2 cm, the anterior cruciate ligament was cut, the medial meniscus was removed, sutured layer by layer and intramuscular injection of penicillin 2 × 10 U/kg per day for a total of 5 days. All rats undergoing surgery or sham surgery were randomly divided into three groups (n=12 in each group): (1) Sham group; (2) PBS group; (3) BMSC-derived exosomes group. At 4 weeks post-operation, rats were injected intra-articularly with 10 ul of PBS or BMSC-derived exosomes (10¹⁰ particles/ml) using a microsyringe for 3 days for 4 weeks.

Micro CT analysis

Knee joint samples were fixed in 4% paraformaldehyde and micro computed tomography (Micro CT) of the fixed knee joint specimens was performed using a photomicrography imaging system (ZKKS MCT Sharp III scanner, China). The small field was selected for scanning and correcting the CT value with a 70kV scan voltage, 30 W power, 429 μA current, and 5 μm scan thickness. 3D-Matic Research 11.0 software was used for 3D knee reconstruction and image capture. The region of interest (ROI) was selected from the periarticular epiphyses using Mimics Research 19.0 software. The ROI size was blindly calculated on all four condyles of the knee (the medial and lateral sides of the tibia and femur) and the mean was used for statistical analysis.

Histological analysis

The knee joint was fixed in 4% paraformaldehyde, decalcified in 20% formic acid, and embedded in paraffin. By collecting 5 μm sections at intervals of 50 μm, continuous sagittal sections, including the entire joint were obtained. Sections were stained with Safranin O-fast green and HE for histological analysis. The middle section was used for immunohistochemistry and immunofluorescence analysis. Synovial activation was assessed by scoring the cell thickness of the synovial lining (0-3) using HE stained slides [37, 38]. The medial and lateral compartments of the joint were scored separately and given the sum of the two scores (maximum site score 6). Cartilage degeneration was graded in Safranin-O/Fast Green-stained sections using the Mankin criteria, modified by the International Association of Osteoarthritis Research (OARSI).

Immunohistochemistry and immunofluorescence

Samples were prepared as previously described. After dewaxing and rehydration, the sections were soaked in citrate buffer at 60° C overnight to reveal the antigen. For immunohistochemical staining, we added 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase activity, and the sections were blocked with 1% goat serum for 1 h at 37° C, and incubated with the following primary antibody overnight at 4° C: anti-Collagen X antibody (Abcam, USA) and anti-Collagen II antibody (Abcam, USA). Samples were then stained with horseradish peroxidase-conjugated secondary antibody (Abcam, USA). Then, 3,3-diaminobenzidine was observed for chromogen and counterstained with hematoxylin. For immunofluorescence, the sections were incubated with primary antibody (anti-ARG1 antibody (Proteintech, China) overnight at 4° C, and then stained with Alexa 488 or Alexa 594 dye-labeled secondary antibody. Nuclei were labeled with DAPI and images were obtained using a fluorescence microscope.

Cytokine determination

The synovial fluid samples of OA rats and the supernatant samples of RAW264.7 cells were collected. Cytokine levels were measured using the following ELISA kits, according to the manufacturer's protocol (All from Abcam, USA): Mouse IL-6 ELISA Kit, Mouse IL-1 beta ELISA Kit, Mouse TNF alpha ELISA Kit, Mouse IL-10 ELISA Kit, Rat IL-6 ELISA Kit, Rat IL-1 beta ELISA Kit, Rat TNF alpha ELISA Kit, and Rat IL-10 ELISA Kit. The results were tested using a multifunctional enzyme marker.

Statistical analysis

IBM SPSS Statistics v20.0.0 software was used to process and analyze the data and images. Data are expressed as the mean ± SEM. Statistical differences were assessed using the Student's t-test, or one-way or two-way ANOVA. A P-value <0.05 was considered significant.

Ethics approval

All animal experiments were approved by the Animal Care and Use Committee of the Institutional Laboratory of Nanjing Medical University (30^th 2019 with ethics code: IACUC-1903035).