The experimental protocol related to rats was in accordance with the US National Institutes of Health’s Guidelines of Laboratory Animal Use and approved by the Ethics Committee of Nanjing First Hospital.
Isolation and culture of MSCs
Bone marrow-derived MSCs (BMSCs) were obtained from the bone marrow of eight-week-old male Sprague-Dawley (SD) rats, which were acquired from the Qinglongshan Experimental Animal Center of Jiangsu Province, China. The MSCs were identified based on their surface phenotypes and multipotency, as described previously[14, 15]. After the SD rats were euthanized, the bone marrow from the femur was washed out in a Petri dish with low-glucose Dulbecco’s modified Eagle’s medium (DMEM; KeyGEN, Nanjing, Jiangsu, China), containing 10% fetal bovine serum (FBS; KeyGEN), without exosomes. Then, the cells were cultured in culture dishes with 5% CO2/95% air at 37°C. After 72 h, the medium was changed, and the cells were resuspended with 0.25% trypsin until the cells reached 80%–90% confluence. Thereafter, the cells were re-seeded at the density of approximately 2 ×106 cells per dish. The follow-up experiment was performed using the third-generation cells.
Activation and inhibition of autophagy
To determine the role of autophagy in MSC exosome release, the cells were treated with the autophagy inhibitor 3-methyladenine (3-MA; Selleck, Houston, TX, USA) (10 μM) or the agonist rapamycin (Selleck) (10 μM) for 24 h. The cells of the control group were treated only with the solvent dimethylsulfoxide (DMSO).
Analysis of autophagy in MSCs
Transmission electron microscopy (TEM) (JEM-1011, JEOL, Akishima, Tokyo, Japan) was used to observe the autophagosomes of MSCs, as described previously[14]. The morphological characteristics of autophagosomes include crescent or cup-shaped, bilayer or multilayer membrane, with a tendency to enclose cytoplasmic components. Immunofluorescence analysis was performed to detected a specific autophagy marker LC3, as described previously[14]. Briefly, the cells were washed with phosphate-buffered saline (PBS) (KeyGEN) and fixed with 4% paraformaldehyde. Then, the cells were again washed with PBS and incubated with 0.5% Triton X-100 (KeyGEN). Subsequently, the cells were incubated with a blocking solution (10% goat serum in PBS) and then treated with the anti-LC3B antibody (Novus Biological, Littleton, CO, USA) overnight at 4℃. Finally, the cells were incubated with secondary antibodies after washing with the blocking solution. The MSCs were stained with diamidine phenylindole (DAPI) (Molecular Probes, Waltham, MA, USA) and observed under a confocal microscope (Dmi 6000-B, Leica, Brunswick, Germany). Western blot analysis was used to detect the autophagy makers Beclin1 and LC3, as described previously[16].
Isolation and analysis of exosomes
The culture medium of MSCs was collected and centrifuged in a 50-mL centrifuge tube at 4℃ for 10 min, and the dead cell precipitate was removed. The supernatant was collected and placed in another 50-mL centrifugation tube. The cell debris and macromolecular proteins were further removed through centrifugation at 4℃ and 2000 ×g for 20 min. The supernatant was collected again in a 50-mL centrifuge tube and then centrifuged at 4℃ and 10000 ×g for 30 min; the supernatant was collected again and passed through the 0.22-μm cell filter. The supernatant was collected in an ultracentrifugation tube with a diameter greater than 0.22 μm and then subjected to ultracentrifugation for 70 min at 4℃ and 12000 ×g. The supernatant was discarded, and the precipitate was resuspended with PBS and centrifuged again at 4℃ and 12000 ×g for 70 min. Finally, the exosome precipitate was obtained, resuspended with 50 μL PBS, and stored at −80℃. The exosome morphology was observed through TEM, and then, the exosomes were quantified through the nanoparticle tracking analysis (NTA) (NanoSight NS300, Malvern Instruments Ltd, UK). Proteins were extracted from MSCs and exosomes by using a total protein extraction kit (KeyGEN), and the exosome surface marker proteins, namely CD63, ALIX, and TSG101, were analyzed through western blotting.
OA chondrocyte isolation and culture
The OA chondrocytes were isolated from the OA rat model. The rat OA model was generated through anterior cruciate ligament transection (ACLT), as described previously[17]. The cartilage was obtained from the femoral condyle of OA rat right knee joint and placed in a Petri dish filled with PBS. The cartilage was cut into small fragments and digested with 2 mL of 0.25% trypsin for half an hour, followed by incubation with 0.2% type II collagenase at 37℃ for 4 h. After the cartilage pieces were digested, they were washed with DMEM and collected through centrifugation at 1000 rpm for 10 min. The isolated chondrocytes were cultured in culture dishes with complete DMEM in 5% CO2/95% air at 37°C. When the chondrocytes reached 80%–90% confluence, they were split and cultured to approximately 2 × 106 cells per culture dish.
OA chondrocyte co-culture with MSCs
The OA chondrocyte and MSC co-culture was established in a 0.4-μm diameter co-culture chamber (Millipore, Massachusetts, USA), as shown in Figure 4A. In the co-culture chamber, MSCs (1 × 106 cells/well) were placed into the apical chamber, whereas OA chondrocytes (1 × 106 cells/well) were placed into the basolateral chamber, and the co-culture chamber was placed in a six-well plate[18]. The culture medium was changed every four days, and the morphological changes in the cells were observed under a microscope.
Immunocytochemistry
COL2 expression in the OA chondrocytes was detected through immunocytochemistry, as described in our previous study[19]. The OA chondrocytes in the transwell co-culture system were fixed with 4% paraformaldehyde for 30 min, washed with PBS, and incubated with a 3% H2O2–methanol solution at room temperature for 10 min. Subsequently, the chondrocytes were washed thrice with PBS and blocked and incubated with goat serum (50–100 µL) at room temperature for 20 min. The cells were then incubated with COL2 antibodies (Abcam, Cambridge, UK) at 37°C for 2 h and washed thrice with PBS. Thereafter, 50 µL of an intensifier was added, and the cells were incubated at room temperature for 30 min. Then, the cells were washed with PBS and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit-(Fab)2 antibodies (Santa Cruz,Dallas, TX, USA) at 37°C for 30 min. Finally, the cells were washed with PBS again and subjected to diaminobenzidine (DAB) staining for color development. COL2 expression was observed under a microscope, and the images were captured.
Toluidine blue staining
To determine the expression of AGG, the OA chondrocytes in the transwell co-culture system were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 20 min. Then, the cells were again washed with PBS and stained with toluidine blue for 30 min. Finally, the cells were observed under a microscope, and the images were photographed.
GW4869 treatment
In vitro study: to inhibit exosome release by MSCs, the secretory-specific inhibitor of exosomes, GW4869 (Selleck, Houston, TX, USA) (10 μM), was added to the co-culture system in the transwell chamber. The cells of the control group were treated with only the solvent DMSO.
In vivo study: GW4869 (10 μM) was added to the MSC suspension and injected into the right knee joint of the OA rat.
LIPUS stimulation
In vitro study: the LIPUS transducer (HT2009-1, Ito Corporation, Tokyo, Japan) was placed under a Petri dish and coated with a coupling agent. Then, the LIPUS (50 mW/cm2, on–off ratio of 20%, frequency of 3 MHz) waves were transferred through the bottom of the Petri dish, as described previously[14]. The control group was subjected to sham LIPUS stimulation with no ultrasound irradiation. The cells were stimulated for 20 min once a day for 0, 3, 7, and 10 days in 5% CO2/95% air at 37℃.
In vivo study: the knee joint of the rats was exposed to LIPUS after MSC intra-articular injection (once every 7 days for 4 times). The LIPUS parameters were similar to those in the in vitro study: 20 min/day for 4 weeks (28 days).
Western blotting
The extracted total protein was subjected to 10% sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Primary anti-Beclin1 and anti-β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-LC3 and anti-AGG antibodies were purchased from Novus Biological; anti-CD63, ALIX, and TSG101 antibodies were purchased from Santa Cruz; anti-COL2 antibody was purchased from Abcam. Following treatment with the goat anti-mouse secondary antibody (Santa Cruz), blot signals were observed using the electrochemiluminescence (ECL) western blotting substrate (KeyGEN).
Animal experiments
A total of 30 eight-week-old male SD rats (weighing 250–300 g) were used to establish OA models, and the OA rats were divided into the following five groups(six rats in each group): control group, MSC group, MSCs + GW4869 group, MSCs + LIPUS group, and MSCs + GW4869 + LIPUS group. The OA rat model was generated through ACLT, as described in our previous study[17]. The control group OA rats received vehicle injections of 0.9% normal saline and a sham LIPUS stimulation, whereas the MSC group OA rats were administered an intra-articular injection of MSCs (1 × 106 MSCs were resuspended with 50 μL normal saline) through their right knee joint and a sham LIPUS stimulation. In the MSCs + GW4869 group, the OA rats received an intra-articular injection of MSCs, with addition of GW4869 and a sham LIPUS stimulation. In the MSCs + LIPUS group, the OA rats received LIPUS stimulation after an intra-articular injection of MSCs. In the MSCs + GW4869 + LIPUS group, the OA rats received an intra-articular injection of MSCs, with addition of GW4869 and LIPUS stimulation.
All the rats were euthanized four weeks after treatment and then subjected to histopathological examination for observation of the femoral condylar cartilage. Safranin-O/fast green staining was used to detect pathological changes in the cartilage, including surface irregularities and crack formation. In addition, we used the Mankin scores to evaluate the extent of fibrosis, matrix distribution, cartilage loss, and chondrocyte colonization (Table 1). Finally, we extracted the protein from the tibial plateau articular cartilage and determined the expression levels of COL2 and AGG through western blotting.
Table 1
Mankin scoring scale
|
Subgroup 1: fibrillation;
1.Even surface
2.Uneven surface
3.Fibrillated and fissured within superficial zone only
4.Fissures and erosions extending below the surface zone, without extending beyond the radial zone
5.Fissures and erosions extending into the deeper zone
Subgroup 2: matrix distribution
1.Normal staining
2.Moderate loss in staining
3.Severe loss in staining
4.No staining
Subgroup 3: chondrocyte loss
1.Loss extending into superficial zone
2.Loss extending into midzone
3.Loss extending into radial zone
Subgroup 4: chondrocyte cloning
1.No clusters
2.Chondrocyte clusters in superficial zone
3.Chondrocyte clusters in superficial to midzone(less than four cells)
4.Chondrocyte clusters of more than four cells located in superficial to midzone, or chondrocyte clusters in deeper zone
|
| Grading was performed separately for the medial femoral condyle, lateral femoral condyle, medial tibial plateau and lateral tibial plateau. The minimum total score was 4, and the maximum total score is 16. |
Statistical analysis
All data are reported as the mean ± standard deviation (SD) and analyzed using SPSS 23.0 software (IBM, Armonk, NY, USA). Statistical comparisons between the groups were performed using single-factor analysis of variance. The Mankin scores were analyzed using the Wilcoxon signed rank test. A p value of 0.05 was considered to indicate statistical significance.