4-week-old male SD rats with SPF grade, body weight about 100g, purchased from Changzhou Cavens Experimental Animal Co., Ltd. The rats were free to move in the cage and eat normally at (25 ± 2) ℃.
Cell extraction and cell culture
Culture of chondrocytes. The rats were killed by neck removal, and the knee joint of the rats was separated aseptic. The connective tissue of cartilage surface was removed, and hyaloid cartilage was taken, washed with PBS (Thermo Fisher Scientific), and the tissue was cut into 1 mm3 pieces. 0.25% trypsin was predigested at 37℃ for 30 minutes, and the discard solution was added with 0.2% type II collagenase for digestion at 37℃ for 4h. Chondrocytes collected from digestion were suspended in DMEM medium (Thermo Fisher Scientific) containing 10% PBS and cultured at 37℃ and 5% CO2 for 8-10 days.[24, 25].
Culture of BMSCs. The rats were sacrificed by neck removal, disinfected with 75% alcohol and rinsed with PBS. The right tibia and femur of the rats were taken, the bone ends were removed, and the bone marrow cavity was exposed. The bone marrow cavity was fully rinsed with DMEM medium and the bone marrow was collected, centrifuged at 1500 r/min for 10 minutes. Cells were collected and suspended again, and cultured in DMEM medium containing 15% FBS.
Extraction and identification of Exosomes
The cultured BMSCs grew to about 90% of the surface area of the culture dish. The culture medium was discarded, washed with PBS for 3 times, and the serum-free medium containing 1% penicillomycin DMEM was added. After 24 hours of culture, the conditioned culture medium was collected and stored at 4℃. Cell conditioned culture medium was collected to 300ml and centrifuged by supernatant centrifuge. After supernatant was removed, the cell conditioned culture medium was cleaned and suspended by PBS. 10μl exosome suspension was dropped onto the copper wire, and the samples were dried at room temperature. Morphological characteristics of exosome were observed under transmission electron microscope. The exosomes added in this experiment were all Exo-100 μg/ml.
Cell Counting Kit-8 (CCK-8) was performed to test cell viability. The chondrocytes were inoculated on a 96-well plate, and the cells were treated in groups after adherence, and cultured for 48 h. Then CCK-8 reagent was added and incubated for another 4h. The absorbance value (OD value) of the cells was measured at 560 nm of the microplate.
Wound healing assay
The chondrocytes were cultured in 6-well plates to 70-80% confluency and were wounded with a 200-μl sterile pipette tip. After washing with PBS, the cells were cultured in serum-free medium. Images were acquired at each time point (0 and 24 h).
The apoptosis rates were determined by using Pharmingen annexin V-FITC Apoptosis Detection Kit I (BD, USA) according to the manufacturer’s instructions. Chondrocytes were inoculated with 6-well plates. When the cell density was about 80%, corresponding complete culture medium was added to each group, and cells were collected after 24 and 48 hours of culture. FITC and PI staining were performed according to the instructions of the kit, and the cell death was analyzed and quantified by flow cytometry within 1 hour (BD Biosciences, USA).
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Thermo Fisher, USA) and reversely transcribed into cDNA according to manufacturer’s instructions (Takara Biological, Japan). Real-time PCR with gene-specific primers was performed on the resulting cDNA using a Step One Plus real-time PCR system (Applied Biosystems) in the presence of SYBR Green PCR Master Mix (Applied Biosystems). The relative expression level of mRNA was calculated using 2-ΔΔCt method.
Oil red O staining
The saturated solution of oil red O (O8010; Solarbio, China) isopropyl alcohol was mixed with distilled water in a ratio of 3:2 to prepare the dyeing solution and soaked for 20 minutes. The treated cells were washed twice by PBS and fixed with 4% paraformaldehyde at room temperature for 30 min. For lipid identification, cells were stained with the prepared solution for 30 minutes and the absorbance was measured at 490 nm.
Toluidine blue staining
The slides were sterilized, treated with polylysine, placed on plates and inoculated into 6-well plates with low density chondrocytes. The cells were placed in a 5% CO2 37°C incubator and then fixed with 4% paraformaldehyde for 30 minutes. The fixate was rinsed with double steam water and dyed with 0.04% toluidine blue for 2h. The dye was removed with anhydrous ethanol and the cells were rinsed 3 times with PBS, followed by xylene cleaning and sealing. Tissue is viewed and photographed under a light microscope.
Expression and transfection of HIF-1α
The third generation rat BMSC with cell density of 1×106/ml were transfected with 5 ml human medium and adenovirus vector carrying HIF-1α, and the medium was changed every 24 h after transfection. The transfection efficiency was observed by fluorescence microscope 3 days after transfection.
Chondrocytes were cultured in 6-well plates, treated with inflammatory factors and exosomes, and proteins were extracted from cell lysates RIPA buﬀer (Thermo Fisher, USA). BCA protein assay kit (P1511-1) (Applygen Technologies Inc. China) was applied to measure the protein concentration in lysates according to the manufacturer’s instructions. Electrophoresis was performed by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Then, the primary antibody was added and incubated overnight at 4℃, and the membrane was washed 3 times every other day. The second antibody was added and incubated for 1h at room temperature, and then the membrane was washed for 3 times before exposure. β-actin and GAPDH were used as internal reference in the detection of proteins on exosomes and chondrocytes individually. Image J software was adopted to measure the gray value of protein bands.
Formalin fixed tissue, paraffin-embedded tissue or paraformaldehyde fixed cells were stained by immunohistochemistry. The fixed tissues or cells were infiltrated by blocking solution (0.1% Triton-x), then incubated with primary antibody overnight at 4°C, and washed with PBS for 3 times before incubating with secondary antibody. The photos were collected with a digital camera (Nikon).
All data was analyzed by SPSS 20.0 and GraphPad Prism 6.0 (GraphPad Software, San Diego, CA). The experimental results are presented as the means ± standard deviation (SD). Student’s t-test and one-way analysis of variance were used to assess differences between groups. P < 0.05 was considered statistically significant.