Human umbilical-cord-derived MSCs (hMSCs)
hMSCs were isolated from umbilical cords of five healthy donors. The experimental design was approved by the Institutional Ethical Committee (Zhengzhou Central Hospital, Henan, China). Samples were obtained from donors after they provided informed consent according to the Helsinki Declaration of 1975, as revised in 2013.
Samples were washed with phosphate-buffered saline (PBS) and minced and digested at 37 °C for 1 h with 2% type I collagenase (Gibco, Life Technologies, Madrid, Spain). Digested tissue was filtered through a 100-µm cell strainer (BD Biosciences, Durham, NC, USA). Cells were then washed with Dulbecco's Modified Eagle Medium (DMEM)/Ham's F12 (Sigma-Aldrich, St. Louis, MO, USA) containing penicillin and streptomycin (1%), seeded into tissue culture flasks (1–2 × 106 cells/mL) in DMEM/Ham's F12 medium with penicillin and streptomycin (1%), supplemented with 15% extracellular vesicle-free human serum, and incubated at 37 °C in a humidified atmosphere of 5% CO2. Human serum was obtained from whole-blood donations of AB blood-group-typed donors according to the criteria of Valencia Transfusion Centre. To eliminate the extracellular vesicle fraction, serum was centrifuged for 18 h at 120,000 × g and 4 °C using a SW-28 swinging-bucket rotor (Beckman Coulter, Brea, CA, USA). When cells reached semiconfluence, culture plates were washed and the MSC phenotype confirmed by flow cytometry (Cytoflex, Beckman Coulter) using the specific antibodies anti-CD73-PE, anti-CD90-APC, anti-CD105-APC-A750, anti-CD34,anti-CD45, anti-CD11b, anti-CD19, and anti-HLA-DR-FITC (Biolegend, San Diego, CA USA), and measuring cell viability with propidium iodide staining. Finally, conditioned medium (CM) was collected from cultured cells at passage 0 every 48 h of culture. CM was pooled, centrifuged, and stored under sterile conditions at -80 °C prior to further use.
Isolation Of Exosomes
Exosomes were obtained from hMSC CM using a filtration/centrifugation-based protocol34. Cellular debris was eliminated by centrifugation at 300 × g for 10 min. Vesicles were then collected from the supernatant through differential centrifugation steps. CM was filtered through an 800-nm filter (Merck, Darmstadt, Germany) and centrifuged at 12,200 × g for 20 min at 4 °C to pellet microvesicles. Then, supernatants were filtered through a 200-nm filter (Merck) and centrifuged at 100,000 × g for 90 min at 4 °C. Pellets were washed once with sterile PBS, resuspended in 15 µL PBS, and stored at -80 °C until further use.
Human Chondrocyte Cultures
The knee specimens were obtained from three females and two males, 69.4 ± 7.2 years of age (mean ± standard error of the mean [SEM]), with diagnosis of advanced OA undergoing total knee arthroplasty. Diagnosis was based on clinical, laboratory, and radiological evaluation. The study design was approved by the Institutional Ethical Committee (Zhengzhou Central Hospital, Henan, China). Samples were obtained with patient’s consent according to the Declaration of Helsinki. Knee articular cartilages samples were obtained and cut into about 1mm3 pieces. Tissues were digested with 0.25% trypsin-EDTA for 30 min and collagenase II for 4 h and then filtered. After rinsing, the chondrocytes were cultured in DMEM with high-dose (4.5 g/L) glucose, 10% fetal bovine serum, and 1% penicillin/streptomycin at 37 °C with 5% CO2. Second-passage chondrocytes were used in the experiments to eliminate the influence of dedifferentiation on experimental results.
Toluidine Blue Staining For Morphological Identification Of Chondrocytes
The chondrocytes were inoculated into a six-well plate and, when the cells reached 50–60% confluence, the culture medium was discarded. The chondrocytes were then fixed in 4% paraformaldehyde for 30 min, stained with 1% toluidine blue at room temperature for 10–30
min, washed with absolute ethyl alcohol until the cells were colorless, and observed under an inverted microscope (Olympus, Tokyo, Japan). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).
Experimental Design
The H+-ATPase inhibitor bafilomycin A1 (0.1 µM) was used to measure the role of hMSC-Exos or hMSC-Exos combined with 50 ng/mL TNF-α in autophagic flux. Then, the NF-κB pathway involved in autophagic activation was investigated. When cells were treated with 50 ng/mL TNF-α and hMSC-Exos or specific COX-2 inhibitor NS398 (10 µM), hMSC-Exos and NS398 were always added to the medium 1 h prior to TNF-α addition. In all experiments, chondrocytes were treated with TNF-α for 24 h. During the autophagic flux assay, 0.1 µM bafilomycin A1 was added to the medium 1 h prior to addition of hMSC-Exos or hMSC-Exos combined with 50 ng/mL TNF-α, followed by co-incubation for 24 hours.
RNA extraction and quantitative (q)PCR
Total RNA was isolated from tissues or cell lines using Trizol reagent (Invitrogen, USA). RNA was reversed transcribed into cDNA using the PrimeScript™ one step
RT-PCR kit (TaKaRa, China) according to the manufacturer’s protocol. The mRNA level was measured using the SYBR® Premix DimmerEraser™ kit (TaKaRa, China)
and the ABI7500 system (Applied Biosystems, Foster City, CA, USA). Relative mRNA expression was calculated using the 2−ΔΔC(T) method and normalized to β-actin. The primer sequences are listed in Additional file 1: Table 1
Table 1
Primer sequences for MMPs, ADAMTSs, chemokines, and GAPDH. GAPDH = glyceraldehyde phosphate dehydrogenase; MMP = matrix metalloproteinase; ADAMTS = a disintegrin and metalloproteinase with thrombospondin motifs; CCL = CC chemokine ligand; CXCL = CXC chemokines ligand.
Gene
|
|
Sequences (5′–3′)
|
Product size (bp)
|
Accession no.
|
MMP3
|
forward
|
ATGAACGATGGACAGATGA
|
19
|
NM_133523.3
|
TGAGAGAGATGGAAACGG
|
18
|
MMP9
|
forward
|
GTCTTCCCCTTCGTCTTC
|
18
|
NM_031055.1
|
AAACCCCACTTCTTGTCAG
|
19
|
MMP13
|
forward
|
ATGAAACCTGGACAAGCA
|
18
|
NM_133530.1
|
GGACCATAGAGAGACTGGATT
|
21
|
ADAMTS4
|
forward
|
CGTGGTGTGTGTGTGTGT
|
18
|
NM_023959.1
|
AGAGGAAAGTAGGGCAGGT
|
19
|
ADAMTS5
|
forward
|
GTGTGTGGAGGGGATAACT
|
19
|
NM_198761.1
|
TCTGGTCTTTGGCTTTGA
|
18
|
CCL2
|
forward
|
TGCTGACCCCAATAAGGAATG
|
21
|
NM_031530.1
|
TGCTGACCCCAATAAGGAATG
|
22
|
CCL5
|
forward
|
GACACCACTCCCTGCTGCTT
|
20
|
NM_031116.3
|
ACACTTGGCGGTTCCTTCG
|
19
|
CXCL1
|
forward
|
GAAGATAGATTGCACCGATG
|
20
|
NM_030845.1
|
CATAGCCTCTCACACATTTC
|
20
|
GAPDH
|
forward
|
CAACGGGAAACCCATCACCA
|
20
|
NM_017008.3
|
ACGCCAGTAGACTCCACGACAT
|
22
|
Western Blotting
Chondrocytes in 10-cm dishes were washed with cold PBS and incubated with RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride, followed by cell scraping and centrifugation. Nuclear protein was isolated as recommended by the Beyotime (Shanghai, China). After measuring protein concentration with the bicinchoninic acid (BCA) assay, 40 µg total protein in loading buffer were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were immersed in 5% nonfat milk for 2 h to block nonspecific binding and then incubated with primary antibody (anti-LC3, 1:1000; anti-NF-κB, 1:500,abcom༌USA) overnight at 4 °C. After incubation with secondary antibody (1:2000), an enhanced chemiluminescence (ECL) detection system (Perkin Elmer, USA) with an ECL reagent was used to visualize proteins on the membrane. A semi-quantitative analysis of protein bands was performed using AlphaEaseFC 4.0 software (Alpha Innotech, San Leandro, CA USA).
Stable expression of stubRFP-sensGFP-LC3 and StubRFP-SensGFP-LC3-mut in chondrocytes
Lentiviral vector containing the stubRFP-sensGFP-LC3 and StubRFP-SensGFP-LC3-mut reporter was purchased from Genechem (Shanghai, China). Cells stably expressing stubRFP-sensGFP-LC3 were selected by treatment with puromycin (2 µg/ml). After different treatments, cells were fixed and analyzed by fluorescence microscopy (Olympus BX51, Japan).
Transmission Electron Microscopy (TEM)
Chondrocytes cultured in 10-cm dishes were treated with TNF-α and/or hMSC-Exo for 24 h and then harvested by manual scraping. After centrifugation, chondrocytes were fixed with 2.5% glutaraldehyde overnight and post-fixed with 1% osmium tetroxide for 2 h at 4 °C. After staining with 2% uranyl acetate, chondrocyte pellets were dehydrated through an acetone series and then embedded in Epon 812. After semi-thin sectioning, chondrocytes were stained with toluidine blue and observed under a light microscope. Finally, ultrathin sections were prepared based on light microscopic observations. Cellular ultra-structures were visualized using a transmission electron microscope (Hitachi, Japan)
Statistical analysis
Statistical assay was performed using GraphPad Prism 7.0 (GraphPad, San Diego, CA USA). All data in this study are shown as the mean ± standard error of the mean (SEM) of three independent experiments. The significance of the differences in mean values between and within multiple groups was examined by one-way ANOVA followed by Duncan’s multiple range test. P value < 0.05 was considered statistically significant.