The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University. All participants signed the informed consent documentation prior to specimen collection. All animal experimental manipulations were carried out in strict accordance with the guidelines for Animal Experiments published by the National Institutes of Health.
DPMSCs were obtained from CAR-T Biotechnology (Shanghai, China) and grown at 37°C in α-MEM supplemented with amino acids, vitamins, inorganic salts, and ribonucleosides in a 5% CO2 incubator. DPMSCs were seeded in tissue culture flasks at a density of 4 × 103 cells/cm2 overnight and then replaced with fresh medium containing 0-75 ng/ml recombinant human IL-17A (Bai ao lai bo, Beijing,China). After IL-17A treatment for 5 days, the cytokine was removed and cells were rinsed with Hank’s balanced salt solution and detached for experiments as indicated elsewhere.
Peripheral blood mononuclear cells (PBMC) in peripheral blood samples from healthy volunteers were isolated by Ficoll-Paque (TBD, China) density gradient centrifugation. T cells (> 80 purity) were enriched by using nylon wool fiber columns (company, city, country). Articular cartilage was harvested from the hip joints of Sprague-Dawley rats as described below and chondrocytes were isolated by digesting at 37°C in medium containing 0.2% collagenase. For transwell co-culture, T lymphocytes and chondrocyte were cultured onto the membrane of transwell insert and DPMSCs were cultured on the bottom of transwell cell culture system, then incubated for different hours at 37°C.
Characterization of DPMSCs
The immunophenotypes of DPMSCs were determined by flow cytometry analysis. Cells were harvested via trypsinization and incubated with monoclonal antibodies against CD73, CD90, CD29, CD44, CD105, CD146, CD34 and CD45 or isotype controls at 4°C for 30 min, then cell surface markers were analyzed using a BD flow cytometry.
When DPMSCs reached 80% confluence, medium was removed and replaced with osteogenic induction medium. Cells continued incubation in the induction medium for 4 weeks with medium refreshed every 3 days. After the formation of mineralized nodules, silver nitrate staining was performed and mineralized nodules, indicative of osteogenic differentiation, were observed under an inverted microscope and photographed.
To initiate adipogenic differentiation, DPMSCs at 80% confluency were cultured in adipogenic induction medium for 4 weeks and the induction medium was refreshed every 3 days. After the appearance of lipid droplets, cells were stained with 0.3% Oil Red O solution and then observed under an inverted microscope and photographed.
For chondrogenic differentiation, chondrogenic induction medium was added after DPMSCs achieved 80% confluence. The induction medium was changed every 3 days. After 4 weeks of culture in the induction medium, chondrogenic cells were viewed using the toluidine blue staining method.
Rat model of osteoarthritis
A total of 26 healthy female Sprague-Dawley rats were purchased from the Department of Zoology, Jiangxi University of Traditional Chinese Medicine and maintained at 22-24°C, with a 12-h light/dark cycle and ad libitum access to food and water. At 12 weeks of age, all rats were injected with 4% papain (0.1 ml each on days 1, 4 and 7) into the knee joint cavity for establishing an OA animal model. After the last injection of papain, the rats were monitored for another week for general health and behavior. Two OA rats were sacrificed for primary culture of chondrocytes. The remaining 24 rats were randomly divided into 3 groups, 8 each. Group 1 was a control group which was injected with 0.1 ml saline into the joint cavity once a week for four weeks. Group 2 was injected with 0.1 ml saline containing 1 × 106 DPMSCs into the joint cavity once a week for four weeks. Group 3 was injected with the same number of DPMSCs once a week for four weeks, but DPMSCs had been pretreated with 75 ng/ml IL-17A for 5 days.
After DPMSCs treatment, the rats were sacrificed by xxx. The injured knee joints were harvested for ultrasonography and histopathology analysis of cartilage damage and blood was collected for determining inflammatory mediators.
Color Doppler ultrasonography
The knee joints were subjected to color Doppler ultrasonography for assessing synovial inflammation, joint effusion, and cartilage damage (surface roughness).
The rat joints were fixed in 10% buffered paraformaldehyde for 48 h. Prior to paraffin embedding, decalcification was performed for 6 h using 10% EDTA (pH7.4) . After paraffin embedding, articular cartilage specimens were cut into 3-μm thick sectios and stained with hematoxylin and eosin (HE). The International Society for the Study of Osteoarthritis (OARSI) scoring system was used to assess the severity of cartilage damage .
Chemiluminescence immunoassay (CLIA)
Rat serum and cell culture supernatants were collected and stored at –80°C until assayed. IL-2, IL-4, TNF-α, and IL-1β levels were detected by CLIA according to the manufacturer’s instructions.
Cell proliferation assay
Chondrocyte proliferation was assayed using the Cell Counting Kit-8 (CCK-8) kit (Keygen Biotech, Jiangshu, China) according to the manufacturer’s instructions. Briefly, after 24, 48, and 120 h of co-culture, the chambers were removed and the CCK8 working solution was added to 10% of the total volume of culture medium of each well and cells continued incubation at 37℃ for 1-4 h. The absorbance was quantified at the wavelength of 450 nm.
All statistical analyses were performed by using SPSS 22.0 (IBM, Chicago, IL, USA). Data are expressed as the mean ± standard deviation (SD). Unpaired Student’s t test was used to compare two groups. For more than two groups, we used one-way analysis of variance (ANOVA) for statistical comparison followed by Bonferroni’s post-test. A p value < 0.05 was considered statistically significant.