Materials
Chitosan (molecular weight:150 kDa, deacetylation:98%), Hyaluronic acid (molecular weight:500-730 kDa), sodium tripolyphosphate (STPP), and IL-1β ELISA kit were provided by Sigma-Aldrich. Cytokine response modifier A (CrmA) was purchaseded from PeproTech. Trypsinase, type II collagenase, DMEM/F12 Medium were purchased from Gibco. All the other chemicals used were of the highest available commercially grade.
Microsphere preparation and characterization
2 g of chitosan was dispersed into the acetic acid (100 mL) under vigorous stirring for 3 h at ambient temperature (below 20°C) to obtain a transparent chitosan solution (2% w/v), and the hyaluronic acid solution (0.1%, w/v) was obtained using the same method. Then, a desired amount of chitosan solution (10 ml) and hyaluronic acid fluid (5 ml) were immediately dispersed with vigorous stirring to obtain a stable mixture of HA-CS solution. A well-mixed suspension containing 100 mL of paraffin oils and 1 g of Span 80 was dispersed in a reactor and and stirred at 1000rmp for 1 h. 6 mL of HA-CS solution prepared was dropped into the suspension with a speed of 1 ml/min. The suspension in the vessel was stirred at the same speed and temperature for an additional 2 h. Next, 10 ml sTPP solution (10% w/v) was added and kept for an additional 1 h at the same stirring speed. After removing the supernatant liquid paraffin, HA-CS microspheres deposited in the bottom were collected. The microspheres were washed with alcohol and acetone several times to completely remove the residual paraffin oil and Span 80. Microspheres of CS-CrmA, HA-CS-CrmA were prepared. Finally, the microspheres were freeze-dried. The sizes and shapes of the microspheres were examined under a scanning electron microscope (SEM).
In vitro release profiles
Approximately 20 mg of microspheres, containing CrmA, was dispersed into a phosphate-buffered saline (PBS, PH = 7.4) solution containing 104 U/ml of lysozyme. Next, this solution was placed in a shaking water bath at 37°C at 135 rpm for various time periods, up to ten days. Periodically, the microsphere suspension was centrifuged to collect the supernatant for analysis, followed by the resuspension of microspheres in a fresh PBS containing lysozyme. Samples were assayed for CrmA concentration using ELISA kits (PeproTech, USA) according to the manufacturer’s protocols.
Chondrocytes isolation and culture conditions
Seven-day-old rats were obtained from the Experimental Animal Center of Wuhan University, China, and were fed under standard conditions (temperature:21 ± 1°C; humidity:55–60%) with food and water continuously available. The care and use of animals followed the recommendations and guidelines of the National Institutes of Health and were approved by the Wuhan University Animal Care and Use Committee.
The cartilage tissues were harvested under sterile conditions from the knees of seven-day-old rats. Then, the cartilage tissues were cut into small pieces (<1 mm3) and digested with 0.2% trypsin and 0.2% type II collagenase for 30 min and 2 hours respectively. After washing twice with DMEM, isolated chondrocytes were suspended in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C with 5% CO2. Cell viability was determined using cell viability analyzer (viability > 90%). Primary cells were maintained in monolayer culture throughout the study. After the cells reached confluence, the medium was changed to DMEM/F12 with 0.5% FBS and antibiotics for 6 hours. Then IL-1β (10 ng/ml) was added to the culture medium without rinse for a further 48 hours. The blank group was cultured in DMEM/F12 with 10% FBS without IL-1β, while the control group was only treated with IL-1β. Chondrocytes were divided into five groups cultured in DMEM/F12 containing 10%FBS without antibiotics and incubated for a period of 4 h: A. blank group, B. controls, C. chondrocytes cultured with CS microspheres, D. chondrocytes cultured with HA-CS microspheres, E. chondrocytes cultured with CS-CrmA microspheres, F. chondrocytes cultured with HA-CS-CrmA microspheres. There were 5 samples in each group and each experiment was repeated 5 times.
Determination of cell viability and GAG synthesis
After 72 hours of co-culture, the microspheres solution was discarded, and the chondrocytes were removed to a fresh media. After standardizing cell samples to one million in different groups using a cell counter, 0.5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-dippphenyltetrazolium bromide (MTT) was added to chondrocytes incubated at 37°C with 5% CO2 for 4 h. The resulting formation was dissolved in dimethylsulphoxide and absorbance was measured at 570 nm with a microplate reader.
The GAG content in cell supernatants was assessed using Blyscan assay kit (Biocolor, UK), and the chondrocytes were normalized to one million cells in each group. Briefly, cell supernatants were digested enzymatically using proteinase K. Following digestion, a desired amount of supernatant was reacted with Blyscan dye for 30 min. GAG-dye precipitate was obtained by centrifugation and the resulting formation was dissolved in 1 ml dissociation reagent and incubated for 10 minutes. Finally, samples were transferred to a 96-well-plate and absorbance at 656 nm was measured on the spectrophotometer. A standard curve was derived from mixed-isomer shark chondroitin sulfate, and the GAG content was calculated.
Western blot for type II collagen, Aggrecan, type I collagen and IL-1β
Proteins were extracted from harvested chondrocytes. Protein concentrations were determined using a BCA Protein Assay Kit. After adjusting to equal amounts (50 ul protein per lane) of proteins, they were separated by sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked in phosphate buffered saline (PBS), pH 7.4, containing 5% nonfat dry milk, and incubated with anti-IL-1β, -type II collagen, Aggrecan, and -type I collagen, respectively. Then incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG), followed with visualization by the enhanced chemiluminescence kit.
Statistical analysis
SPSS 20.0 software was applied for data analysis. Data were presented as the means ± S.D. of five separate experiments. Each experimental condition was represented by triplicate wells, with replicates from each culture averaged and used as one value for analysis. Significant differences among the mean values of multiple groups were evaluated by one-way analysis of variance and Student-Newman-Keuls q-test. P < 0.05 was considered as being significant.