Human chondrocyte culture
The concise experimental flow chart is shown in Fig 1. The study protocol was approved by the Ethics Committee of XiangYa Hospital, Central South University. Each participant or the legally authorized representative of the participant was aware of and agreed to the study. Only cartilage samples from unique donors were employed for each culture. Thus, for each experiment, cells were obtained from the same subject. Normal human articular cartilage was obtained from five subjects (aged 14-50 years) who underwent knee amputation because of severe trauma. OA human articular cartilage was obtained from 11 patients (aged 59-75 years) with knee OA who were undergoing total knee replacement surgery. After washing twice with phosphate-buffered saline (PBS), the cartilage tissue was ground with a scalpel blade into 1-5-mm3 sections. The cartilage tissue was subsequently digested with 5-8 ml of 0.2% collagenase II (Sigma-Aldrich, St. Louis, MO, USA) for 12 16 h at 37°C in a constant temperature shaker. Digestion was terminated with 8 10 ml of Dulbecco's modified Eagle's medium/F12 (DMEM/F12; HyClone, Logan, UT, USA). The released cell pellets at the bottom of the centrifuge tube were aspirated and transferred to a culture flask following centrifugation at 1000 rpm for 5 min. Cell pellets were resuspended in 5 ml of DMEM/F12 containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin solution (Gibco) and incubated for 24 h at 37°C with 5% CO2 in a plastic culture flask. The nonadherent cells were subsequently washed out. The growth medium was changed every 3 days prior to trypsinization, and cells were then passed to a new 6-well plate at a density of 5×105 cells per well. Cells at passages one through two were used for experiments.
Total RNA isolation, quantification and reverse transcription; real-time quantitative PCR assays
Total cellular RNA was extracted from cultured normal and osteoarthritic chondrocytes using TRIzol reagent (Invitrogen, Life Technologies, Paisley, UK). RNA was further purified using an RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Preservation of 28S and 18S ribosomal RNA (rRNA) species was used to assess RNA integrity. All samples included in the study had prominent 28S and 18S rRNA components. The total RNA yield was quantified by spectrophotometry. One microgram of total RNA was converted to cDNA using a Revert Aid™ First Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA). First, all components. Including template RNA (1 μg) and oligo (dT)18 primer (1 μl) were mixed, and nuclease-free water was added to a total volume of 12 μl. The mixture was mixed gently, centrifuged briefly and incubated at 65°C for 5 min. The mixture was chilled on ice, spun down and placed back in a vial on ice. The following components were added, to a volume of 20 μl: 5X reaction buffer (4 μl), RiboLock™ RNase Inhibitor (20 u/µl) (1 μl), 10 mM dNTP Mix (2 μl) and Revert Aid™ M-MuLV Reverse Transcriptase (200 u/µl) (2 μl). This reaction was then incubated for 60 min at 42°C and terminated by heating at 70°C for 5 min. The resulting cDNA products were stored in aliquots at –80°C until needed.
The primers were synthesized by Shanghai Sangon Bioengineering Corporation. All primers used are shown in Table 1. For all real-time quantitative PCR reactions, Maxima® SYBR Green/ROX qPCR Master Mix (2×) (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA) was used (12.5 µl of Maxima® SYBR Green/ROX qPCR Master Mix (2×), 2.5 μl of forward primer (0.3 µM), 2.5 μl of reverse primer (0.3 µM), 2 μl of template DNA and 5.5 μl of nuclease-free water were added to a volume of 25 μl). The ABI 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used for all real-time Q-PCRs. The PCR thermal cycling protocol applied consisted of 1 step for 2 min at 50°C for UDG pretreatment and 1 step for 10 min at 95°C for initial denaturation followed by 40 cycles consisting of a denaturation step for 15 s at 95°C, an annealing step for 30 s at 30°C and an extension step for 30 s at 72°C. A melting curve analysis was performed after the final amplification period with a temperature gradient of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s.
Table 1
Oligonucleotide primers used in real-time PCR assay
Gene | Forward primer sequence | Reverse primer sequence |
β-catenin | 5’-GAGGAGATGTACATTCAGCAG-3’ | 5’-GTCTCCGACCTGGAAAAC-3’ |
TCF-4 | 5’-CTTTCCCTAGCTCCTTCTTC-3’ | 5’-CTACGATGGAAAGTGGACAT-3’ |
OPN | 5’-GTGGGAAGGACAGTTATGAA-3’ | 5’-CTGACTTTGGAAAGTTCCTG-3’ |
GAPDH | 5’-CAATGACCCCTTCATTGACC-3’ | 5’-GACAAGCTTCCCGTTCTCAG-3’ |
Real-time Q-PCR test results: The relative expression level of mRNAs was calculated as the ratio of each mRNA expression level to that of GAPDH mRNA as the reference housekeeping gene. The relative expression levels of genes of interest were calculated and expressed as 2-△△CT values. All quantities were expressed as n-fold relative to a calibrator.
Transient transfection with TCF-4 shRNA in chondrocytes
siRNAs specific to TCF-4 based on the coding sequence of human TCF-4 were designed and synthesized by GenePharma Corporation (China, Shanghai). The three shRNA sequences and scrambled shRNA sequences constructed are shown in Table 2. Normal and osteoarthritic chondrocytes were seeded in six-well plates at a density of 5×105 cells/well in medium without antibiotics. After overnight incubation, when cells reached 70% confluence, they were transfected with specific shRNAs against TCF-4 with Lipofectamine TM 2000 reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instructions. No cell toxicity from the transfection agent was detected. The transfection efficiency was evaluated with fluorescein by fluorescence microscopy 24 h after transfection. In each experiment, the results from three of the six-well plates were averaged and considered n = 1. No significant variance was observed among the individual wells in each averaged group. After 24 h, total RNA and protein were isolated, and the expression levels of TCF-4 and OPN were detected by real-time Q-PCR and Western blot analyses.
Table 2
TCF-4 siRNA sequences used in Transient transfection
| sense | antisense |
siRNA-1105 | 5’-GCCATGGAGGTACAGACAAAGTTCA-3’ | 5’-GAGACTTTGTCTGTTACCTCCATGGCTT-3’ |
siRNA-1791 | 5’-GGATGATGCTATTCATGTTCTTTCA-3’ | 5’-GAGAAGAACATGAATAGCACTACATCCTT-3’ |
siRNA-1859 | 5’-GGGACATGCATGGAATCATTGTTCA-3’ | 5’-GACACAATGATTCCATGCATGTCCCTT-3’ |
NC-siRNA | 5’-GTTCTCCGAACGTGTCACGTCAAG-3’ | 5’-GATTACGACACGTTCGGAGAATT-3’ |
Cell treatment with recombinant DKK-1
Normal and OA chondrocytes were seeded on six-well plates at 1x106 cells/well, and 3 days after seeding, cells were treated with recombinant DKK-1 (100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, and 500 ng/ml) for 12, 24, 36, and 48 hours; each experiment was conducted with triplicate wells.
Cell viability was determined by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Culture medium was removed, and 20 μl of MTT solution (5 mg/ml in PBS) was added to each well and incubated at 37°C with 5% CO2 for 4 h. The supernatant was then carefully aspirated, and the formazan reaction products were dissolved in 150 μL dimethylsulfoxide (DMSO) (Sigma, St Louis, MO, USA) solution and shaken for 15 min. The spectrophotometric absorbance at 570 nm was measured in an enzyme-linked immunosorbent assay (ELISA) plate reader (Multiskan MK3-Thermo Labsystems; Thermo Fisher Scientific, Waltham, MA, USA) or an ELISA reader (Bio-Rad, CA, USA).
Western blot analysis
Chondrocytes were lysed by using RIPA buffer and a cocktail of protease and phosphatase inhibitors. The protein concentration was quantified by using a BCA Protein Assay Kit (Thermo Scientific Company, Prod#23225, Rockford, USA) with bovine serum albumin as the standard. Cell lysates from normal and OA chondrocytes were electrophoresed and separated on a 4% to 20% Tris-HCl gel (Bio-Rad, Hercules, CA, USA), and the separated proteins (25 µg) were electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Nonspecific proteins on the membranes were blocked with 5% skim milk powder in PBS. The membrane was probed with an anti-osteopontin antibody (ab8448, 1:1,000 dilution, Abcam, Cambridge, MA, USA) and an anti-TCF-4 antibody (sc-57040; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. A mouse monoclonal anti-actin antibody (Sigma) was used as the loading control. The membranes were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 dilution). Immunoreactive proteins were visualized with western blotting luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The polyvinylidene fluoride membranes were then exposed to photographic film, which was scanned, and the intensities of the protein bands were determined with computerized densitometry. The results were normalized by using an anti-GAPDH polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA).
DAPI staining
β-Catenin nuclear accumulation was observed by nucleic acid staining with DAPI. Chondrocytes were treated with or without rDKK 1, and the treated chondrocytes were collected and fixed in 4% paraformaldehyde for 5 minutes. The fixation solution was discarded, and the cells were rinsed 3 times in 1× PBS. The buffer was discarded, and the cells were incubated in 10 μM DAPI solution in the dark at 37°C for 15 minutes. The DAPI solution was removed, and the stained cells were rinsed 3 times in 1× PBS and supplemented with fresh 1× PBS buffer. The stained cells were examined for cellular morphology and nuclear profiles under a Leica laser confocal microscope.
Statistical analysis
All statistical calculations were performed using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Data are expressed as the means ± standard errors of the mean. Student's t-test was used to analyze statistical differences between two groups, and one-way ANOVA was performed to determine the statistical differences among groups. A P-value of less than 0.05 was considered statistically significant.