Preparation and Culture of chondrocyte/agarose constructs
Embryonic mouse chondrocytes were isolated from the ventral parts of the rib cages of 17.5 dpc mice. For each experiment, cells were pooled from all embryos from 2 or 3 OF-1 mouse litters. Mouse care and treatment were conducted in conformity with institutional guidelines in compliance with national and international laws and policies (Authorization n°69387416 given by the French Prefecture du Department du Rhone). Agarose hydrogels were prepared by mixing 2.5% low melting agarose (Seaplaque, Cambrex BioScience) with 5 × Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F-12) containing 10% FBS, 50 mM Hepes and 500 U/mL Penicillin and 500 μg/mL Streptomycin (all products from Gibco). Cells were suspended in this agarose solution with a density of 2 × 106 cells/mL. 700 μL of the mixture was poured into wells of 24-well plates, and chondrocyte/agarose constructs were allowed to gel at room temperature. Constructs were then punched to obtain cylindrical gels (13 mm diameter and 3 mm height) that were placed into wells of Biopress™ compression plates (Flexcell international), within the 13 mm diameter foam ring (Figure ). A volume of 4 mL DMEM/F-12 culture medium containing 10% FBS and 100 U/mL Penicillin and 100 μg/mL Streptomycin was added to each well. The cell/agarose constructs were maintained in culture for 6 days in 5% CO2 at 37°C. The culture medium was changed every day with a progressive deprivation of serum as detailed in Table .
Samples were submitted to load with FX-4000C™ Flexercell® Compression Plus™ System (Flexcell International). The strain regimen consisted either in (i) cyclical compression with pulses of 20 kPa at a frequency of 0.33 Hz (2 sec on, 1 sec off) superimposed on 20 kPa static offset pressure for 30 min, or in (ii) intermittent compression of 30 kPa using a sinusoid waveform at 0.2 Hz for 30 min, as indicated (Figure ). As control, cell/agarose constructs were maintained under uncompressed conditions.
The chondrocyte/agarose constructs were fixed in 4% formaldehyde for at least 24 h, after which they were embedded in Paraffin, sectioned at 7 μm, and then deparaffinised. Prior to antibody treatment, the sections were sequentially incubated with hyaluronidase (Sigma, type I-S, 800 U/mL) for 1 h at 37°C and 0.1% Triton X-100 for 20 min at room temperature. The sections were then incubated overnight with 2B1 anti-type II collagen primary mouse antibody (kind gift from Richard Mayne) [29
] at 4°C. After washing in Phosphate Buffer Saline, the sections were incubated with Cy™2-conjugated AffiniPure donkey anti-mouse IgG (Jackson Immunoresearch) for 1 h at room temperature.
Protein Extraction and Analysis
At the different time points studied the chondrocyte/agarose constructs were frozen in liquid nitrogen, freeze-dried and stored at -20°C until analysis. For protein extraction, 4 × Laemmli buffer was prepared (250 mM Tris-HCl, 20% glycerol, 10% SDS and bromophenol blue) and extemporaneously diluted to 1 × and supplemented with 3% 2-mercaptoethanol. A volume of 200 μL of this extraction buffer was added to each freeze-dried construct and the mixture was boiled immediately for 5 min. The lysates were allowed to gel at room temperature before being transferred on paper filter mini-spin columns (Pierce) and centrifuged at 12,000 g for 1 h at room temperature. The exudates were collected in mini-tubes and stored at -20°C until further analysis by Western blotting. The antibodies used in this study are described in Table .
Antibodies used for Western blot analysis
The protocol of RNA extraction was modified from the RNeasy-mini kit (Qiagen) procedure. Briefly, each chondrocyte/agarose construct was diced in a mixture of 1.5 mL QG buffer (Qiagen) and 2 mL RLT buffer (Qiagen kit) and the samples were kept at room temperature until complete dissolution, and then stored at -20°C. After defrosting, the samples were homogenized and supplemented with 2 mL 70% ethanol. The subsequent RNA purification steps were performed as described by the manufacturer. RNA quantity and quality were assessed by using a capillary electrophoresis system (RNA Nano kit and 2100 BioAnalyzer, Agilent).
Reverse transcription and Real time PCR
Reverse transcription (RT) was performed as described [20
]. For real time PCR amplification, a 20 μL reaction contained 1.3 μL of RT product, 10 μL SYBR green Supermix (Bio-Rad) in the presence of 300 nM of specific primers (see Table ). Amplification was performed in an iCycler iQ (Bio-Rad), using the following conditions: an initial denaturation step of 2 min at 95°C, followed by 40 cycles of 30 sec at 95°C, 30 sec at 54°C, and an extension step of 3 min at 72°C. Levels of gene expression were determined by using the comparative Ct
method with RPL13a
gene as endogenous control.
Primers used for real time PCR analysis
Prior to transfection, chondrocytes were amplified in monolayer culture for one week. After trypsinization, 3 × 106 cells were suspended in 100 μL of electroporation buffer and mixed with 4 μg plasmid of interest and 1 μg plasmid encoding β-galactosidase. The plasmids were transfected by using the Human Chondrocyte Nucleofector kit (Amaxa) according to the manufacturer's protocol (Program U-24). Although nucleofection leads to about 60% cell mortality, this method provides high transfection efficiency (around 80%) for the viable cells. This transfection efficiency was assessed by monitoring synthesis of green fluorescent protein in chondrocytes observed under a fluorescence microscope, after transfection of the corresponding expression vector (data not shown).
Measure of COL2A1 promoter activity
Immediately after nucleofection, chondrocytes were embedded in agarose and the cell/agarose constructs were submitted to compression the following day. At the end of the compression regimen (20 h post-transfection), the constructs were frozen in liquid nitrogen and stored at -80°C. The frozen samples were then freeze-dried for 12 h before being grinded in TissueLyser (Qiagen) for 1 min at 20 Hz. The resulting powder was rehydrated in 200 μL of the kit lysis solution for 1 h at 4°C and reporter assays were performed using Dual-Light® combined reporter gene assay system for detection of luciferase and β-galactosidase (Applied Biosystems), as described by the manufacturer, and MLX™ Microtiter® plate luminometer (Dynex). COL2A1 promoter activity was obtained as a ratio of luciferase to β-galactosidase luminescence.
Quantification and statistical analysis
For Western-blotting analysis, band intensity was quantified by densitometry using ImageQuant software (v5.2, Molecular Dynamics). The ratio of phospho-MAPK to total-MAPK band intensity was calculated and mechanically-induced phosphorylation was normalized to uncompressed controls. For the analysis of the compression effects on mRNA levels and promoter activity, data represent the mean and standard deviation values of 3 independent experiments. Statistical analysis was performed using a Student's paired, two-tailed t-test. Significance was reported at the 90% (*) or 95% (**) confidence level.