Primary chondrocyte cultures.
Normal human chondrocytes were obtained from ankle cartilage of organ donors within 24 hours postmortem. Normal-appearing cartilage (Collins grade 0–1) (15) was used for these experiments. OA cartilage was obtained from the distal femur and the proximal tibia of tissue from patients undergoing total knee replacement surgery for OA. Full-thickness cartilage slices were digested in a 5% CO2 incubator at 37°C with continuous agitation for 1 hour in Dulbecco's modified Eagle's medium (DMEM) /F-12 (1:1) culture media containing 0.2% pronase (Calbiochem, San Diego, CA), and then overnight with 0.025% collagenase P (Roche, Indianapolis, IN) in DMEM/F-12 supplemented with 5% fetal bovine serum (FBS). After isolation, the cells were counted, and initial viability was assessed using trypan blue exclusion and was determined to be >90%.
Cells were cultured under serum-free conditions by either plating on coated coverslips or by placing the cells in suspension in alginate beads. For the coverslip experiments, sterile coverslips (13 mm; Nunc, Naperville, IL) were placed in wells in a 24-well plate and preincubated overnight at 4°C with 10 μg/ml FN (Gibco, Grand Island, NY) or 10 μg/ml poly-L-lysine (Sigma, St. Louis, MO). The wells were gently washed 3 times with phosphate buffered saline (PBS) and then 7.5 × 104 chondrocytes were plated in 400 μl per well of serum-free DMEM/F-12 media with antibiotics, for a final cell density of 3.75 × 104 cells/cm2. Alginate beads were prepared at a density of 2 × 105 cells/bead and were cultured in serum-free DMEM/F-12 supplemented with “mini-ITS+” (consisting of 5 nM insulin [Calbiochem], 2 μg/ml transferrin [BD Biosciences, Bedford, MA], 2 ng/ml selenous acid, 420 μg/ml/2.1 μg/ml bovine serum albumin [ BSA]/linoleic acid [Collaborative Biomedical Products, Bedford, MA]) plus 25 μg/ml ascorbate (Wako Pure Chemical Industries, Richmond, VA) and antibiotics as previously described (16). In experiments testing the effects of IGF-1 and FBS on survival, 100 ng/ml human recombinant IGF-1 (a gift from Chiron, Emeryville, CA) was added to DMEM/F-12 with mini-ITS+, or 10% FBS (Gibco) was added to DMEM/F-12 containing antibiotics. For the 7-day cultures, the medium was changed every other day.
Inhibition of specific integrin subunits. Monoclonal antibodies known to block α1-, αv-, and α5-integrin subunits were used, and included anti-α1 (5E8D9) from Upstate Biologicals (Lake Placid, NY), anti-α5 (NKI-SAM-1) from Chemicon (Temecula, CA), and anti-αv (M9; from Chemicon). Integrin inhibition was also tested using echistatin (Bachem, King of Prussia, PA), a disintegrin that has been shown to be a potent inhibitor of αvβ3 (17, 18). Controls included cultures incubated without antibodies, with nonspecific isotype control IgG (Sigma), or with a nonblocking anti-α5 antibody (HA5; Chemicon). In addition, because some of the antibodies were provided in a buffer containing a preservative (0.001% merthiolate or 0.1% azide), controls were tested with equal final concentrations of the preservative alone. Neither preservative, at the concentrations used in this study, caused chondrocyte death.
For treatment of chondrocytes plated on coated coverslips in 24-well plates, antibodies were added in the media with the cells as they were plated. The plates were gently shaken for 15 minutes at room temperature to evenly distribute the cells and antibody, prior to overnight incubation at 37°C in a 5% CO2 incubator. For treatment of cells cultured in alginate, the freshly made beads were transferred to 96-well plates with 1 bead per well in 100 μl of serum-free DMEM/F-12 media supplemented with mini-ITS+. Integrin and control antibodies or echistatin were added 30 minutes later to each well in duplicate, and then cells were incubated overnight at 37°C in a 5% CO2 incubator. Cell survival was assessed the next day, as described below.
Chondrocyte survival assay. Cell survival was measured by incubating cultured cells with calcein AM and ethidium bromide homodimer 1 (Molecular Probes, Eugene, OR). Calcein AM is a nonfluorescent cell-permeable dye that is cleaved by a ubiquitous intracellular esterase present in live cells to produce a highly green-fluorescent, cell-impermeable product. Ethidium bromide homodimer 1 is a cell-impermeable dye that enters cells following the compromise of plasma membrane integrity and stains nuclear DNA red. Media from cells cultured on coverslips were gently removed so as not to disturb any loosely attached cells and replaced with 400 μl of PBS containing 1 μM calcein AM and 4 μM ethidium bromide homodimer 1. The coverslips were then incubated at room temperature and protected from light. After 45 minutes, the coverslips were placed on a microscope slide and the numbers of dead and live cells were counted using an Olympus (New Hyde Park, NY) fluorescence microscope. Calculations of the percentage of live cells were determined by a total cell count performed in triplicate, with the number of cells counted always exceeding 100 for each data point.
For alginate cultures, after overnight incubation with integrin antibodies or echistatin, the media were removed and replaced with 100 μl of PBS containing 1 μM calcein AM/4 μM ethidium bromide homodimer 1 and incubated at room temperature and protected from light. After 30–45 minutes, the staining solution was removed and the beads were dissolved by shaking for 10 minutes in 20 μl 55-mM sodium citrate with 0.15-mM NaCl. Finally, 15 μl of the dissolved bead was placed on a microscope slide for counting, as described above.
FN immunocytochemistry. Chondrocytes were cultured overnight on poly-L-lysine–coated coverslips in serum-free media as described above. The cells were gently rinsed with PBS and then fixed for 20 minutes with 4% paraformaldehyde at 4°C followed by washing with Tris buffered saline (TBS). Slides were blocked for 3 hours with TBS plus 1% high-purity BSA (Sigma) at 4°C, and then incubated overnight at 4°C with anti-FN monoclonal antibody (P1H11; Chemicon) at 1:100 in TBS plus 0.1% Tween plus 1% high-purity BSA. The slides were then washed with TBS plus 0.1% Tween and incubated for 90 minutes with fluorescein-conjugated anti-mouse secondary antibody (ICN/Cappel, Aurora, OH) at 1:100 in TBS plus 0.1% Tween plus 0.1% high-purity BSA at 4°C. The slides were then washed again with TBS plus 0.1% Tween followed by double-distilled H2O and then mounted with anti-fading aqueous mounting medium (Dako, Carpinteria, CA) and viewed on an Olympus BX 60 microscope equipped for detection of fluorescence.
Apoptosis detection. The presence of apoptotic cells was based on the detection of histone-associated DNA fragments (mono- and oligonucleosomes) using the Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Boehringer Mannheim, Indianapolis, IN) and confirmed using electron microscopy on cells from a selected experiment. Ten beads (∼2 × 106 cells) in 400 μl of media per well of a 24-well plate were treated overnight with anti-α5 antibodies as described above. For the ELISA, the media were removed and the cells were released from the alginate beads by shaking in 1.5 ml 55-mM sodium citrate in 0.15-mM NaCl. The cell suspension was centrifuged to obtain a cell pellet which was then lysed with buffer from the kit. The samples were then used for the ELISA following the manufacturer's directions. The assay is a sandwich enzyme immunoassay for detection of histone-associated DNA fragments using anti-histone (H2A, H2B, H3, and H4) monoclonal antibodies coated onto a 96-well plate, cell-lysate samples from control and treated cultures, and anti-DNA monoclonal antibodies conjugated to peroxidase for detection.
For electron microscopy, the alginate beads were fixed with 2% glutaraldehyde in 0.1M sodium cacodylate buffer and postfixed in 1% osmium tetroxide in 0.1M sodium cacodylate. The cells were dehydrated in ethanol and propylene oxide and embedded in epoxy. Thin sections of ∼600 angstroms were mounted on grids and stained with 0.2% Reynolds lead hydroxide stain plus 2.5% uranyl acetate. Cells were imaged on a JEOL (Palo Alto, CA) 100-CX electron microscope, and representative cells were photographed.
Statistical analysis. Where indicated, results were compared by analysis of variance using StatView software (SAS Institute, Cary, NC). Significance was determined as a P value less than 0.05, and post hoc testing was performed using Fisher's protected least significant difference test.