Vimentin contributes to changes in chondrocyte stiffness in osteoarthritis.

Cartilage Osteoarthritic human articular cartilage was obtained from femoral and tibial condyles resected during total knee arthroplasty (TKA) surgeries performed at Scripps Hospital, healthy human cartilage was obtained from tissue banks, both in accordance with HIPPA guidelines and with IRB approval. Samples were obtained from 8 TKA patients. Fibrillated and diseased cartilage (OA group) was dissected away from cartilage with normal appearance (Normal group). In addition, healthy cartilage was obtained from 1 male and 2 female tissue donor without cartilage pathology (Healthy group). The average TKA donor age was 62 (±14 range 40–84), the average healthy tissue donor was 24 ( Supplemental Table 1 ).

Cell culture

Unpassaged primary human articular chondrocytes were isolated and cast into 6mm diameter discs of 2% alginate as follows: chondrocytes were released from the cartilage by overnight digestion with type IV collagenase as described previously( 22 ). Chondrocytes were counted, centrifuged, and resuspended at 5×10 5 cells/ml in a 2% solution of sterile-filtered and endotoxin-free alginate (FMC BioPolymer, Sandvika Norway) in HBSS. The alginate-cell suspension was pipetted into a polysulfone casting frame 1cm × 4cm × 2.4mm thick sandwiched between Whatman 3mm filter paper held in place by stainless steel mesh and clamps (modified from Ragan et al ( 23 )). The assembly was submerged in 102mM CaCl 2 for 45 minutes to polymerize the alginate. Four discs were created using 6mm dermal biopsy punches as shown in Figure 1A , and rinsed several times in culture media (DMEM with 10% calf serum, 1% penicillin/streptomycin, and 30μg/ml ascorbic acid). To disrupt vimentin, monomeric acrylamide (Sigma) was added at a final concentration of 4mM to the culture media of two alginate discs, control discs were cultured without acrylamide. At this concentration, acrylamide causes collapse of intermediate filament structures but does not noticeably affect either microtubules or actin filaments ( 24 – 26 ). In preliminary studies we observed that 40mM acrylamide was cytotoxic within 12 hours, but that 4mM acrylamide monomer caused a collapse of vimentin filaments without significantly affecting chondrocyte viability up to 72 hours when compared to untreated control cells from the same donors. Mechanical Loading Mechanical loading was performed 24 hours after cell seeding in alginate. Pilot studies indicated that at this timepoint the primary chondrocytes do not produced sufficient pericellular matrix to affect the cytosolic stiffness measurements. Gels were cut in half radially and labeled for 30 minutes with calcein-AM and 10 minutes with ethidium homodimer (Live-dead stain, Invitrogen). Gels were placed onto a coverslip on the stage of a Zeiss LSM510 inverted confocal microscope custom-fitted with a linear actuator with 1μm resolution (SMAC, Carlsbad CA) as shown diagrammatically in Figure 1B . The actuator was used to measure the exact dimensions of each gel and then compress them to 80% of their original thickness (20% nominal strain). Strain was applied in the Y-axis of the gels relative to the microscope stage. Strain was applied at a rate of 50 microns/sec. While strained, at least 50 cells were imaged in each gel by taking a single confocal z-slice of the cell at the widest diameter. Images were acquired after stress relaxation was complete: approximately 10 minutes after compression. The calcein fluorescence in the cytosol of living cells was imaged with a 63x water immersion lens and saved as 512x512 pixel tiff files. Image Processing and Statistical Analysis A Matlab (MathWorks, Natick MA) subroutine was developed to automate measurements of the maximum cell diameter in the X and Y direction and calculate the ratios of the Y and X axes, based conceptually on the techniques described by Lee et al( 27 ). Whenever possible, at least 50 cells were imaged from each condition, and 4 conditions were measured for each TKA patient (untreated normal cells, untreated OA cells, acrylamide treated normal cells, and acrylamide treated OA cells). For cells from healthy donors without arthritis, 2 conditions were measured (untreated and acrylamide-treated). A total of ~2000 individual cells were imaged from the ten donors, representative cells are shown in Figure 1C . Data were imported into a JMP 7.1 database for statistical analysis. After testing for normal distributions, cell aspect ratios of the groups were compared by one-way ANOVA with Tukey-Kramer’s post-hoc correction for multiple comparisons, statistical significance was set at p