Isolation and expansion of human chondrocytes
Healthy chondrocytes were harvested from normal cadaver femoral condyles and OA chondrocytes from tissue taken during total joint replacement surgery (n = 5 each; for representative images, see Additional file 1). Human tissue was obtained as discard tissue without patient identifiers. The Oregon Health & Science University Institutional Review Board approved the study as being exempt from the requirement for consent. Cartilage was scraped from the condyles and finely minced before enzymatic digestion. Cartilage digestion was initiated with 1% protease (wt/vol) from Streptomyces griseus in low-glucose Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Grand Island, NY, USA) supplemented with 1% penicillin-streptomycin (P/S). After 1 h at 37°C, protease was removed and replaced with 1,300 U/ml collagenase II (Worthington Biochemical, Lakewood, NJ, USA) in DMEM + P/S for 3 h at 37°C. The cell suspension was passed through a 40-μm cell strainer and centrifuged at 500 × g for 5 min, then the cells were resuspended in low-glucose DMEM supplemented with 10% fetal bovine serum and 1% P/S. Chondrocytes were plated at 7,000/cm2 and expanded in monolayer culture in a standard tissue culture incubator with atmospheric oxygen and 5% CO2.
The storage conditions for osteochondral allografts may affect chondrocyte viability and metabolic activity, although the time at which these effects become evident depends on the storage solution, and in some medium solutions no change is observed even after 2 wk [21]. We obtained OA specimens just hours after total joint replacement, and healthy cartilage was collected from discarded osteochondral allografts of proprietary storage conditions. For the chondrocytes used here, however, we found no significant difference in either the number of viable cells obtained per gram of tissue digested (5.1 × 106 ± 3.3 × 106 versus 6.2 × 106 ± 3.1 × 106, for healthy and OA cells, respectively) or in the doubling time from initial plating to first passage (6.9 ± 2.1 versus 6.4 ± 1.5 for healthy and OA cells, respectively).
Pellet culture redifferentiation of chondrocytes
Chondrocytes were redifferentiated between the first and third passages with serum-free chondrogenic induction medium consisting of high-glucose DMEM (Life Technologies) containing 10 ng/ml transforming growth factor β1 (Peprotech, Rocky Hill, NJ, USA), 10-7 M dexamethasone, 37.5 μg/ml ascorbic acid 2-phosphate, 1 mM sodium pyruvate, 40 μg/ml L-proline, 1 × ITS+ Universal Culture Supplement Premix (BD Biosciences, San Jose, CA, USA) and 1% P/S. Pellet cultures were formed by centrifuging 1 × 105 cells at 500 × g in 250 μl of medium in Nunc polypropylene V-bottom 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) and maintained in a hypoxic chamber (BioSpherix, Lacona, NY, USA) set at 2% oxygen, 5% CO2 or a standard tissue culture incubator with 5% CO2. Although CO2 displacement actually lowers the oxygen level, hereafter we refer to the standard condition as 20% oxygen according to convention. Medium was changed every 2 or 3 days, with that of the hypoxic cultures being done at 2% oxygen.
Biochemical assays
All biochemical assays were conducted on at least triplicate pellets. Pellets were rinsed with phosphate-buffered saline (PBS) and digested overnight at 60°C in 4 U/ml papain (Sigma-Aldrich, St Louis, MO, USA) in PBS containing 6 mM Na2-ethylenediaminetetraacetic acid and 6 mM L-cysteine (papain buffer, pH 6.0). For samples to be assayed for hydroxyproline, iodoacetic acid was added to a final concentration of 10 mM after papain digestion.
DNA content of all papain-digested pellets was quantified using 2 μg/ml Hoechst dye with calf thymus DNA diluted in papain buffer used to prepare standard curves. Briefly, 50 μl of samples, standards and blanks were added to black 96-well plates with 200 μl of Hoechst dye before fluorescence emission was measured with a multiwell plate reader (excitation 355 nm, emission 455 nm).
The sulfated glycosaminoglycan (sGAG) content of pellets and culture medium was quantified using 1,9-dimethymethylene blue (DMMB) dye. Supernatant collected at each medium change was used to quantify the total amount of sGAGs produced and lost into the medium. Shark chondroitin sulfate diluted in either DMEM or papain buffer was used to prepare standard curves. For microplate assays, 50 μl of samples, standards and blanks were added to clear 96-well plates with 200 μl of DMMB dye (18 μg/ml in 0.5% ethanol, 0.2% formic acid, 30 mM sodium formate; pH 3.0) before absorbance was measured (575 nm).
Hydroxyproline content of pellets was quantified using an adaptation of the chloramine-T hydrate oxidation/p-dimethylaminobenzaldehyde development method with solid-phase hydrolysis on Dowex 50WX8-400 ion exchange resin (Thermo Fisher Scientific) [22]. Trans-4-hydroxy-L-proline in papain buffer was used to generate a standard curve. Absorbance of samples was measured at 560 nm in a multiwell plate reader.
RNA isolation and real-time quantitative polymerase chain reaction
The RNeasy Mini Kit (QIAGEN, Germantown, MD, USA) was used to collect total RNA from four to six pellets of each condition. Pooled pellets were snap-frozen in liquid nitrogen and crushed, then immediately lysed with Buffer RLT lysis buffer (QIAGEN) containing 40 mM dithiothreitol (DTT). RNA isolation then proceeded as per the manufacturer's instructions.
RNA samples (250 ng) were reverse-transcribed using qScript cDNA SuperMix (Quanta BioSciences, Gaithersburg, MD, USA) as per the manufacturer's instructions. Quantitative polymerase chain reaction (qPCR) analysis was performed using a MyiQ iCycler thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) with PerfeCTa qPCR FastMix (Quanta BioSciences) and TaqMan assays (Life Technologies) for ACAN (Hs00153936_m1), COL2A1 (Hs00264051_m1), COL1A1 (Hs00164004_m1), COL10A1 (Hs00166657_m1), MMP1 (Hs00899658_m1), MMP2 (Hs00234422_m1), MMP3 (Hs00968305_m1), MMP9 (Hs00234579_m1), MMP13 (Hs00233992_m1), MMP14 (Hs00237119_m1), TIMP2 (Hs00234278_m1), ADAMTS4 (Hs00192708_m1), ADAMTS5 (Hs00199841_m1), HAS2 (Hs00193435_m1), HIF1A (Hs00153153_m1) and EPAS1 (Hs01026149_m1). The cycling parameters were 45°C for 2 min, 95°C for 1 min, then 95°C for 5 s and 60°C for 30 s for a total of 40 cycles. Results were analyzed using the 2-ΔCt method relative to the housekeeping gene 18S (Hs99999901_s1).
Gelatin zymography
Gelatin zymography was performed on 10% SDS-PAGE gels containing 10 mg/ml gelatin. Gelatin was dissolved in 1.5 M Tris, pH 8.8, and displaced a corresponding volume of 1.5 M Tris during gel preparation. Medium samples from days 9 to 14 of pellet culture were pooled for each oxygen condition. Pooled samples were prepared in Lammeli sample buffer without reducing agents or boiling, and 20 μl of this solution was used for zymography. Samples were run alongside PageRuler Plus marker (Thermo Fisher Scientific), then gels were incubated in renaturing buffer (2.7% Triton X-100) two times for 30 min each before equilibrating in developing buffer (50 mM Tris base, 5 mM CaCl2, 40 mM HCl, 200 mM NaCl, 0.02% (wt/vol) Brij-35) for 30 min at room temperature. The developing buffer was then changed, and the gels were incubated at 37°C for 24 h. Developed zymograms were stained with Coomassie Blue (0.1% Coomassie R-250, 10% acetic acid, 40% methanol) for 45 min, then destained in a solution of 10% acetic acid and 20% methanol until bands were resolved.
Immunohistochemical analysis
Pellets were fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned onto slides. Sections were deparaffinized and treated with 1 mg/ml protease in PBS for 30 min at room temperature. For collagen I and collagen II double-staining, protease treatment was followed by 1 mg/ml hyaluronidase in PBS for 30 min at 37°C. Slides blocked with 5% bovine serum albumin (BSA) were probed overnight at 4°C with primary antibodies to collagen I (rabbit polyclonal antibody, 1:200; kind gift from A Hollander, University of Bristol, UK), collagen II (II-II6B3 mouse monoclonal antibody, 1:200; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), and collagen X (mouse polyclonal antibody, 1:300; kind gift from GJ Gibson, Henry Ford Hospital, Detroit, MI, USA) diluted in 1% BSA. Secondary antibodies (Oregon Green-conjugated anti-rabbit (1:250) and Alexa Fluor 596-conjugated anti-mouse (1:500) for double-staining; fluorescein isothiocyanate-conjugated anti-mouse diluted 1:500 for collagen X) were added in 1% BSA for 45 min at room temperature. Slides were mounted with ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (Life Technologies) and imaged using an Olympus BX51 microscope (Olympus America, Melville, NY, USA).
Western blot analysis
Whole-cell lysates were obtained by pooling 20 pellets from each condition at days 2, 7 and 14. Pellets were rinsed with PBS, snap-frozen in liquid nitrogen and crushed. The powdered sample was immediately resuspended in an enhanced radioimmunoprecipitation assay buffer with 1% SDS and 100 mM DTT [23]. Hypoxic cultures were maintained in low oxygen until directly before freezing.
Lysates were prepared in Laemmli sample buffer with 100 mM DTT and boiled for 3 min. Samples were run on 12% SDS-PAGE gels for histone H3 and on 6% SDS-PAGE gels for HIF blots using PageRuler Plus Prestained Protein Ladder (Molecular Weight Marker) (Thermo Fisher Scientific). Gels were transferred to polyvinylidene difluoride and blocked for 1 h with 2% milk-PBST (1 × PBS, 0.1% Tween 20) at room temperature. Primary antibody incubations were done in blocking solution overnight at 4°C. All washes were done three times for 10 min each in PBST.
The quantity of nuclear protein loaded between samples was normalized to histone H3. The relative signal of bands from a blot with equal volumes of lysates loaded were determined using an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA), and lysate volumes were adjusted accordingly for HIF protein blots. Anti-histone H3 primary antibody (ab24834; Abcam, Cambridge, MA, USA) was used at a 1:500 dilution, and IRDye 800CW secondary antibody (catalog no. 926-32210; LI-COR Biosciences) was used at a 1:400 dilution. HIF blots were probed with anti-HIF-1α (1:250 dilution, catalog no. 610958; BD Biosciences) and anti-HIF2α antibodies (1:500 dilution, sc-46691; Santa Cruz Biotechnology, Santa Cruz, CA, USA). To visualize blots, horseradish peroxidase-conjugated secondary antibody (1:2,500 dilution, catalog no. W4021; Promega, Madison, WI, USA) and Western Lightning Plus-ECL (PerkinElmer, Waltham, MA, USA) were used with a Kodak RP X-OMAT Developer (Eastman Kodak Health Imaging, Rochester, NY, USA).
Statistical analysis
SPSS version 19.0 software (SPSS, Chicago, IL, USA) was used to assess statistical significance, which was defined as P < 0.05. Normality was evaluated using the Shapiro-Wilk test. Two-tailed independent t-tests or paired t-tests were used for normally distributed data, and Mann-Whitney U or Wilcoxon tests were used for nonparametric data. The unpaired tests were used to compare means for healthy chondrocytes with those of OA chondrocytes at a given oxygen level, and paired tests were used to compare means between oxygen levels within a given disease condition. The correlation coefficient, Pearson's r, for the ratios of mRNA expression of hyaluronic acid synthase 2 (HAS2) and sGAG retention at 2% oxygen over 20% oxygen was determined using bivariate analysis.