Harvest of bovine articular cartilage and isolation of the cells
Surface zone of bovine articular cartilage was obtained from the calf knee joint as described previously in our lab by Khalafi et al [13
]. The bovine articular cartilage was divided into three zones (surface, middle and deep). The total thickness of articular cartilage was about 5mm. The surface zone of approximately 100μm thickness was obtained by dermatome. After washing with medium, they were minced and digested with 0.2% collagenase P [Roche Molecular Biochemicals] in DMEM/F12 containing 3% fetal bovine serum (FBS) for 3 hours at 37°C. The superficial zone, the middle and deep zones were obtained by using a custom device.
The gap interval was 1.25mm. The deep zone consists of the first 1.25mm close to subchondral bone. Next 1.25mm was designated as middle zone. The middle and deep zone cartilage was digested with collagenase (0.2%) for 6 hours. The dispersed cells were filtered through a 70μm cell strainer to minimize cell aggregates and washed with DMEM/F12 containing 10% FBS and penicillin and streptomycin twice. The cells were placed in same medium for 1 hour before counting the cells.
Hoechst 33342 staining
Isolated cells were plated in monolayer on 100 mm culture dish at 4×106 in DMEM/F12 containing 10% FBS and 1% penicillin/streptomycin [Gibco]. After 5 days monolayer culture, they were released by trypsin treatment, counted, and resuspended in prewarmed Hanks’ Balanced Salt Solution (HBSS) containing 2% FBS, 10mM HEPES buffer (HBSS+) at a density of 1×106 cells/ml. 5μg/ml Hoechst 33342 [Sigma Chemical Co] was added and incubated for 60 min at 37°C. When the verapamil was used, the cells were stained in presence of 50μM verapamil [Sigma Chemical Co]. After that, cells were washed once excess ice-cold HBSS+ and pelleted through a serum cushion. The cells were resuspended in ice-cold HBSS+ containing 2μg/ml propidium iodide (PI). After Hoechst staining, the cells were maintained at 4°C before FACS analysis.
Flow cytometry analysis
Analysis and cell sorting were performed on a MoFlo cell sorter [DakoCytomation] using a Coherent Enterprise laser [Coherent] for excitation of Hoechst 33342 and propidium iodide. The Hoechst dye was excited at 350 nm and Hoechst emission was measured using a 450/65 BP filter (Hoechst blue) and a 670/30 EFLP (Hoechst red) optical filter [Chroma Technology Corp]. A 510 DCLP (510 nm long pass dichroic mirror) was used to separate the emission wavelengths. PI fluorescence was measured through a 580/30 BP (having been excited at 488nm). Hoechst “blue” measured through the 450 BP filter, represents the standard analysis wavelength for Hoechst 33342 DNA content analysis. Cells positive for PI were seen on a plot of PI vs FSC (forward scatter) and excluded. The addition of PI did not affect the Hoechst staining profile, but permitted exclusion of dead cells. Both Hoechst blue and red fluorescence were performed on a linear scale. The gating on forward and side scatter was not stringent; only erythrocytes and debris were excluded. The side population sorting gates were established as follows. A live gate was defined on the flow cytometry using a two-dimensional profile with PI vs FSC. We established PI negative populations as a live gate and apply this gate to following operations. After collecting 105 events within this live gate, the Hoechst blue vs red profile was displayed. The SP cells were able to be defined. A new gate was established on this profile. The sorted cells were cultured to amplify for 7 days in monolayer culture on 100mm dish. And also, main population (non-SP, NSP) was sorted at the same time as a control.
Cell culture was performed using micromass culture technique. Briefly, 10μl (containing 105 cells) of a concentrated cell suspension (1×107/ml) were plated onto the center of each well of a twelve-well culture plate and the cells were allowed to attach at 37°C for 1 hour. Chondrogenic medium (DMEM/F12 supplemented with 1% FBS, 50μg/ml ascorbate phosphate [Sigma Chemical Co] 1% penicillin/streptomycin [Gibco], and 50mg/ml ITS+Premix [Becton-Dickinson; a final concentration of 6.25μg/ml bovine insulin, 6.25μg/ml transferrin, 6.25ng/ml selenous acid, 1.25mg/ml bovine serum albumin, and 5.35μg/ml linoleic acid]was gently overlaid from the periphery so as not to detach the cell nodules, and cultures were maintained in chondrogenic medium for 7 days prior to analysis. The day of plating in micromass culture was designated as day 0. Starting on day 0, recombinant human Bone Morphogenetic Protein (BMP)-4, BMP-7, and transforming growth factor β1 (TGFβ1) [R&D Systems] was added to the each well at a final concentration of 300ng/ml for BMPs, 10 ng/ml for TGF-β1. The chondrogenic medium was changed at day 4 and the BMPs and TGF-β1 were re-added during change of culture medium.
Alcian blue staining
After 7 days, the micromass cultures were rinsed twice with PBS, fixed with 10% neutral buffered formalin (pH7.4) including 1% CPC (Cetylyridinium Chrolide) for 15 minutes at room temperature, washed with PBS, and covered with alcian blue (pH 1.0) (1% alcian blue 8 GS [Sigma Chemical Co] in 0.1M HCl). After 1 hour staining, cultures were washed extensively with distilled water and photographed. To quantify alcian blue intensity, cultures were rinsed with 50mM Tris-HCl (pH7.4), incubated with 300μl 4M Guanidine-HCl, 50mM Tris-HCl, 0.1% Chaps, pH7.4 for 2 hours at room temperature. Optical density of the extracted dye was evaluated at 595nm in a microplate reader [Bio-Rad].
For immunoblotting, the proteins secreted in media were electrophoretically resolved by SDS-PAGE and transferred onto PVDF membrane. The blots were then washed once in sterilized water, blocked with 5% non-fat dry milk in TBST for 1 hour at room temperature, and incubated with a mouse anti-bovine SZP antibody (generous gift of T. Schmid) diluted 1:5000 in blocking buffer at 4°C over night. The blots were then washed three times, 20 minutes each, in TBST. The blots were then incubated with a 1:3000 dilution of secondary antibody conjugated to horseradish peroxdase (goat anti-mouse IgG antibody) for 1 hour at room temperature in 1% non-fat dry milk in TBST. After washing three times, 20 minutes each, in TBST, the immunoblots were then processed for detection using the chemiluminescence kit. The immunoblots were exposed to X-ray film for 60 seconds.
Histology and Immunohistochemistry
After 7 days, the micromass cultures were fixed with 10% neutral buffered formalin, peeled off the wells, embedded in paraffin, and sectioned at 7 μm. For histologic evaluation, sections were deparaffinized and stained with toluidine blue.
For immunohistochemical analysis, after deparaffinization, nonspecific binding was blocked with 3% goat or horse serum albumin. The slides were incubated over night at 4°C with a rabbit anti-bovine type II collagen antibody [Chemicon International], diluted 1: 150 in normal blocking serum or mouse anti-bovine SZP antibody diluted 1:1000 in normal blocking serum. Slides were then incubated for 60 minutes at room temperature with biotinylated goat anti-rabbit IgG or goat anti-mouse IgG diluted in normal blocking serum. The antibody-biotin conjugates were detected with a streptavidin–biotin–horseradish peroxidase complex (vectastain elite ABC kit) [Vector] applied for 3 minutes at room temperature, using diaminobenzidine [Vector] as a substrate. Negative controls were sections of micromass cultures incubated with normal rabbit IgG instead of primary antibody.