Articular cartilage tissue was obtained from the metatarso-phalangeal joints of freshly slaughtered 6-month-old pigs in a slaughterhouse. Articular cartilage slices were cut into smaller pieces, and the cartilage specimens were washed well in PBS (pH 7.4) and digested with 0.25% trypsin–ethylenediamine tetraacetic acid (Gibco, Grand Island, NY, USA) for 20 minutes. The resultant chondrocyte preparations were washed again with PBS to remove the trypsin, and were then incubated for about 8 hours in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 0.1% type-II collagenase (Worthington Biochemical Co., Lakewood, OH, USA), 10% heat-inactivated FBS (Equitech-Bio, Inc., Kerrville, TX, USA) and antibiotics. The chondrocytes were subsequently isolated and washed with culture medium, collected by centrifugation (2,000 rpm, 37°C, 5 min), and then mixed with 0.2% atelocollagen gel (type-I collagen derived from bovine tendons; Koken Co., Tokyo, Japan) containing culture medium (DMEM; Gibco).
Twenty-four-well plates containing type-I honeycomb collagen sponges (discs with a diameter of 15 mm and thickness of 2 mm; Koken Co.) at the bottom of each well were used as 3D carriers of the chondrocyte culture . Chondrocytes in the atelocollagen gel and also chondrocytes in the culture medium composites were added to each sponge and were incubated at 37°C for 1 hour. The final cell density was adjusted to 2 × 106 cells/well/ml . After the collagen sponge and cell–collagen gel composites became stiff, they were then incubated with 2 ml complete DMEM in 5% CO2/95% air at 37°C, and the cultured medium was replaced with fresh DMEM containing L-ascorbic acid (50 μg/ml) twice weekly.
Low-intensity pulsed ultrasound stimulation
The sonic accelerated fracture healing system (Exogen Inc., Piscataway, NJ, USA), a LIPUS apparatus, was used to deliver an ultrasound signal. The sonic accelerated fracture healing system is one of the instruments in current clinical use in cases of delayed repair of a fracture. The temporal average intensity was 30 mW/cm2 and the frequency was 1.5 MHz with a 200-μs tone burst repeated at 1.0 KHz. LIPUS was applied to the chondrocytes after 24 hours in culture through the bottom of the culture dish (24-well plate) via a coupling gel and silicon rubber that had been placed between the LIPUS transducer and the dish. LIPUS was administered for 20 minutes every day in a span of this experiment. Control samples were prepared in the same manner without LIPUS. Thereafter, the cultured tissues and their supernatant medium were harvested at days 3, 7, 10 and 14.
The cartilage tissues were harvested 1, 3, 7, 10 and 14 days after culture (2 hours after the last LIPUS) and were cut into smaller pieces. Each sample was then incubated for about 8 hours in DMEM (Gibco) supplemented with 0.1% type-II collagenase (Worthington Biochemical Co.), 10%-heat-inactivated FBS (Equitech-Bio, Inc.) and antibiotics. The chondrocytes were then isolated, washed with culture medium, and collected by centrifugation (2,000 rpm, 37°C, 5 min). After the supernatant medium was removed, a solution containing 0.1 M. citric acid and 0.1% crystal violet was added to the cells and then the cells were counted using a hemocytometer (Burker-Turk, Tokyo, Japan).
Histological evaluations of the specimens were conducted at weeks 1 and 2 post culture. The specimens were fixed overnight in 4% paraformaldehyde in PBS, paraffin-embedded, sectioned to a 5 μm thickness, and were stained with Alcian blue and Safranin O. For each sample, at least two different section levels and two histological sections for each level were analyzed. The sections were analyzed and photographed using an Olympus photomicroscope BX-50 (Olympus Co., Tokyo, Japan).
Immunohistochemical analyses were conducted with antibodies raised against anti-type-II collagen antibody (1:100; Fuji Pharm. Lab., Toyama, Japan) and against anti-type-IX collagen (1:100; Chemicon International, Billerica, MA, USA) using week 1 and week 2 postcultures to evaluate the expression of the chondrocyte phenotype and also to assess the type-II and type-IX collagen production levels. The specimens from the 1-week and 2-week postcultures that were harvested 2 hours after the last LIPUS were fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4), and 16-μm cryostat sections were made.
For further confirmation of chondrocyte growth, Ki67 staining was performed because this factor has been shown to be a very reliable proliferation marker . The monoclonal mouse anti-human antibody Ki67 (MIB1; DAKO, Glostrup, Denmark), which also shows cross-reactivity with porcine tissues, was used to determine the extent of proliferation. The sections cultured at day 7 were incubated with this Ki67 primary antibody, followed by a secondary biotinylated anti-rabbit antibody and horseradish peroxidase–avidin complex (DAKO). The color reaction was developed by 3,3'-diaminobenzidine substrate, followed by counterstaining with hemalaun (Merck, Frankfult, Germany). Chondrocytes showing a definite nuclear staining pattern were scored as positive. All slides were reviewed by two investigators without any prior knowledge of the experiment. Five different randomly chosen areas were reviewed in five different specimens, and the number of Ki67-positive cells per 100 chondrocytes was counted in each slice. The percentages of positive cells (MIB1 index) were then calculated.
Quantitative evaluations were conducted using specimens stained with an anti-β-catenin antibody (Acris, Herford, Germany). The nuclear translocation of β-catenin was visible by brown staining. After counting 100 cells, the ratio of the cells whose nuclei were stained brown was compared between the ultrasound group and the control group. All slides were reviewed by two investigators without any prior knowledge of the experiment. In five different randomly chosen areas in five different specimens, the number of β-catenin antibody-positive stained cells per 100 chondrocytes was counted in each slice. The percentages of positive cells were then calculated.
Western blotting analysis
For the western blotting analysis of the specimens cultured for 1 week, cartilage tissues specimens were harvested 2 hours after the last LIPUS and were cut into smaller pieces. Each sample was then incubated for about 8 hours in DMEM (Gibco) supplemented with 0.1% type-II collagenase (Worthington Biochemical Co.), 10%-heat-inactivated FBS (Equitech-Bio, Inc.) and antibiotics. The chondrocytes were then isolated, washed with culture medium, and collected by centrifugation (2,000 rpm, 37°C, 5 min). After the supernatant medium was removed, the cells were rinsed with 200 μl PBS, filtered by centrifugation, and added to a 200 μl aliquot of 2× sample buffer (62.5 mmol/l Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mmol/l dithiothreitol, 0.01% bromophenol blue). The cell lysates were then boiled for 10 minutes at 75°C.
Equal amount of the proteins were separated on a 10% SDS–polyacrylamide gel at 200 V, 25 mA for 80 minutes and were transblotted to nitrocellulose membranes (Millipore, Billerica, MA, USA) using a wet transfer system (BIO-RAD, Hercules, CA, USA) at 200 V, 150 mA for 60 minutes. The membranes were blocked with blocking buffer (5% skimmed milk in TBS and 0.05% Tween 20 and Blocking One–P; Nacalai Tesque Inc., Kyoto, Japan) and were incubated with the following antibodies: anti-Akt (Rockland, Gilbertsville, PA, USA), anti-phospho-Akt (Cell Signaling Technology, Beverly, MA, USA), anti-MAPK and anti-phospho-MAPK (Cell Signaling Technology), anti-cyclin D1 (Biosource, Camarillo, CA, USA), anti-cyclin B1 and anti-focal adhesion kinase (anti-FAK; Upstate Cell Signaling Solutions, NY, USA), anti-phospho-FAK (Rockland), anti-collagen-II (Chemicon International), and anti-collagen-IX (Cell Signaling Technology).
After incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies (dilution: 1/5,000), membranes were finally incubated with a chemiluminescent reagent (NEL103; Perkin Elmer Life Science, Fremont, CA, USA) and the signals produced were recorded on X-ray film (BIOMAX XAR Film, Rochester, Minesota, USA) for a densitometric analysis. The effects of PI3K inhibitor (LY294002; Cell Signaling Technology) and MEK1 inhibitor (PD98059; Cell Signaling Technology) for cell growth were studied. Chondrocytes were pretreated with MEK1 inhibitor (250 μM/ml) and PI3K inhibitor (250 μM/ml) for 12 hours and 24 hours, followed by stimulation with LIPUS for 20 minutes. Each sample was harvested 2 hours after LIPUS stimulation and the influence of these inhibitors was judged by western blotting analysis of proliferating cell nuclear antigen (PCNA; DAKO).
Data are expressed as the mean ± standard deviation. Quantitative evaluations of Ki67-positive cells and β-catenin-positive cells were assessed by Mann–Whitney's U test. The change in the number of chondrocytes was assessed using repeated-measures analysis of variance. P < 0.05 was considered significant.