Chondrocyte migration in enzymatically treated cartilage explant
Full-thickness cartilage was harvested from the patella of a 3-month-old (young adult) pig; cartilage was obtained as rectangular blocks, each measuring 2 mm × 2 mm × 1 mm in size, that were generated by manually sawing. A 0.5-mm-diameter cartilage gap was created in each cartilage block. The cartilage blocks then were treated enzymatically with 0.2% actinase (Catalog 650,164, Kaken, Tokyo, Japan) and 0.02% collagenase P (Catalog 11,213,865,001, Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco’s modified essential medium (DMEM; Sigma-Aldrich D5796) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), 0.2% ascorbic acid, and 0.5% antibiotics (streptomycin 1.0 mg/mL, penicillin 10,000 unit/mL), at 37 °C in a 5% CO2 atmosphere for 15 or 60 min. Blocks without enzyme treatment served as controls. The blocks with or without enzyme treatment were washed three times in serum-free DMEM. To permit visualization of the number and the migration distance of the migrating chondrocytes, the washed blocks were encapsulated in collagen gel using a collagen gel culturing kit (Nitta Gelatin, Osaka, Japan) and then incubated at 37 °C in a 5% CO2 atmosphere for 1 week (Fig. 1A). The blocks then were fixed with 4% paraformaldehyde and observed using a phase-contrast microscope (ECLIPSE TS 100, Nikon, Tokyo, Japan). Chondrocyte migration was examined at the outer cut edge or within the gap of the cartilage blocks. Next, the samples were embedded in a tissue-freezing medium and stained with toluidine blue. Some samples also were subjected to immunohistochemical analysis (see below).
Migration of isolated chondrocytes using Boyden chambers
Chondrocytes were isolated from the knee of a 3-month-old pig. Superficial-zone (shallowest one-third) and deep-zone (deepest one-third) cartilage samples were dissected away from the femoral condyle. Cartilage samples were digested with actinase as above but for 2 h with stirring, followed by digestion (overnight at 37 °C in a 5% CO2 atmosphere) with collagenase (Sigma-Aldrich) at a final concentration of 0.02% in serum-free DMEM (1 mL per well). The density of the chondrocytes was determined by counting with a hemocytometer and set to 105 cells per mL. Migration assays were performed as follows. A modified Boyden chamber (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was prepared using a 12-well cell culture insert companion plate and cell culture insert containing polyethylene terephthalate membrane with 8-μm pores. The lower chambers were filled with serum-free DMEM, and the upper chambers (inserts) were placed over the lower chambers. Chondrocytes in serum-free DMEM were placed into the upper inserts and the plates were incubated overnight at 37 °C in a 5% CO2 atmosphere to permit the cells to attach to the membrane. Next, the upper inserts were transferred to fresh lower chambers containing serum-free DMEM (control) or DMEM supplemented with 10% FBS, PDGF-AA (50 ng/mL), PDGF-AB (50 ng/mL), or PDGF-BB (50 ng/mL). Cells were allowed to migrate toward the lower side of the insert membrane during a 6-h incubation at 37 °C in a 5% CO2 atmosphere. After incubation, cells remaining on the upper surface (having not migrated through the membrane) were removed from the upper surface by wiping with a cotton swab, and membranes then were removed, fixed with 4% paraformaldehyde, stained with Meyer’s hematoxylin, and mounted on glass slides. The number of migrated cells was counted in three randomly selected fields per membrane using a light microscope at 20× magnification, and the mean and standard deviation (SD) were calculated for each treatment.
Gene expression
Total RNA was extracted from various portions of cartilage using a RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, USA) according to the manufacturer’s instructions. An aliquot of each resulting total RNA preparation (in a reaction volume of 20 μL) was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems [ABI], Foster City, CA, USA). The resulting single-stranded cDNA products then were analyzed by real-time (RT) PCR using TaqMan gene expression assays (ABI) and an ABI 7900HT Fast Real Time PCR System (ABI). Specifically, cDNA samples (1 μL in a 21-μL reaction volume) were analyzed for the genes of interest. The master-mix consisted of 1 μL of 20x TaqMan® Gene Expression Assays (ABI), 9 μL of water, 10 μL of 2x TaqMan® Gene Expression Master Mix (ABI), and 1 μL (18 μM) of PCR primers (Invitrogen, Waltham, MA, USA). The level of expression of each target gene was normalized to that of GAPDH (encoding glyceraldehyde dehydrogenase, a housekeeping protein) in the same sample, and expression then was compared between samples. Analysis of each sample was repeated 5 times for each gene of interest. RT-PCR was performed using a program as follows: samples were initially denatured at 95 °C for 20 s, then subjected to 40 cycles of denaturation at 95 °C for 1 s, annealing at the primer-specific temperature for 30 s, and elongation at 60 °C for 20 s. The gene of interest was PDGFRB, which encodes PDGFR-B. The sequence of the pig PDGFRB gene transcript was retrieved from GenBank as Accession No. AK394805.1, and primers and probes for the target were designed using Primer3 (http://primer3.sourceforge.net/). To avoid genomic DNA amplification in the PCR results, the exon-intron boundaries were confirmed by comparison of the RNA sequence with the genomic sequence from Blat at UCSC (http://genome.ucsc.edu). The probe and primer sequences for PDGFRB were as follows: probe, 5′-AAT GAA GTC AAC ACC GCC TC-3′; forward primer, 5′-CAA CGA GGG TGA CAA CGA CT-3′; and reverse primer, 5′-CTC TGG CTC TGG CTC CTC TT-3′. The difference in threshold cycle (ΔCt) for target sequences versus GAPDH (ABI Ss03375435)101 was measured for each sample.
Western blot analysis
Approximately 200 mg wet weight of various portions of cartilage were incubated for 24 h at 4 °C on a rocker after addition of 600 μL of cold buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% (w/v), NP40, 0.5% (w/v) deoxycholic acid, and 1 tab of proteinase inhibitor (Complete Mini, Catalog 11,836,153,001, Roche, Basel, Switzerland). The samples were cleared by centrifugation at 10,000–15,000 rpm at 4 °C for 20 min, and each cleared supernatant was combined with 3 volumes of cold absolute ethanol and incubated overnight at − 20 °C. The samples then were centrifuged at 10,000–15,000 rpm at 4 °C for 20 min, and each resulting pellet was washed, dried, and resuspended in 1 mL of chondroitinase ABC (CHase) buffer (50 mM sodium acetate, 50 mM Tris, 10 mM EDTA, pH 7.6). Each extract then was combined with 15 μL (60 mU) of CHase and the mixtures were incubated for 2 h at 37 °C to dissolve the samples. For western blotting, protein extract samples were separated on a 10% SDS-PAGE gel and electroblotted onto nitrocellulose membranes. After blocking with 1% Membrane Blocking Agent (Catalog RPN2125V; GE Healthcare, Boston, MA, USA) membranes were incubated overnight at 4 °C with 1000-fold diluted rabbit monoclonal anti-PDGFR-B antibody (Catalog #4564, Clone No. C82A3; Cell Signaling Technology, Danvers, MA, USA) or 2000-fold diluted anti-tubulin antibody (Catalog #9280-0050G, BIO-RAD, Hercules, CA, USA). After washing, membranes were incubated for another hour with the secondary antibody (1000-fold diluted rabbit monoclonal anti-PDGFR-B antibody or 2000-fold diluted anti-tubulin antibody). Membranes were washed and rinsed with ECL™ Prime Western Blotting Detection Reagent (Catalog RPN2236; GE Healthcare). Bands were imaged using LAS-3000 (Fujifilm, Tokyo, Japan), followed by quantification of band intensity using Image J (ver. 5.0, National Institute of Health, USA).
Immunohistochemistry
Staining was carried out using anti-lubricin antibody (Catalog 5C11; Sigma-Aldrich), anti-PDGFR-B antibody (as above), anti-keratan sulfate antibody (Catalog RIT-M001, Cosmo Bio Co., Ltd., Tokyo, Japan), and anti-chondroitin 4 sulfate antibody (Catalog PRPG-BC-M02, Cosmo Bio Co., Ltd.) for immunostaining. Cartilage explants (for staining with anti-lubricin antibody, anti-keratan sulfate antibody, and anti-chondroitin 4 sulfate antibody) were obtained from the medial condyle of a 3-month-old pig. Similarly, whole osteochondral tissues (for staining with anti-PDGFR-B antibody) were harvested from the medial condyle of the tibia of a 3-month-old pig and consisted of tissue from the peripheral region that was covered with meniscus to the central region that was not covered with meniscus and close to the intercondylar eminence of the tibia. Cartilage explants were treated enzymatically and cultured for 1 or 2 weeks as described above. These explants then were fixed in 10% neutral buffered formalin for 3 days and embedded in paraffin wax. Before embedding, osteochondral tissues were decalcified by incubation for 14 days in 5% ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS). Sections of the resulting samples were deparaffinized in xylene and dehydrated in ethanol. Endogenous peroxidase activity was blocked by incubation in 0.6% H2O2. Peroxidase-labeled polymer (Catalog K4003; DAKO, Santa Clara, CA, USA) and diaminobenzidine (DAB) substrate solutions were used for visualization of immunoreactivity. Samples were observed using a light microscope (AXIO Imager A1, Carl Zeiss Microscopy, Jena, Germany).
Statistical analysis
The Mann-Whitney U test was used for comparisons between two groups. Multiple comparisons were performed by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test.:p < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS software (version 17.0; SPSS, Chicago, USA).
Ethics statement
All procedures were conducted in accordance with Institutional Guidelines on Animal Experimentation at Keio University and Institutional Guidelines on Animal Experimentation at the National Defense Medical College. Experimental approval was obtained from an institutional review board of Keio University and the National Defense Medical College. This study was carried out in compliance with the ARRIVE guidelines.