Cartilage harvest
Cartilage biopsies were harvested with a curved chisel from macroscopically affected and unaffected surplus cartilage from seven patients with OA (age 64 to 83 years), with OA grades 3 to 5 on the Ahlbäck scale [16], undergoing total knee replacement. The affected side was considered to be the femoral condyle on the concave side of the knee deformity; that is, the medial condyle in varus deformity and the lateral in valgus knees. In all patients the hip–knee–ankle angle was determined from standing whole-leg radiographs (an angle of more than 180° indicates a valgus knee deformity). The harvested biopsies were transported to the cell culture laboratory in sterile saline solution (0.9% NaCl; Fresenius Kabi, Uppsala, Sweden) supplemented with gentamicin sulphate (50 mg/l; Gibco, Paisley, Renfrewshire, UK) and amphotericin B (250 μg/ml; Gibco). Part of the cartilage biopsy was processed for histology, blinded and scored by two independent experienced researchers in accordance with a modified (biopsies without subchondral bone) Mankin scale [17], with a maximum score of 13. The rest of the biopsy was used for cell culture as described below. The donation of surplus cartilage was approved by the ethical committee at the Medical Faculty at Gothenburg University.
Cell culture
The chondrocytes were isolated from the surrounding matrix by mechanical mincing of the tissue with scalpel followed by enzymatic treatment overnight with collagenase (0.8 mg/ml; Worthington Biochemical Corp, Lakewood, NJ, USA) in Ham's F-12 medium (Invitrogen, Lidingö, Sweden), at 37°C in 7% CO2/93% air. The isolated cells were seeded at 104 cells/cm2 in culture flasks (Costar; Corning Incorporated, Corning, NY, USA) in DMEM/F12 medium (Invitrogen) supplemented with L-ascorbic acid (0.025 mg/ml; Apotekets produktionsenhet, Umeå, Sweden), gentamicin sulphate (50 mg/l; Gibco), amphotericin B (250 μg/ml) and L-glutamine (2 mM; Gibco) with the addition of 10% human serum [18]. In brief, the human serum was collected in transfusion bags (dry pack; JMS, Singapore) from healthy blood donors. The serum was left to coagulate overnight at 4 to 8°C, centrifuged, sterile filtered, divided into aliquots and frozen until use. The first medium change was made on day 6 and thereafter twice a week. When the cells reached 80% confluence, they were subcultured and frozen. Thawed cells were subcultured into new flasks (Costar) at a density of 4 × 103 cells/cm2.
Three-dimensional pellet culture
After passage 1, the cells were cultured in a 3D pellet culture system as described previously [15, 19]. On days 7 and 14, the pellets were fixed in Histofix™ (Histolab Products AB, Göteborg, Sweden), dehydrated and embedded in paraffin. Sections 5 μm thick were cut and placed on microscope slides (Superfrost Plus; Menzel-Gläser, Braunschweig, Germany), deparaffinized and stained with Alcian blue/van Gieson or immunohistochemically with anti-type I collagen and anti-type II collagen antibodies.
Immunohistochemistry of pellets
Deparaffinized sections were digested with hyaluronidase, 8,000 units/ml (Sigma, St Louis, MO) in 0.1 M PBS for 60 min at 37°C and blocked with 3% BSA (Sigma) in PBS for 5 min. The primary antibodies (anti-type I and II collagen; ICN Biomedicals, Aurora, OH, USA), diluted 1:150 in PBS containing 3% BSA, were incubated with the sections for 1 hour at room temperature (20–22°C). The secondary antibody, peroxidase-conjugated goat anti-mouse (1:150; Jackson Immunoresearch Laboratories, West Grove, PA, USA) were applied to the sections for 1 hour at room temperature. A substrate kit (Vector VIP; Vector Laboratories, Burlingame, CA, USA) was used for visualization and the results were analysed with a Nikon Optiphot2-pol microscope (Nikon Instruments Inc, Melville, NY, USA). Goat cartilage and bone explants were used as a positive control; for a negative control the primary antibodies were omitted.
Biochemical analysis of pellets
On days 7 and 14, pellets were digested in papain (Sigma) solution (0.3 mg/ml in 20 mM sodium phosphate buffer, pH 7.4, containing 1 mM EDTA and 2 mM dithiothreitol) for 60 min at 60°C. The digested pellets were then mechanically dissolved by vortex-mixing and further analysed for DNA, glycosaminoglycan and hydroxyproline content as described previously [15]. All biochemical analyses was performed on triplicate pellets.
Cells in scaffold
Culture-expanded cells (passage 2), 106/cm2 or 5.0 × 106/cm2, were seeded on human serum precoated Hyaff-11 scaffolds (thickness 2 mm; Fidia Advanced Biopolymers, Abano Terme, Italy) in 100 μl in Ham's F12 medium (Invitrogen) supplemented with 20% human serum. After incubation overnight at 37°C in 7% CO2/93% air, the scaffolds were cultured in serum-free medium [15] in non-adherent dishes (Falcon four-well IVF; Becton Dickinson, Le Pont De Claix, France) for 14 days. After fixation, the scaffolds were embedded in paraffin, sectioned (10 μm thickness), stained with Alcian blue/van Gieson and analysed immunohistochemically for type II collagen as described above.
Isolation of total RNA
Total RNA was isolated from cells cultured in a monolayer (passage 1) and from day 7 pellets with the use of an RNeasy mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's description. Before RNA isolation, the pellets were collected in a 1.5 ml micro-tube (Sahrstedt, Nümbrecht, Germany) containing RLT buffer (Qiagen) and disrupted by sonication. To remove cell debris and cartilage matrix proteins a QIAshredder column (Qiagen) was used. Contaminating genomic DNA was removed from the isolated RNA by using a DNA-free kit (Ambion, Huntingdon, UK) and total RNA content and purity were determined spectrophotometrically at 260 and 280 nm. In general, A260/A280 ratios of about 2 were considered to indicate acceptable purity of the samples [20].
Real-time PCR
Expression patterns of four cartilage genes were analysed by real-time PCR with an ABI PRISM 7000 (Applied Biosystems, Foster City, CA, USA) sequence detector and software system. TaqMan MGB probes (FAM dye-labelled) and primers for type I collagen (Hs00164004_m1) and type X collagen (Hs00166657_m1) were ordered from Applied Biosystems assays-on-demand (20× assay mixes). The gene-specific primers and probes for type II collagen 5'-TGG TGT CAA AGG TCA CAG AGG TTAT-3', antisense 5'-GGA ACC ACT CTC ACC CTT CACA-3', probe 5'-TCC CTT AGC ACC GTC CAG GCC TG-3', were designed by using Primer Express Software version 2.0 (Applied Biosystems). All genes were designed to amplify fragments of 70 to 150 base pairs; as endogenous control, 18S rRNA labelled with VIC/TAMRA was used (Applied Biosystems).
Reverse transcription in vitro was performed with 500 ng of total RNA with the use of random hexamer primers and TaqMan Reverse Transcription reagents (Applied Biosystems). Real-time PCR was performed with 5 μl of diluted (1:10) cDNA corresponding to 10 ng of RNA, 15 μl of TaqMan Universal PCR master mixture (Applied Biosystems), 1× assay-on-demand mixes of primers and TaqMan MGB probes. All samples were analysed in triplicate and PCR was performed in optical 96-well microtitre plates (Applied Biosystems). After an initial denaturation step at 95°C for 10 min, the cDNA products were amplified with 40 PCR cycles consisting of a denaturation step at 95°C for 15 s and an extension step at 60°C for 1 min.
To analyse the real-time PCR data, a standard curve method was used. The data were analysed with ABI Prism 7000 SDS software (Applied Biosystems). For each sample, the Ctsample values were determined as the cycle number at which all samples were in the exponential phase of amplification. By using the formulas below, a value (Y) was obtained as a measure of the gene expression correlated to the standard curve for that particular gene: X = (Ctsample - Intercept value)/Slope value; X10 = Y. The Y value for each cDNA sample and target sequence was divided by the Y value from the housekeeping gene (18S) for that particular sample to derive a ΔCt value (PE-ABI; Sequence Detector User Bulletin 2).
Statistical analysis
Biochemical differences between donors and chondrocytes isolated from affected and unaffected were analysed with a two-sided Student's t-test (two-sample equal variance). P < 0.05 was considered significant. All analyses were performed with cell samples from at least three separate donors unless otherwise indicated; as a comparison, surplus cells from three or four donors undergoing ACT were used [15].