Articular cartilage samples were harvested from the metacarpo-phalangeal joints of calves aged 6–12 months. Full-thickness cartilage explants of 8 mm diameter and with a thickness of 0.9–1.2 mm were prepared using a dermal biopsy punch and scalpel. The explants were then randomly divided into two groups. From the centre of the explants, 3-mm cores were punched out, using a custom built device to ensure punching in the exact middle of the explant. Group 1 (n = 12) specimens (both outer ring and inner core) were incubated for 24 hours in 0.1% hyaluronidase type I-S from bovine testes (Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) followed by 24 hours in 10 U/ml highly purified collagenase VII (Sigma-Aldrich Chemie BV), both in Dulbecco's modified eagle's medium/Hams' F12 with 2% foetal calf serum. Specimens from group 2 (controls; n = 12) were incubated in Dulbecco's modified eagle's medium/Ham's F12 culture medium (Gibco, Grand Island, NY, USA) supplemented with 2% foetal calf serum at 37°C for 48 hours (controls). The choices of enzymes, enzyme concentrations and treatment times were based on the findings from our previous in vitro study [22]. After 48 hours the samples were washed three times for 10 min in culture medium, and the 3-mm inner cores were reimplanted in their accompanying 8-mm outer rings. Constructs were then implanted in four subcutaneous pockets on the backs of six nude mice (BALB-C nu/nu; Harlan, Horst, The Netherlands), for which approval was obtained from the local animal ethical committee (DEC no.126-01-01). Each mouse carried two enzyme-treated constructs (group 1) and two control constructs (group 2). After 5 weeks the mice were killed by cervical dislocation and constructs were harvested.
Histology
From each mouse, one control and one enzyme-treated construct were processed for histology. Constructs were divided into two halves. One half was fixed in 4% phosphate-buffered formalin and embedded in paraffin, and the other half was frozen in liquid nitrogen and stored at -80°C for later cryosection preparation. Sections (6 μm) were cut using a standard microtome (paraffin) or cryomicrotome and mounted on Starfrost® slides (Knittel, Braunschweig, Germany). Paraffin sections were haematoxylin and eosin stained as well as immunostained for collagen type II. Cryosections were used for thionin (proteoglycan) stain, picro-Sirius Red stain and immunohistochemical stains for procollagen type I, collagen type I and collagen type III.
Evaluation of chondrocyte viability
The number of vital chondrocytes was counted in surface, middle and deep zones in both wounded and unwounded areas using haematoxylin and eosin coloured slides at 400× magnification using a 50 × 50 μm boxed grid. Nuclear and cytoplasmic changes, as described by Kim and coworkers [23], were analyzed to assess cell viability/death. Only cells with visible nuclei were evaluated. Values for wounded cartilage were scored from a 150-μm broad band on both sides of the lesion, and the values for both sides of the interface were then averaged to obtain one value per interface. Furthermore, the cartilage was divided into superficial (100 μm from the surface down), deep (150 μm from the bottom up) and middle (between superficial and deep) zones, as previously described [22]. For comparison, values from unwounded tissue were obtained from the middle part of the outer ring, which is more then 1 mm from the wound edge. Chondrocyte densities are represented as vital cells/mm2.
For each explant the amount of viable chondrocytes was calculated from the values obtained from two to four sections. Subsequently, the averages for the control and enzyme-treated groups were calculated and used for statistical evaluation.
Evaluation of integration
Cryosections were fixed in acetone and stained with 0.04% thionin in 0.01 M sodium acetate for 5 min. For each sample we assessed the percentage of total interface length that had a matrix–matrix connection using a microscope with a 50 μm square grid. A clear distinction could be made between parts with a matrix connection and parts of the cartilage touching each other but without a clearly connected matrix, which were scored as parts with a gap. Interface integration percentages were obtained from measurements of two to four different sections from each sample, resulting in one average value for each interface.
Picro-Sirius Red stain
Cryosections were fixed in acetone and stained with 0.1% Sirius Red F3BA (Direct Red 80; Fluka Chemie, Zwijndrecht, The Netherlands) in a saturated picric acid solution for 1 hour. Brief washing in 0.1% acetic acid was followed by rapid dehydration in 100% alcohol (three changes for 3 min each), after which a xylene bath (two changes for 5 min each) was used to prepare the slides for mounting with Entellan (Merck, Darmstadt, Germany). Slides were analyzed using a polarized light microscope (Dialux 20; Leitz, Wetzlar, Germany) to evaluate fibre orientation in the interface area. The relative sign of birefringence was determined using the analyzer filter. For semiquantitative analyses, samples from both groups were classified as follows: 0 = no fibres crossing; 1 = occasional fibre crossing; and 2 = many fibres crossing.
Collagen immunostaining
Cryosections were fixed in acetone for procollagen type I, and collagen types I and III staining. For collagen type II staining paraffin sections were deparaffinized using xylene and rehydrated through a graded series of ethanol, after which they were incubated with 0.2% pronase for 30 min to retain antigenicity. Treatment with 1% hyaluronidase (Sigma-Aldrich Chemie BV) was used to unmask the epitopes. Nonspecific binding was blocked using 10% normal goat serum (CLB, Amsterdam, The Netherlands) followed by incubation with the respective antibody for 2 hours. Antibodies used were M38 and II-6B3 (both 1:100; Developmental Studies Hybridoma Bank) for procollagen type I and collagen type II, respectively; ab6308 (mouse monoclonal IgG antibody, 1:500; Abcam Ltd, Cambridge, UK) for collagen type I; and ab6310 (mouse monoclonal IgG antibody, 1:500, Abcam Ltd) for collagen type III. All primary antibodies were previously complexed with goat Fab fragment against mouse conjugated with alkaline phosphatase (GAMAP, 1:400; Immunotech, Marseilles, France) at 4°C overnight. After coupling, 0.1% normal mouse serum was used for 2 hours before usage to capture the unbound GAMAP, after which the antibody solution was used on the slides. Sections were subsequently incubated for 30 min with alkaline phosphatase anti-alkaline phosphatase (APAAP, 1:100 for procollagen I and collagen II, 1:75 for collagen types I and III; Dakopatts, Copenhagen, Denmark). New Fuchsine substrate (Chroma, Kongen, Germany) was used for colour development and haematoxylin for counterstaining, after which slides were mounted using Vectamount (Vecto Laboratories Inc., Burlingame, CA, USA). Negative controls were subjected to the same protocol with omission of the primary antibody.
Mechanical testing
After harvesting of the constructs, the surrounding fibrotic tissue was carefully removed. From each of the six mice, one control and one enzyme-treated construct were frozen using liquid nitrogen and stored in airtight tubes at -80°C for later mechanical testing. Immediately before testing constructs were slowly thawed in airtight tubes. Thickness of the sample was measured to an accuracy of 50 μm using calipers. Constructs were then mounted in a specially designed push-out setup (Fig. 1) on a materials testing machine (LRX; Lloyd Instruments, Fareham, UK) equipped with a 500 N load cell. Push-out tests were performed by leading the push-out rod on top of the 3 mm inner core through the specimen at 10 μm/s. During the test constructs were kept moist by adding a few drops of phosphate-buffered saline on top before starting the test, which on average took 4–5 min. During the test both displacement and load were monitored at a sample frequency of 18 Hz and the output of these values was read out and stored on a desktop computer. For each specimen the peak load-to-failure (maximum observed load) was used to calculate the interface stress-to-failure (maximum load normalized to interface area) as a representative marker of interfacial strength. Furthermore, we also performed push-through tests of intact cartilage for comparison (n = 4) and push-out tests of constructs immediately after reassembly of core and annulus (n = 8) to determine the friction component of our setup; all this was done in a manner similar to that described above.
Statistical analysis
Values shown are mean ± standard deviation unless otherwise specified. Statistical analyses for both viable cell count and mechanical testing were done using Student's t-test for independent samples. Matrix integration scores and results from polarized light microscopy were analyzed using the Mann–Whitney U test. P ≤ 0.05 was considered statistically significant.