Culture and degradation protocols
Cartilage–bone cores of 5mm or 7mm in diameter were harvested from young bovine femoropatellar groove articular cartilage within 24 hours of slaughter. After removing the articular surface, three or four 1-mm-thick discs were sliced parallel to the articular surface. A flat edge was made on some of the discs to ease orientation and registration during imaging and analysis. The discs were weighed, and then immediately placed in 2ml sterile culture media in 24-well culture plates. All explants were incubated at 37°C throughout the study.
Culture medium was prepared with low-glucose Dulbecco's modified Eagle medium with 10mmol/l HEPES (GIBCO BRL, Grand Island, NY, USA), 0.1mmol/l nonessential amino acids (Sigma Chemical, St. Louis, MO, USA), 0.4 mmol/l L-proline (Sigma Chemical), and 1mmol/l gadolinium-DTPA (Magnevist, Berlex Imaging, Wayne, NJ, USA). Supplements including: 1% fetal calf serum (GIBCO BRL, Grand Island, NY), 1% l-glutamine (Sigma Chemical), 1% ascorbic acid (Sigma Chemical) and 1% penicillin and streptomycin (Sigma Chemical) were added immediately prior to use. Media was was collected daily and analyzed for GAG release by the dimethylmethylene blue (DMMB) assay using purified shark chondroitin sulfate (Sigma Chemical) as the reference standard.
All samples were cultured in basal media for at least 3 days after harvest. To create samples that have heterogeneously-degraded ECM, samples were exposed to IL-1β (Cistron Biotechnology, Pine Brooks, NJ, USA), which was added each day to the culture medium. Two series of experiments were conducted, each with its own set of controls. In the first series, samples were exposed to 10 ng/ml IL-1 for 3 and 6 days (incurring 'mild' degradation; n=4). The addition of IL-1 to the media began at days 6 and 3, respectively, after harvest, such that all samples began the 'recovery phase' on day 9 after harvest. Control samples were cultured in basal media for the entire experiment. In the second series, samples were exposed to 20 ng/ml IL-1 (n=10) for 6 and 9 days and incurred 'moderate' degradation. In this group the addition of IL-1 addition to the media for all samples started at 3 days after harvest, and therefore samples began the recovery phase at day 9 or 12 after harvest. Again, control samples were cultured in basal media for the entire experiment. Control samples are referred to as 'mild controls' or 'moderate controls', according to the IL-1 series with which they were cultured (n=2 and 2).
Following IL-1 exposure, the samples were transferred to sterile flat-bottomed 10mm nuclear magnetic resonance (NMR) tubes (Wilbur Scientific, Boston, MA, USA) that were custom cut to a length of 5cm, where they were cultured in basal media for the duration of the recovery experiment.
In summary, the experiment comprised three periods: an initial period (before any exposure to IL-1), an exposure period (during which tissue was exposed to IL-1 in preparation for the recovery phase), followed by a recovery period (during which there was no IL-1 present).
Imaging protocols
For imaging, the shortened NMR/culture tubes were joined to full-length NMR tubes via a sterilized rubber stopper inserted into the open ends of both tubes.
All images were acquired on a Bruker 8.45 T magnetic resonance microimaging system (Bruker Instruments, Billerica, MA, USA) with a standard 10mm radiofrequency coil. T1-weighted images in the axial orientation with respect to explant cylindrical geometry were measured weekly post-IL-1 exposure with either an inversion recovery sequence ('mild' series) or a saturation recovery sequence ('moderate' series). The saturation recovery protocol consisted of 10 T1-weighted images measured with time-to-repeat times of 25, 75, 125, 175, 275, 375, 475, 600, 900, and 1800ms. For inversion recovery measurements, images were acquired with nine inversion delays of 16.7, 33.3, 50, 66.7, 100, 150, 250, 400, and 600ms. Both pulse sequences used a time-to-echo of 8.5ms, section thicknesses of 0.5mm, in-plane resolutions of 100μm, and two averages, for a total imaging time of less than 1 hour per sample.
Analysis of GAG release throughout these experiments suggested that, within the range of sensitivity provided by our dimethylmethylene blue assay, GAG release patterns were unaffected by removal from incubation at 37°C to room temperature for 3–6 hours of imaging each week (because all the plugs were out of the incubator for the imaging session).
Tissue T1s without contrast agent were determined spectroscopically using an inversion delay pulse sequence with 12 delays ranging from 0.2 to 10s and a 10mm broadband radiofrequency probe. At the conclusion of each experimental series, two samples (one control and one treated) were equilibrated in gadolinium-free medium, then extracted from media and blotted dry, and placed in an NMR tube. As had previously been observed, we found very little difference in T1 between samples of the same series (<5%) or across series (<10%) [12]. Therefore, the T1 times in the absence of contrast agent of samples from the same series were averaged and these averages used as the reference tissue T1 for all other samples within the same experiment series.
Image processing
MATLAB (The Math Works, Natick, MA, USA) was used to create a T1 map by curve-fitting each T1-weighted image series on a voxel-by-voxel basis. T1 maps were then processed into GAG maps with MATLAB using equations derived from a modified ideal Donnan theory. This dGEMRIC method of relating measured T1 and cartilage [GAG] was previously validated and reported [12, 13, 22].
The mean [GAG] for a sample at a given time point was computed as the mean [GAG] calculated across all cartilage-containing pixels of the image. The rate of [GAG] accumulation (i.e. the tissue's recovery rate) was calculated as the difference in the mean [GAG] values at specified time points divided by the elapsed time.
As expected, qualitative assessment of images from samples exposed to IL-1 exhibited the characteristic heterogeneity in degree of GAG depletion. In order to quantitate objectively the time course of GAG recovery relative to the degree of GAG depletion, GAG maps were registered using Adobe Photoshop (Adobe Systems, Inc, San Jose, CA, USA) in order to allow chosen regions of interest to be automatically analyzed across multiple images from successive imaging sessions. Registered images were segmented so that tissue regions of relatively high, medium, or low [GAG] were identified in images taken after 3 weeks of recovery. The [GAG] and location of these pixels were tracked in time. High, medium, and low [GAG] regions of the 3-week images were discerned according to the following definitions:
Pixel assigned to 'high' GAG region if:
[GAG]pixel > (mean [GAG]all pixels + 0.5 × SDall pixels)
Pixel assigned to 'low' GAG region if:
[GAG]pixel < (mean [GAG]all pixels - 0.5 × SDall pixels)
Pixel assigned to 'medium' GAG region if not assigned to high or low region
Statistical analysis
Magnetic resonance imaging derived group mean [GAG] changes were assessed using repeated-measures oneway analysis of variance with a compound symmetry variance structure using SAS (SAS Institute Inc., Cary, NC, USA) to test the hypothesis that mean [GAG] in a given sample or regions of a given sample did not change with time. This technique analyzed the significance of time as an effect on weekly [GAG] measurements (or weekly changes in [GAG]) taken from the same samples (or same region of a sample) each week. Paired two-sample Student's t-tests (Microsoft Excel) were used to determine the degree of [GAG] recovery observed with respect to initial [GAG], before exposure to IL-1.