Twenty-week-old female New Zealand white rabbits were obtained from Nita Bell Laboratories (Hayward, California, USA). Ketamine hydrochloride was from Parke Davis (Morris Plains, New Jersey, USA), and xylazine was from Rugby Lab (Rockville Center, New York, USA). Lactalbumin hydrolysate, α-casein, β-estradiol-17-valerate, pepsin, papain, chondroitin sulfate A sodium from bovine trachea, Safranin-O, Fast Green, cetylpyridinium chloride, and other reagents were from Sigma (St Louis, Missouri, USA). 1,9-Dimethylmethylene blue (DMMB) was from Molecular Probes (Eugene, Oregon, USA), and 35S was from Amersham (Arlington Heights, IL, USA). Protein assay kits, gelatin (EIA grade), and nitrocellulose membrane were from Bio-Rad (Hercules, California). α-Minimal essential medium, trypsin, penicillin–streptomycin, and Fungizone® were from Gibco (Grand Island, New York, USA). All other standard chemicals were from Sigma or Fisher Scientific (Pittsburg, Pennsylvania, USA).
Rabbit anti-human collagenase-1 polyclonal antibody and rabbit anti-mouse stromelysin-1 monoclonal antibody, horseradish peroxidase-conjugated secondary antibodies, and the MMP inhibitor GM6001 and its control analog were from Chemicon International (Temecula, California, USA). Rabbit anti-human-TIMP-1 antibody that cross-reacts with the rabbit inhibitor  was from Triple Point Biologics (Forest Grove, Oregon, USA). Enhanced chemiluminescence reagent for western blotting was from Amersham International (Little Chalfont, Bucks., UK). Sircol collagen assay kit was from Accurate Chemical and Scientific Corporation (Westbury, New York, USA), and fluorescein isothiocyanate (FITC)-labelled collagen was from Chondrex (Seattle, Washington, USA). Recombinant human relaxin was kindly provided by Connetics Corporation (Palo Alto, California, USA).
Retrieval and culturing of TMJ discs, pubic symphysis, and articular cartilage
All procedures on rabbits were approved by the Committee on Animal Research of the University of California, San Francisco, and conducted in accord with accepted standards of humane animal care. Rabbits were anesthetized with ketamine hydrochloride (40 mg/kg) and xylazine (3–5 mg/kg), and the TMJ discs were harvested bilaterally under sterile conditions and immediately placed in calcium-free and magnesium-free phosphate-buffered saline (PBS) containing antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, and 100 U/ml Fungizone). After removal of the synovium under a dissecting microscope, each disc was washed three times in PBS and bisected longitudinally such that four samples from each rabbit were available (three for hormone treatments and one for control). The hemisections were weighed, placed in wells of a 24-well culture plate, covered with 1 ml of serum-free medium (phenol-free α-minimal essential medium with 0.2% lactalbumin hydrolysate, glutamine, nonessential amino acids, 100 U/ml penicillin, and 100 mg/ml streptomycin) with or without hormones, and cultured at 37°C in air containing 5% CO2.
For determination of MMPs and GAG staining, 32 hemisections from eight rabbits were exposed to medium alone, β-estradiol (20 ng/ml), relaxin (0.1 ng/ml), or both hormones at the same doses for 48 hours. The conditioned medium was collected and stored for MMP assays, and the discs were processed for GAG staining. To assess the contribution of relaxin-induced MMPs to the loss of collagen and GAGs, 24 hemisections from six rabbits were cultured with the MMP inhibitor GM6001 or its control analog 2 hours before and during the hormone treatments. The inhibitor was used at 10 μM, because this concentration was shown to inhibit collagenase activity induced by 0.1 ng/ml relaxin in dose–response experiments to baseline levels. The conditioned medium was collected and stored at -70°C for total protein and MMP assays. The discs were dried in a SpeedVac®, weighed, digested, and used for the determination of GAG and collagen content.
To determine whether the observed induction of collagenase by relaxin is specific to fibrocartilage, experiments were performed with pubic symphysis fibrocartilage, which is a known target site for β-estradiol and relaxin as a positive control, and with articular cartilage from the knee. For retrieval of articular cartilage, the joint was shaved, the articular surfaces were exposed, and the cartilage was scraped from the articular surfaces of the femur and tibia and incubated in PBS with antibiotic as described above. Similarly, the pubic bones and symphyseal areas were exposed under sterile conditions and the pubic symphysis (fibrocartilaginous tissues between the pubic bones) was dissected, removed, and incubated in PBS with antibiotics. The tissues were weighed, placed in wells of a 24-well culture plate, and studied as described above.
Hormone-induced changes in collagenase-1, stromelysin-1, and TIMP-1 were determined by western blotting. Disc-conditioned medium was mixed with 4 × sample buffer and subjected to SDS–polyacrylamide-gel electrophoresis with 10% or 18% gels. Equal amounts of protein (determined with a bicinchoninic acid protein assay kit) were loaded in each lane. The proteins were transferred to nitrocellulose membranes, which were blocked, washed, and incubated for 1 hour with antibodies against TIMP-1 (1:250 dilution), collagenase-1 (1:250 dilution in Tris-buffered saline), or stromelysin-1 (1:500 dilution). The membranes were then washed, incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:1000 dilution), and washed again. Bands were revealed by incubation with enhanced chemiluminescence reagent and exposure to radiographic film. The bands for TIMP-1 western blots were quantified by videodensitometry as described . Conditioned medium from pubic symphysis and articular cartilage explants was similarly subjected to western blot analysis for collagenase-1 and stromelysin-1.
Enzyme activities were quantified by substrate zymography of conditioned media from 32 hemisections (mean wet weight 13 ± 9 mg). The samples were standardized by total protein and subjected to SDS–polyacrylamide-gel electrophoresis with 10% gels containing 2 mg/ml gelatin or casein at 15°C as described . The gels were washed in 2.5% Triton X-100 for 30 min with one change of wash buffer, incubated at 37°C for 60–72 hours in incubation buffer (50 mM Tris-HCl buffer pH 8, 5 mM CaCl2, 0.02% NaN3), stained with 5% Coomassie blue, and destained in 10% acetic acid and 40% methanol until proteinase bands were clearly visible. Images of the gels were captured with a charge-coupled device camera and NIH image software. The levels of 53/58 kDa gelatinolytic and 51/54 kDa caseinolytic enzymes and their low-molecular-mass activated forms were quantified by videodensitometry . The substrate zymograms rather than western blots were used to quantify hormone-mediated increases in proteinase levels because zymograms are more sensitive, often display both pro-forms and active forms of proteinases, show a greater linear range of densitometric values and have good reproducibility that together enable a reliable quantification of the enzymes from these gels [23–25]. In addition, gelatin zymograms selectively detect proteinase activity at 53/58 kDa and at 43 kDa attributable primarily to collagenase rather than stromelysin because gelatin is a poor substrate for stromelysin [25, 26].
Histochemical staining and quantification of GAGs
To assess changes in GAG levels, the discs were washed three times in PBS, frozen in OCT compound, and sectioned with a cryostat. The section were defrosted for 30 min, fixed for 10 min in methanol, air-dried for 15 min, stained with 1% Fast Green solution for 3 min, placed in 1% acetic acid for 1 min, stained with 2% Safranin-O for 2 min, dehydrated through successive ethanol and xylene washes, and mounted with coverslips. Ten sections of each hemisection were analyzed by an examiner blinded to the hormone treatment. The stained discs were videodigitized and analyzed with a software program that automatically outlined the total and Safranin-O-stained areas with threshold settings (Photoshop 4.0; Adobe, San Jose, California, USA). These areas were then quantified with NIH Image 1.62, and the percentage of disc staining positive for GAGs was calculated from the ratio of the stained area to the total area in each section. The average of the 10 values for each half disc was used for analysis.
Determination of GAG synthesis by 35S radiolabeling
To quantify GAG biosynthesis, 32 disc hemisections (mean weight 14 ± 4 mg) were incubated at 37°C for 6 hours in 1 ml of phenol-free and serum-free medium with or without hormones and 165 kBq (0.0044 mCi) of 35S as described . The discs were washed three times with medium containing 1 mg/ml sodium sulfate and digested for 24 hours with 20 U/ml papain. The digest (500 μl) was incubated for 30 min with 100 μl of 5% cetyl pyridiuium chloride in 0.3 M potassium chloride at room temperature (20–22°C) to precipitate GAGs. After centrifugation (3000 g for 20 min), the supernatant was removed and the precipitate was dissolved in 600 μl of concentrated formic acid by heating to 70°C for 10 min. Aliquots (20 μl) of this solution were added to 3 ml of scintillation fluid and subjected to liquid scintillation counting. The radioactivity (counts/min) was standardized to the total dry disc weight.
Quantification of GAGs and collagen
Each disc hemisection was digested in 600 μl of 3 mg/ml pepsin in 0.05 M acetic acid and incubated at 37°C for 18–20 hours in a dry bath. DMMB binding assays for GAGs, and Sircol assays for collagen content, were performed in triplicate on 24 disc hemisections. The DMMB reagent was prepared as described . Pepsin digests (200 μl) from each treatment group (GM6001 or analog control) were mixed with 1 ml of DMMB reagent, and absorbance at 525 nm was determined with a spectrophotometer. The GAG concentration (μg/ml) was determined by comparing the absorbance of the sample against a standard curve prepared from bovine chondroitin sulfate A, and the disc GAG content was standardized to the total dry tissue weight.
For the collagen assay, 200 μl of pepsin digest was mixed with 1 ml of Sircol dye reagent, incubated for 30 min at room temperature, and centrifuged at 10,000 g to separate the unbound dye from the collagen-bound dye. After removal of the unbound dye, 1 ml of the alkali reagent was added to the collagen–dye complex and vortex-mixed to dissolve the collagen-bound dye completely. Aliquots (200 μl) were transferred to the 96-well plates, and absorbance at 550 nm was determined with a microtiter plate reader (Molecular Devices, Sunnyvale, California, USA). The collagen concentration (μg/ml) was determined against a collagen standard curve, and the disc collagen content was standardized to the total disc dry weight.
Quantification of collagenase activity
Collagenase activity in conditioned medium from discs cultured with GM6001 or control analog was assessed by FITC–collagen assay. A 96-well plate was coated with FITC–collagen (10 μg per well) overnight at 4°C and washed twice with PBS. Disc-conditioned medium (100 μl) was added to the wells, and the plate was incubated at 35°C for 1 hour. As a reference, 100 μl of blank medium containing 3000 ng of bacterial collagenase was added to one set of wells for complete digestion of FITC–collagen. After incubation, 90 μl from each well was transferred to another 96-well plate, and the fluorescence intensity of degraded FITC–collagen products was determined with a microplate spectrofluorometer (Spectramax Gemini XS; Molecular Devices) with excitation at 494 nm and emission at 518 nm. The data were converted to relative fluorescence units of collagenase activity as described by the manufacturer and standardized to the dry weight of each half disc. The fold differences in collagenase activity in medium from control and hormone-treated discs were determined for each experiment. All assays were performed in duplicate.
Because of inherent variability in matrix content and proteinase activity in discs from different rabbits, three disc hemisections from each rabbit were treated with hormones and one served as control. MMP levels and the GAG and collagen content in each hormone-treated disc hemisection were standardized to the values of the control hemisection within each animal and the fold changes were plotted as histograms. The statistical significance of differences was determined by single-factorial analysis of variance (ANOVA). Intergroup differences were analyzed by Fisher's multiple comparisons test; P < 0.05 was considered statistically significant. Values are expressed as means ± SD.