All common chemicals and reagents were purchased from RPI Corp. (Mount Prospect, IL) or Sigma Chemical Co. (St. Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM), F-12, Fetal Bovine Serum (FBS), and Hanks’ Balanced Salt Solution (HBSS) were from Gibco/Invitrogen (Carlsbad, CA). Methanol-free 16% paraformaldehyde solution was purchased from Thermo Fisher Scientific (Rockford, IL). Goat serum was from Sigma-Aldrich® (St. Louis, MO). Alexa Fluor® 568 F(ab′)2 fragment of goat anti-rabbit IgG (H + L) was obtained from Invitrogen™ (Carlsbad, CA). VECTASHIELD mounting medium with DAPI was from Vector Laboratories, Inc. (Burlingame, CA). Protease inhibitor cocktail set III reagent was purchased from Calbiochem/EMD (Gibbstown, NJ). Bicinchoninic Acid (BCA) Protein Assay Reagent was from Thermo Scientific/ Pierce Biotechnology (Rockford, IL). Total and phosphor-specific p38 (Thr180/Tyr182), JNK1/2 (Thr183/Tyr185), ERK1/2 (Thr202/Tyr204) antibodies and HRP-conjugated goat anti-rabbit IgG were from Cell Signaling Technology (Danvers, MA). The chemical inhibitors of p38 MAP kinase, SB203580 and SB202190, and inhibitors of MEK1/2/ERK1/2, and U0126 were purchased from CalBiochem® (San Diego, CA). Dimethyl Sulfoxide (DMSO) (molecular biology grade, ≥99.9%), 1, 9-Dimethyl-Methylene Blue (DMMB), and papain were obtained from Sigma-Aldrich (St. Louis, MO). Calcein AM and ethidium homodimer-2 (EthD-2) were from Invitrogen™ (Carlsbad, CA). Pure Nitrocellulose Membranes (0.45 μM) were purchased from Bio-Rad Laboratories (Hercules, CA). SuperSignal West Dura Extended Duration Substrate was from Thermo Scientific/ Pierce Biotechnology (Rockford, IL). BioMax XAR Film was purchased from Kodak Film (Rochester, NY). Biopsy punches were from Miltex (York, PA).
Stifle joints were taken from 18–20 month old cows from a local abattoir. 2.5 cm × 2.5 cm (W × L) osteochondral explants with 0.5–1.0 cm-thick subchondral bone were manually sawed from the central loaded area of the lateral tibial plateau. The explants were then washed once with 1 X HBSS containing antibiotics, placed in culture media [DMEM/F-12 (50%/50%)/10% FBS, with antibiotics] and incubated overnight at 37 °C (5% CO2, and 5% O2).
A drop tower was employed to impart loads to an indenter resting on the cartilage surface of the explants. The indenter was a flat-faced 5.0 mm diameter brass rod with rounded edges (r=1 mm). A 2 kg mass was dropped onto the rod from a height of 14 cm resulting in impact energy density of 14 J/cm2. Impacted explants were placed in fresh culture media and incubated at 37 °C, 5% CO2, and 5% O2 for various times. Impact sites were visible to the naked eye immediately after insult (). Safranin-O histology revealed local delamination and superficial/transitional zone fissuring typical of high energy blunt impact injuries ().
Figure 1 Osteochondral Explant Blunt Injury Model. (A) The image shows the gross appearance of a 14J/cm2 blunt impact injury on the surface of a typical osteochondral explant. The center of the 2.5 cm × 2.5 cm explant was struck once with a 5-mm diameter (more ...)
In order to visualize the distribution of phosphorylated kinases, cartilage from explants at 24 hr post-impaction or from non-impacted explants at the corresponding time point was embedded in Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC) and full-thickness saggital sections were cut at 5 μm to 10 μm intervals. The sections were fixed with 4% paraformaldehyde for 15 min at room temperature (RT), washed three times with 1 X PBS, and blocked for 1 hr at RT with 5% goat serum diluted in 1 X PBS containing 0.3% Triton X-100. After blocking, sections were incubated overnight at 4 °C with anti-phospho p38 antibody or anti-phospho ERK1/2 antibody diluted 1:100 in 1% BSA/PBS containing 0.3% Triton X-100. After three washes, sections were incubated with Alexa 568 conjugated goat anti-rabbit IgG diluted 1:400 in 1% BSA/PBS containing 0.3% Triton X-100 for 1–2 hrs at RT. After final washes with 1 X PBS, sections were mounted in VECTASHIELD/DAPI mounting medium. Alexa 568 fluorescence representing phosphorylated MAP kinases and DAPI fluorescence representing cell nuclei were imaged with a Zeiss 710 confocal microscope.
Cartilage for phosphoprotein analysis was harvested at 20 min, 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, and 48 hr post-impact. Non-impacted control cartilage was harvested at the same time points. 6 and 8 mm diameter biopsy punches and an osteotome were used to separate the impacted and annulus cartilage from the bone (). Impacted and annulus cartilage, both cut in half with scalpels, were immediately submerged into cold lysis buffer (150 mM NaCl, 10 mM Tris, pH 7.0, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EGTA, 50 mM NaF, 1 mM Na3VO4, 1 mM glycerol phosphate, 2.5 mM sodium pyrophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM EDTA). Protease inhibitor cocktail set III was diluted in lysis buffer (1:100). The tissue was incubated in lysis buffer with gentle shaking at 4 °C overnight.
After incubation with lysis buffer, supernatants were removed and total protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent. Cartilage tissue lysates were denatured with 2 X sample buffer and reduced with 0.05 M DTT prior to electrophoresis. Lysate volumes containing 5 μg protein were fractionated on 10% acrylamide SDS gels and electrolotted to nitrocellulose membranes. The blots were blocked with 5% nonfat dry milk in 20 mM Tris buffer, pH 7.4, containing 140 mM NaCl (TBS) and 0.1% Tween 20 (TBST) for 1 hr at room temperature and incubated overnight at 4 °C with the anti-total or anti-phospho-specific kinase antibodies diluted 1:1,000 in 5% BSA in TBST (BSA/TBST). This was followed by washing and reaction for 1 hr with HRP conjugated goat anti-rabbit IgG diluted 1:2,000 in 5% BSA/TBST. After washing, the blots were reacted with SuperSignal West Dura substrate for 5 min. Blots were then exposed to KODAK BioMax XAR film. Image J software was employed to measure the integrated density (ID) of each band on a blot. The relative fold increase of ID over controls was calculated as: IDpMAPK (impaction) X ID tMAPK (control) / [IDpMAPK (control) X ID tMAPK (impaction)]. The means and 95% confidence intervals of the relative fold increase were calculated and plotted from at least three individual experiments.
A p38 inhibitor (SB202190) and an ERK inhibitor (U0126) were tested to determine if blocking the p38 pathway inhibits impact-induced ERK activation. Prior to impaction, osteo-chondral explants were pre-treated with either 10 μM SB202190 or 10 μM U0126 for 90 min. After 24 hrs of post-impaction, cartilage discs were dissected from the impact zone and area surrounding the zone with 6-mm and 8-mm biopsy punches. Cartilage discs from non-impacted explants or from impacted explants without inhibitor pre-treatment were also collected at the same time point. Blots of cartilage lysates were probed with anti-total or –phospho ERK1/2 antibody as described above.
Two p38 inhibitors (SB203580, SB202190) and an ERK inhibitor (U0126) were tested for effects on impact-induced changes in viability and proteoglycan content. All three inhibitors were used at a concentration of 10 μM. Controls were incubated with equivalent volumes of solvent (DMSO), which was 0.1% in culture media. Explants were incubated in MAPK inhibitors starting 90 min prior to impaction and were returned to culture media containing the inhibitors immediately after impaction. The media were changed and the inhibitors were reapplied every other day. On day 7 post-impact chondrocyte viability in the impact and annulus sites was evaluated by confocal microscopy. Briefly, Osteochondral explants were incubated with 1.0 μg/ml calcein AM (live cell staining) and 1 μM ethidium homodimer (dead cell staining) for 45 minutes, and then washed with 1 X HBSS once. Z-section images at 20 μm intervals were taken from three different sites in impacted zone and three in the area surrounding the impacted zone, respectively. Live and dead cells were counted with Image J. The ratio of live cell count to total cell count (chondrocyte viability) was calculated and averaged among three individual experiments. Variances are reported in terms of 95% confidence intervals. Effects were analyzed using one-way ANOVA and the Dunnett test.
The effects of p38 and ERK inhibitors on impact induced PG loss were examined with DMMB assays. On day 7 post-impact cartilage samples from impact sites were dissected with a 4-mm biopsy punch and osteotome. Five or six cartilage disks in the area immediately next to the impact site were also recovered with 4-mm biopsy punches. The mass of the harvested cartilage disks was measured to obtain wet weight and the cartilage was incubated in papain digest buffer containing 0.5 mg/ml papain, 5 mM L-cysteine, 5 mM EDTA, 0.1 M Na2PO4 (pH 7.5) for 4 hrs at 65 °C. The total sulfated glycosaminoglycan (PG) concentration in the papain digest was measured using DMMB reagent with shark chondroitin sulfate as a standard. The amount of PG was normalized to the wet weight of each cartilage disk and was averaged among three individual experiments. Uncertainty estimates are reported as 95% confidence intervals. One-way ANOVA and Holmes-Sidak test was employed for statistical analysis.
MMP-13, TNF-α, and ADAMTS-5 expression was examined at the mRNA level with quantitative real time PCR (qRT-PCR). Dermal punches were used to dissect impacted, annulus, and control cartilage from 4 different explants. The cartilage discs were flash frozen in liquid nitrogen and cryosectioned. Total RNA was extracted using TRIZOL® (Invitrogen) and was purified using Qiagen RNEasy Kits (Valencia, CA). The concentration was measured with a Nanodrop spectrometer. Sample volumes containing 50 ng total RNA were assayed by qRT-PCR using Superscript III Platinum SYBR Green One-Step qRT-PCR kits (Invitrogen). Primers for bovine β-actin, MMP-13, TNF-α, and ADAMTS-5 were obtained from Integrated DNA Technologies (Coralville, IA) (sequences available upon request). Data were obtained using an ABI Prism 7700 Sequence detection System (Applied Biosystems, Foster City, CA). Relative gene expression (Impact or Annulus/Control) was calculated using a comparative CT method. Results are reported as fold differences (2−ΔΔCT). Treatment effects (impact versus annulus versus control) were evaluated using one-way ANOVA and the Holmes-Sidak test.