Establishment of cartilage surface injury ex vivo model
Bovine osteochondral explants (2.0–2.5 cm W X 2.0–2.5 cm L X 0.5–1.0 cm H) were sawed under sterile conditions from medial or lateral tibial plateau of stifle joints of 18-month-old cows obtained from a local abattoir (Bud's Custom Meats, Inc., Riverside, IA, USA). Explants were pre-equilibrated for 48 hrs in serum free DMEM/F12 supplemented with 50 U/ml Penicillin, 50 mg/l Streptomycin, and 2.5mg/l Amphotericin B inside a humidified 37 ̊C incubator supplied with 5% CO2 and 5% O2.
To create an impact injury, cartilage surface was subjected to a single blunt impaction with a customized drop-tower reported in our previous studies [11, 28]. Briefly, an osteochondral explant was rigidly fixed in a chamber with a 5.0 mm diameter brass rod resting on the surface of cartilage. A 2-kg mass was dropped from 14 cm height onto the brass rod resulting in impact energy density of 14 J/cm2. This high energy impact created cracks and instant cell death in cartilage surface. To create a scratch injury, a sterile 26 G1/2 needle was dragged over cartilage surface. An X-shaped matrix tear in the superficial zone was generated.
Harvest and monolayer culture of CPCs, non-CPCs and normal articular chondrocytes
As previously reported [11, 29], an impacted or scratched osteochondral explant was firstly incubated with 0.25% trypsin-EDTA for 10 min with cartilage side facing down. CPCs were then collected, counted, and cultured in DMEM/F12 supplemented with 10% FBS, 50 U/ml Penicillin, 50 mg/l Streptomycin, and 2.5mg/l Amphotericin B inside a 37 ̊C incubator supplied with 5% CO2, 5% O2 and 100% humidity.
To harvest non-CPCs, full thickness cartilage slices were shaved from osteochondral explants immediately after CPCs were harvested, and were weighed, minced, and subjected to 0.4% protease (Sigma-Aldrich®, St. Louis, MO) for 90 mins and then to 0.02% collagenase (Sigma-Aldrich®, St. Louis, MO) for 16 hrs to release chondrocytes. Same method was used in isolation of normal articular chondrocytes (ACs) from uninjured cartilage. Passage 1 CPCs, or non-CPCs, or ACs were seeded at a high density (3.0 X 105 cells/cm2). The experimental design was summarized and illustrated in Figure 1.
Light microscopy and calculation of cell population doubling time (PDT)
The growth status of CPCs, or non-CPCs, or ACs was recorded with an Olympus CKX53 light microscope (Olympus Corporation of the Americas, PA, USA) equipped with a camera (Olympus Soft Imaging Solutions GMBH, Olympus LC30, Munster, Germany). PDT of CPCs, or non-CPCs, or ACs was calculated from total cell counts obtained at the seeding time of passage 0 and 1 cells, respectively.
Treating CPCs or non-CPCs with DAMPs or IL-1b
At Day 4 post-seeding, passage 1 CPCs or non-CPCs were switched to serum free media in order to synchronize cell cycle. After 24-hr serum deprivation, cultures were replenished with equal volume of fresh serum free media immediately prior to treatments with DAMPs or IL-1b. Cultures were divided into four treatment groups (each group in triplicates): 1) non-treated control; 2) 10 nM rhHMGB1/HMG-1 (R&D Systems, Minneapolis, MN); 3) MTDs: 10 nM N-formyl-Met-Leu-Phe (fMLF) (Tocris Bioscience, Bristol, UK) + 10 µg/mL CpG DNA (a 22-mer oligonucleotides containing CpG motifs) (InvivoGen, San Diego, CA); 4) 10 ng/mL rhIL-1b (R&D Systems, Minneapolis, MN). After 24-hr stimulation, conditioned media were collected for determination of MMP expression with Western blotting and cells were lysed for total RNA extraction and real-time PCR analysis.
Determination of MMP expression with Western blotting
Conditioned media were dialyzed against MilliQ water and then concentrated to dry powder with a speed vacuum. Equal volume of prepared reduced medium samples was resolved by 10% SDS-PAGE. Proteins were then blotted onto a nitrocellulose membrane. After been blocked with 3% BSA/TBS, blots were incubated with 1% BSA/TBST containing 1:3,000 diluted anti-MMP-3 antibody (Biomol International, Kelayres, PA) or 1:1,000 diluted anti-MMP-13 antibody (Abcam®, Cambridge, MA) overnight at 4 °C. After being washed three times, those blots were then incubated with 1% BSA/TBST containing 1:7,600 diluted goat anti-rabbit IgG antibody conjugated with HRP (Sigma-Aldrich®, St. Louis, MO). Chemiluminescent signals were generated with SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL) and captured with Kodak BioMax MR film (Sigma-Aldrich®, St. Louis, MO) or Blue Classic Autoradiography film (RPI, Mount Prospect, IL).
Expression of MMPs, chemokines, and cytokines determined with real-time PCR
CPCs or non-CPCs were lysed with TRIZOL® (Invitrogen, Waltham, MA) and total RNAs were extracted with Qiagen RNEasy Kits (Qiagen Inc, Valencia, CA). A 50 ng of each RNA sample was firstly reverse transcribed into cDNA with TaqMan™ Reverse Transcription Reagents (Applied Biosystem, Grand Island, NY). Quantitative PCR reactions were performed with SYBR™ Green PCR Master Mix (Applied Biosystem, Grand Island, NY). Primers for bovine β-actin, MMP-3, -13, CCL2, CXCL12, IL-6 & 8, were provided by 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 to β-actin was calculated using a comparative Ct method (2−ΔΔCt). Results are reported as mean ± standard deviation (SD). Relative expression of each target gene in CPCs from impact or scratch injury was compared to that in non-CPCs with Welch's t-test. P (T<=t) two-tail value was reported. P < 0.05 was considered statistically significant. The 95% confidence interval (CI) was also calculated by using this formula: CI = Mean ± t X (SD/√sample size). The sample size was determined by n = (z2 × σ2)/MOE2.