Effects of AdipoRon on knee fibroblasts
Patient derived fibroblasts were treated with TGFß1 and TGFB plus AdipoRon to assess for potential anti myofiborogenic effects of AdipoRon. Overall design: Tissues were collected following patients' consent via an approved Institutional Review Board (IRB) protocol. Knee tissues from the suprapatellar pouch (SP) were collected from patients undergoing primary total knee arthroplasty while fibrotic tissue was collected from patients undergoing revision total knee arthroplasty for arthrofibrosis. Tissues were excised by the operative surgeons, and the samples were immediately delivered to the laboratory. Knee tissue samples were then cut into smaller pieces and subsequently digested with 2 mg/ml type I collagenase (Worthington Biochemical Corporation, Lakewood, NJ, USA) in advanced minimum essential medium (Advanced MEM, Thermo Fisher Scientific, Waltham, MA, USA) for 3 hours at 37°C in an orbital shaker (~120 rotations per minute). The digestion solution was filtered through 70-µm cell strainers, the cell suspension was centrifuged at 1200 rpm for 7 minutes at 4°C, and the resulting cells were cultured in Advanced MEM supplemented with 5% human PLTMax (Mill Creek Life Sciences, Rochester, MN, USA), a clinical grade commercial platelet lysate product, 2?U/mL heparin (hospital pharmacy), GlutaMAXTM (Thermo Fisher Scientific), and antibiotic/antimycotic (Thermo Fisher Scientific) at 37°C, 95% humidity, 21% O2, and 5% CO2. At approximately 100% cell confluency, passage 0 cells were detached using TrypLE Express (Thermo Fisher Scientific) and banked using CryoStorR (Sigma-Aldrich, St. Louis, MO, USA). For experimental purposes, cells were retrieved from frozen stocks, expanded in growth medium (same as above), and passage 3-5 cells were used for all experiments. In this regard, at Day 0, cells were treated with 5 µM AdipoRon or its vehicle (DMSO) in the presence of 10 ng/ml TGFß1 or its vehicle in growth medium. Additional 5 µM final concentration of AdipoRon and equal volume of its vehicle were added on Day 1 and Day 2 without media change. On Day 3, cells were processed and RNA was extracted for downstream analyses.