Clincal And Lab Researches On Cartilage Degeneration

ObjectiveTo evaluate the effect of arthroscopic surgery on knee osteoarthritis (OA) in clinical practice and investigate the michanism of integrin linked kinase(ILK) on the progress of periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis in molecular biology study.MethodsPart Ⅰ Clinical researchWe analyzed a total of 416 patientswith knee osteoarthritis, who underwent arthroscopy between January,2010 and December,2011 in our department. Among the 416 knees, mechanical symptons due to meniscus tear, free bodies, stenosis exsised in 118 knees. Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores and VAS scores before the surgery and at the follow-up of two years were recorded and analyzed for all patients with special attention on knees with mechanical symptoms.Part Ⅱ Lab researchUnder sterile conditions, chondrocytes were harvested from the limb joints cartilage derived from two-week-old SD rats and cultured in DMEM-F12 medium supplemented with 10% FBS in an incubator at 37℃ with 5% CO2. Then second-generation chondrocytes were plated at a concentration of 105 cells/well into six-well plates and allowed to adhere for one day before experiment.The cell morphology was examined under an inverted phase-contrast microscope following staining with collagen type Ⅱ with the conventional ABC method. Meanwhile,second-generation cells were seeded onto glass slides (25 mm x 25 mm) coated with type Ⅱ collagen at a density of 105 cells. Experiments were performed when cells reached 70-80% cell confluence.Firstly, cells were divided into non-pressure and pressure groups and were cultured under static conditions or periodic mechanical stress (0-200 kPa,0.1 Hz) for 0,0.5,1 or 2 h, prior to ILK kinase activity analysis. Secondly,cells were pretreated with shRNA targeted to ILK or control scrambled shRNA respectively and then maintained under static conditions or periodic mechanical stress for 1 h prior to ILK kinase activity analysis and western blot analysis,8 h prior to qPCR analysis (aggrecan and type Ⅱ collagen gene expression) or 3 days (8 h per day mechanical stress) prior to direct cell counting and CCK-8 assay.Finally, cells were pretreated with blocking antibodies against integrin β1 (10 μg/mL each) and ERK1/2 and ILK in chondrocytes under conditions of periodic mechanical stress were explored by ILK kinase activity analysis and western blot analysis.ResultsPart Ⅰ Clinical researchBy comparing the follow-up scores, we found that arthroscopy surgery became useless in three years. However, patients with mechanical symptons had significantly better outcomes than those without such symptoms.Part Ⅱ Lab researchOur results showed that periodic mechanical stress stimulation time-dependently enhanced ILK kinase activity in chondrocytes.It was found that after pretreatment with Lv-shRNA-ILK plasmid, chondrocytes proliferation as well as aggrecan and type Ⅱ collagen gene expression were markedly diminished as compared to the Lv-GFP group under periodic mechanical stress.We further examined the potential role of integrin β1 in periodic mechanical stress-induced ILK kinase activity. Upon treatment of chondrocytes with blocking antibody against integrin β1, ILK kinase activity as well as ERK1/2 kinase activity were strongly attenuated compared with those in the control group subjected to periodic mechanical stress.ConclusionIn the clinical study, we found that generally knee OA patients did not benefit from arthroscopic surgery in the long run, whereas knees with mechanical symptoms were a good indication.In the lab research, we delineated a critical role of ILK in periodic mechanical stress-stimulated mitogenic effects in chondrocytes through integrin β1 initiated ERK1/2 phosphorylation.