Patients and preparation of samples This research has been verified by the Ethics Committee of Xiangya Hospital, Central South University, and all the patients offered informed consent. This study contained 8 normal healthy individuals(6 male,2 female, age: 17–55 years) and 40 patients(12 male at the age of 62-81years, 28 female at the age of 55–76 tears) with major knee OA according to the criteria of the American College of Rheumatology. The samples of osteoarthritic cartilage were gathered from the 40 patients with main OA with knee arthroplasty. The normal samples could be gained from the knees of 8 age matched postmortem donors, without any history of joint pain. Informed consent of ethics could be gained from all the families and donors. After eosin and hematoxylin (HE) staining, a light microscopy was used to evaluate the histological changes of the sections,osteoarthritic changes were classified histomorphologically, using the Mankin score: normal (Mankin score: 0–1), mild lesions (Mankin score: 2–5), moderate lesions (Mankin score: 6–9) and severe lesions (Mankin score: 10–14).
Immunohistochemistry. Cartilage tissues were snap-frozen in liquid nitrogen at -70°C before analysis. In analysis, cartilage were cut to 10 µm thick slice. The slices put into the oven at 60℃for 3 ~ 6 h,then the slices soak in xylene for 10 min twice. And soak in 100%, 95%, 85% and 75% ethanol in sequence,subsquently, add urea (n ≥ 8mol/L) incubate at 37°C for 25min and add pancreatin dropwise for antigen retrieval,after this, 3% H2O2 was added for about 10 min to inactivate endogenous enzymes.Next, the slices were then incubated with diluted αvβ3 antibody (1:50, BA0321, 18309-1-AP, Proteintech), OPN(1:100, Ab8448,Abcam,), SAβ-gal(1:50, 15518-1-AP, Proteintech) for 1 h at 37 C,respectively., overnight at 4°C, and a second antibody reaction was carried out for 20 min using biotinylated anti-rabbit IgG antibody (Proteintech),after wash, horseradish peroxidase/Fab polymer conjugate (PicTureTM-Plus kit, Zymed, South San Francisco, CA) was applied to the slices for 30 min. A negative control was prepared simultaneously by neglecting the major antibody. Lastly, the staining was developed by incubation with DAB Chromogen for 20 min. To evaluate the expression of αvβ3,OPN and SAβ-gal, the sections were examined under a microscope at 40x magnifification. The relative αvβ3,OPN and SAβ-gal distribution of cartilage tissue can be visualized and quantifified as the Average optical density with Image-pro Plus 6.0. All densities were normalized to PBS. The final data, which were applied in all analysis, consisted therefore of a mean of three independent measurements representing the average levels of αvβ3,OPN and SAβ-gal in articular cartilage. The coeffificient of variation (CV) of αvβ3,OPN and SAβ-gal expression in articular cartilage was 2 %.
Western blot. The histones were extracted from cartilage tissue in liquid nitrogen immediately after the cartilage tissue was washed and ground into small sections. Chondrocytes were lysed using 100ul/well SDS with protease inhibitor (100:1), and the protein concentration of the lysate was measured using the BCA protein assay kit (Thermo Fisher Scientific, Boston, MA, USA). 40ug protein was used in the next experiment. Proteins were separated by 12% SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) (genescript, piscataway, USA) at 80v, 30min and then 120v, 60min and transferred to polyvinylidenedifluoride (PVDF) membranes with 300mA ,75min. The membranes were blocked in nonfat dry milk solution 1h, and incubated overnight at 4°C with primary antibody αvβ3 antibody (1: 500,Sc-7985-R,SantaCruz), OPN(1:500, SC-21742, SantaCruz,), SAβ-gal(1:1000, 15518-1-AP, Proteintech) dilution buffer and then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2000) for 1 h at room temperature. β-actin was used as housekeeping protein. After that, the membranes were developed using the enhanced chemiluminescence (ECL) (NCM biotech,China) and exposed and qualified by Bio-Rad chemiDoc-XRS with image lab software (BioRad, Richmond, CA, USA).
Statistical analysis SPSS software for Windows (version 22; IBM SPSS, Armonk, NY, USA) was utilized for data analysis and management. One-way analysis of variance (ANOVA) was applied to study the difference between multiple groups in the mean values between multiple groups. Then Spearman’s correlation and linear regression analysis were used to analyze the correlation between the average optical density of αvβ3, OPN and SAβ-gal in the articular cartilage with the degree of degeneration. The pearson correlation analysis was used to analyze the correlation among the average optical density of αvβ3, OPN and SAβ-gal. P < 0.05 indicated statistical significance.