Aggrecan

The following soluble factors at designated concentrations were used in the study: bone morphogenetic factor 6 (BMP6, R&D systems, 100 ng/ml), 1α,25-dihydroxyvitamin D3 (VD3, Sigma Aldrich, 100 nM), valproic acid (VPA, Sigma Aldrich, 2 mM), Wnt3a (R&D systems, 100 ng/ml), Wnt5a (R&D systems, 100 ng/ml), and Dickkopf1 (Dkk1, R&D systems, 100 ng/ml).
Among ECM components, we used aggrecan, biglycan, fibronectin, matrigel, collagen type I, hyaluronic acid (HA), and chondroitin sulphate A. Multiwell plates were coated with aggrecan (R&D systems) at 0.4 nmol/cm², biglycan (R&D systems) at 0.2 nmol/cm², fibronectin (R&D systems) at 0.4 nmol/cm², chondroitin sulphate A (CS) (Sigma Aldrich) at 0.05 mg/cm², or 0.05 mg/cm² HA (Abcam, MW 1.6 kDa). Type I collagen solution extracted from the rat tail tendons was incubated at 37°C for 30 min. matrigel (BD Biosciences) was diluted at a concentration 0.12 mg/cm² and incubated at 4°С overnight.

ADSCs and ADSCKs were tested for their ability to differentiate into adipocytes, chondrocytes and osteocytes using Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems), according to the manufacturer instructions. Adipogenic differentiation was performed starting from 2.1×10⁴ cells/cm² in SCM up to confluence, when SCM was replaced with Adipogenic Differentiation Medium (and changed every 3–4 days). After 21 days, cells were fixed with 4% paraformaldehyde for 1 hour and visualized by Oil Red O Staining (Sigma Aldrich).
Chondrogenic differentiation was performed with 2.5×10⁵ cells seeded in 15 ml conical tubes in Chondrogenic Differentiation Medium (replaced every 2–3 days); after 21 days, chondrocyte pellet was fixed with 4% paraformaldehyde for 20 min and immunostained for aggrecan (R&D System).
Osteogenic differentiation was done starting form 4.2×10³ cells/cm² in SCM up to 70–80% confluence, when SCM was replaced with Osteogenic Differentiation Medium (changed every 3–4 days). After 21 days, cells were fixed 10 min with 70% ethanol and processed for Alizarian Red Staining (Sigma Aldrich). Images were obtained at 20X magnification, using Nikon Eclipse TE300 equipped with the Axiovision device camera (Zeiss Instr., Oberkochen, Germany). Images were processed using Axiovision release 4.6.3 (Zeiss Instr., Oberkochen, Germany).

Nomilin (purity > 98%) was purchased from Shanghai aladdin Medical Technology Co., Ltd. Recombinant human IL‐1β, dimethylsulphoxide (DMSO) and type II collagenase were purchased from Sigma‐Aldrich. The primary antibody against collagen Ⅱ, Lamin B1, iNOS, COX‐2 and GAPDH was acquired from Abcam, goat anti‐rabbit and antimouse IgG‐HRP were from Bioworld and antibodies against Keap1, Nrf2, HO‐1, COX‐2, IκBα and p65 were purchased from Cell Signaling Technology; Alexa Fluor^®488 labelled and Alexa Fluor^®594 labelled Goat Anti‐Rabbit IgG (H + L) second antibody was purchased from Jackson ImmunoResearch. The 4′, 6‐diamidino‐2‐phenylindole (DAPI) was obtained from Beyotime. The cell culture reagents were purchased from Gibco. Foetal bovine serum (FBS), bovine serum albumin (BSA), Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium and 0.25% trypsin‐ethylenediaminetetraacetic acid (trypsin–EDTA) were purchased from Gibco (Life Technologies Corp.). TRIzol reagent was purchased from Invitrogen. Quanti Tect Reverse Transcription kit was purchased from Qiagen. SYBR Green Master Mix was purchased from Bio‐Rad Laboratories. ELISA kits of PGE2, TNF‐α, IL‐6, Collagen II, Aggrecan, MMP‐13 and ADAMTS‐5 were purchased from R&D systems. Griess reagent was purchased from Beyotime Institute of Biotechnology.