Bullet kit

Normal human knee chondrocytes (NHAC-kn; Cambrex, Charles City, IA, USA) were cultured in a humidified atmosphere of 5 % CO₂ and 95 % air at 37 °C in a BulletKit (Cambrex). NHAC-kn were derived from a single-donor knee articular cartilage and used as an established normal chondrocyte cell line [27 (link), 28 (link)]. To characterize the chondrocytes, we confirmed that NHAC-kn expressed type II collagen and sulfated proteoglycans, but not type X collagen, before use.
Before we performed the experiments, cells were grown to a subconfluent state (3 × 10⁵ cells/well) in a 6-well plate and were then plated onto a silicon chamber coated with fibronectin (Sigma-Aldrich) at a density of 3 × 10⁵ cells/well in DMEM/F-12 supplemented with 10 % FBS and 100 U/ml penicillin-streptomycin. Cyclic tensile strain experiments were performed using an ST-140 cyclic tensile strain system (STREX, Osaka, Japan). Cyclic tensile strain was enforced at 3 %, 5 %, 8 %, and 10 % elongation for 3 h (0.5 Hz) according to previous studies [23 (link), 29 (link), 30 (link)]. We used 5 % tensile strain as normal joint loading and 10 % tensile strain as excessive loading to investigate the relationship between mechanical stress and cartilage metabolism.

BEAS-2B cells—which were isolated from normal human bronchial epithelium obtained from autopsy of non-cancerous individuals and immortalized and are widely used to study the functions of bronchial epithelial cells—were purchased from the American Type Culture Collection (Manassas, VA. USA). Normal human bronchial epithelial (NHBE) cells were purchased from Takara Bio Inc. (Tokyo, Japan). The cells were seeded in culture dishes coated with collagen type 1 (Iwaki, Tokyo, Japan) and cultured at 37°C in a humidified 5% CO₂ atmosphere in serum-free epithelial growth medium (BEGM; Cambrex, Walkersville, MD, USA) supplemented with Bullet Kit (Cambrex) to contain 0.5 ng/mL human recombinant epidermal growth factor, 0.5 μg/mL hydrocortisone, 10 μg/mL transferrin, 0.5 μg/mL epinephrine, 5 μg/mL insulin, 50 μg/mL bovine pituitary extract, 0.1 ng/mL retinoic acid, 6.5 ng/mL triiodothyronine, 50 μg/mL gentamicin and 0.1 ng/mL amphotericin B. Cells were then grown in a starved medium beginning from the day before stimulation.

BEAS-2B cells, an immortalized cell line established from normal human bronchial epithelium obtained by autopsy of non-cancerous individuals and widely used to study the functions of lung bronchial epithelial cells [21] , were purchased from the American Type Culture Collection (Manassas, VA). Primary normal human bronchial epithelial cells (NHBE) were purchased from TAKARA BIO INC. (Tokyo, Japan). The cells were cultured in serum-free epithelial growth medium (BEGM; Cambrex, Walkersville, MD) supplemented with Bullet Kit (Cambrex) to contain 0.5 ng/ml human recombinant epidermal growth factor, 0.5 µg/ml hydrocortisone, 10 µg/ml transferrin, 0.5 µg/ml epinephrine, 5 µg/ml insulin, 50 µg/ml bovine pituitary extract, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine, 50 µg/ml gentamicin and 0.1 ng/ml amphotericin B at 37°C in a humidified 5% CO₂ atmosphere. THP-1 cells, a human monocytic cell line, were purchased from the American Type Culture Collection. Cells were cultured in 10-cm culture dishes in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified 5% CO₂ atmosphere.