Hfob cell line

The human normal osteoblastic hFOB cell line and the human osteosarcoma MG63, U2OS, HOS and Saos2 cell lines were purchased from the American Type Culture Collection and cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator containing 5% CO₂ at 37˚C.

The human osteosarcoma MG-63 and SAOS-2 cell lines, and the normal bone hFOB cell line were obtained from the American Type Culture Collection (ATCC). The MG-63 and SAOS-2 cells were cultured in Eagle's minimum essential medium (cat. no. 30-2003; ATCC) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.). hFOB cells were cultured in McCoy's 5a modified medium (cat. no. 30-2007; ATCC) containing 10% FBS. All cells were cultured at 37°C with 5% CO₂. HAND2-AS1 small interfering RNA (siRNA; 5′-CCGAGGUGCUCCAAUAUUATT-3′) and negative control siRNA (5′-UUCUCCGAACGUGUCACGUdTdT-3′); were purchased from Shanghai GenePharma Co., Ltd. All three cell lines were cultured to 80–90% confluence, and 5×10⁵ cells/sample were transfected with 50 nM siRNA using Lipofectamine^® 2000 reagent (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to measure the expression levels of HAND2-AS1, and to confirm a <50% reduction prior to subsequent experiments. Subsequent experiments were performed at 24 h after transfections. The control group for transfection was un-transfected cells, and the negative control group was negative control siRNA-transfected cells.

HA paste was purchased from 3DCeram Sinto, France. Sylgard 184 silicone elastomer and curing agent were purchased from Dow Corning Toray, Japan. Doxorubicin hydrochloride (DOX) was purchased from Sigma-Aldrich and was of analytical grade and used without further purification. Fluorescein isothiocyanate isomer I (FITC) and rhodamine were purchased from Sigma-Aldrich, USA. Cell Tracker Green CMFDA (5-chloromethylfluorescein diacetate) and other cellular fluorescent stains were purchased from Thermo Fisher, USA. Dulbecco's modified Eagle's medium (DMEM), phenol red-free DMEM/F12 (DMEM-F12), foetal bovine serum (FBS), phosphate-buffered saline (PBS) and trypsin-EDTA were purchased from Gibco, Invitrogen. The UMR-106 cell line and the hFOB cell line were purchased from the American Type Culture Collection (ATCC).