Ols4100

For the immunofluorescence analyses, the cells were cultured in 6-cm dishes. The cells were mounted with cytospin on polylysine-coated glass slides and fixed with 4% paraformaldehyde for 15 min, followed by the addition of 100% ice-cold acetone for 10 min at 4°C. To detect the protein expression of DUSP2, immunofluorescence analysis was performed with DUSP2 antibody (dilution, 1:100; cat. no. LS-B14289; LifeSpan BioScienes, Inc.), incubated for 24 h at 4°C, followed by incubation with anti-IgG-PE (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 37°C for 30 min in the dark and mounting with DAPI mounting medium. The samples were investigated by laser scanning confocal microscopy (OLS4100; Olympus Corporation, Tokyo, Japan).

An in vitro degradation test was performed to evaluate the changes in the mechanical properties as a function of the degradation time. The mechanical stability after degradation was assessed over an immersion period of 12 and 40 weeks. Dog bone samples with a thickness of 450 μm were used for the degradation and mechanical test in accordance with American Society for Testing and Materials (ASTM) E8/E8M − 08. All samples were immersed in SBF solution, and the temperature was maintained at 37 °C. To create an environment for accelerated degradation, the SBF was replaced every 2 days for 40 weeks. After 12 and 40 weeks of immersion, all samples were extracted from the SBF solution and ultrasonically cleaned with ethanol to prevent further degradation. The surface roughness of the samples was examined by laser scanning microscopy (OLS 4100, Olympus, Japan). For the tensile test, a universal testing machine (Instron 5580, Instron Corp., Canton, MA) was used with a fixed loading rate of 1 mm/min, and the thickness loss of samples was measured by a vernier calipers at three different regions (upper, middle, and lower) of each sample after specific time interval of degradation test.

ROS levels were assessed using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Molecular Probes; Thermo Fisher Scientific, Inc.), which is converted to highly fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of ROS. F98 cells (3×10⁵) were incubated at 43°C for 1 h before incubation at 37°C for 0, 3, 6 or 12 h; cells were then incubated with 5 mM DCF-DA at 37°C for 30 min in darkness. H₂O₂ was used as a positive control; H₂O₂ (200 µM) was added to F98 cells and incubated at 37°C for 1 h. In addition, cells were pretreated with or without MnTBAP for 1 h at 37°C and were then incubated at 43°C (HS) or 37°C (control) for 60 min. Analysis of the fluorescence intensity of DCF was subsequently performed using a flow cytometer, and images were obtained via laser scanning confocal microscopy (OLS4100; magnification, ×40; Olympus Corporation, Tokyo, Japan) at an excitation of 488 nm and emission of 530 nm.