Eclipse ti imaging system

To investigate cell metabolic activity samples were stained with MTT 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide reagent. Briefly, MTT reagent (Sigma, UK) was dissolved in phosphate buffered saline (PBS) to prepare a MTT stock solution (5 mg/mL). A one tenth dilution of MTT stock solution and serum‐free DMEM was added to the samples. Fibers were incubated for 3 h at 37°C and 5% CO₂ following which the staining solution was aspirated and DMSO added to elute the formazan. After an incubation of 1 h at room temperature, the absorbance was read at 570 nm. MTT was performed on days 1 and 14. Three samples were prepared for each condition and each sample was read five times. hBMSC cell numbers were obtained from an MTT standard curve, which was prepared from hBMSC numbers ranging between 0 and 2 × 10⁶ cells (passage 2). Live/Dead stain was performed to assess cell viability on days 1, 7, and 14. The medium was aspirated and cells were washed with PBS. Live/Dead stain (Sigma, UK) was prepared following the suppliers instructions. Cells were incubated for 30 min at room temperature and then washed with PBS. Images were taken using a Nikon eclipse Ti imaging system.