Rat bone marrow-derived mesenchymal stem cells (rBMSCs) (Sprague-Dawley, Fisher Scientific, PA) were maintained in the low glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 0.5% penicillin-streptomycin (Pen-Strep, Gibco) within a 37 °C cell incubator (5% CO2, 95% relative humidity). For the live/dead study, the click-ON bone cement disks and the clinically used PMMA bone cement disks were sterilized by 70% alcohol, and rBMSCs were seeded with a density of 15, 000 cells per cm2. After 3 days of culture, the cells were stained with LIVE/DEAD® Cell Imaging Kit (Thermo Fisher Scientific, Waltham, MA) and scoped on inverted laser scanning confocal microscope (Carl Zeiss, Germany).
To further confirm the cytotoxicity, the click-ON bone cement disks and the clinically used PMMA bone cement disks were placed in transwell chambers (mesh size 3 μm) and co-cultured with rBMSC cells in six-well TCPS plates. After 1, 3, and 7 days, the rBMSC cell numbers in each well were determined using the MTS assay and cell viability was calculated by normalizing to non-treatment TCPS positive control (set as 100%). The cell apoptosis after co-culturing with the two bone cements was determined by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Sigma).
To further confirm the cytotoxicity, the click-ON bone cement disks and the clinically used PMMA bone cement disks were placed in transwell chambers (mesh size 3 μm) and co-cultured with rBMSC cells in six-well TCPS plates. After 1, 3, and 7 days, the rBMSC cell numbers in each well were determined using the MTS assay and cell viability was calculated by normalizing to non-treatment TCPS positive control (set as 100%). The cell apoptosis after co-culturing with the two bone cements was determined by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Sigma).