2.6 In vitro biological evaluation of bilayer scaffolds
To evaluate the biological properties of bilayer scaffolds, BMSCs extracted from healthy adult rats were used in in vitroexperiments. BMSCs were propagated in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) penicillin-streptomycin (Gibco). The cell culture work was performed in a CO2 incubator supplemented with 5% CO2 and 95% humidity at 37 ℃.
The attachment, survival, proliferation and morphology of BMSCs on PTMC/TPU (designated as “S”), S+F and S+F-PDA@E2 scaffolds were studied through SEM observation, live/dead assay (Invitrogen, Thermo Fisher Scientific), MTT assay (Invitrogen, Thermo Fisher Scientific), and confocal laser scanning microscopy (LCSM, Leica, Germany) after phalloidin/DAPI staining (Invitrogen, Thermo Fisher Scientific), respectively. BMSCs at the density of 5×104 cells per well were seeded on S, S+F and S+F-PDA@E2 scaffold samples in a 24-well cell culture plate and cultured in DMEM at 37 ℃. After being immobilized by 4% paraformaldehyde for 30 min, samples were dehydrated by a serial gradient ethanol solution (50%, 60%, 70%, 80%, 90%, and 100%) for 10 min and then dried in a vacuum oven overnight. The dried samples were then sputtered with a thin layer of gold, and cell morphology was observed under SEM.
For cell survival analysis, BMSCs at the density of 5×104 cells per well were seeded on S, S+F and S+F@-PDAE2 scaffolds in a 24-well cell culture plate and cultured in DMEM at 37 ℃. After culture for 24 h and 48 h, respectively, live/dead assay was used to stain BMSCs. Living cells were stained green and dead cells were stained red when observed under a fluorescence microscope (Leica DMi8, Germany). Moreover, cell survival rate was calculated using Image J software. On the other hand, BMSCs at density of 1×104 cells per well were seeded on scaffold samples to study the BMSC proliferation rate and cell morphology. After culture for 1, 3 and 7 days, respectively, BMSC viability on S, S+F and S+F-PDA@E2 scaffolds was determined using MTT assay. Furthermore, BMSCs were visualized under CLSM after phalloidin/DAPI staining.