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.