C2C12 cells (10 000 cells/cm2) are seeded on Cross-linked (PLL-PGA)14 films (non-porous) and cross-linked ([PLL-PGA]5-PLL-NP)2-(PLL-PGA)2 films treated with THF (porous) loaded or not with 500 ng of BMP-2 (as described above) and cultured for 7 d in DMEM high glucose/Glutamax (Invitrogen) supplemented with 2% horse serum (Invitrogen), 100 U/mL penicillin G and 100 µg/mL streptomycin (Eurobio).
Cell differentiation is investigated following the expression of a late myoblastic marker (myosin heavy chain) and of an early osteoblastic marker (osteopontin) using specific antibodies (Millipore). The immunostaining is performed following the protocol described in the Cell proliferation and morphology section. Succinctly, cells are labeled with mouse anti-myosin heavy chain (MHC, 1:1000), and rabbit anti-osteopontin (1:500), (both from Millipore). Primary antibodies are revealed using Alexa Fluor 488 or Alexa Fluor 633-conjugated goat anti-mouse or anti-rabbit antibodies (1:400, Invitrogen) as secondary antibodies. Samples are examined using a LSM710 confocal microscope (Carl Zeiss).
Cell differentiation is investigated following the expression of a late myoblastic marker (myosin heavy chain) and of an early osteoblastic marker (osteopontin) using specific antibodies (Millipore). The immunostaining is performed following the protocol described in the Cell proliferation and morphology section. Succinctly, cells are labeled with mouse anti-myosin heavy chain (MHC, 1:1000), and rabbit anti-osteopontin (1:500), (both from Millipore). Primary antibodies are revealed using Alexa Fluor 488 or Alexa Fluor 633-conjugated goat anti-mouse or anti-rabbit antibodies (1:400, Invitrogen) as secondary antibodies. Samples are examined using a LSM710 confocal microscope (Carl Zeiss).