Bone Mechanical Stimulation with Piezoelectric Materials - Natural Polymers, Biopolymers, Biomaterials, and Their Composites, Blends, and IPNs - page 285

respectively at 24 and 48 hr post-seeding. The nitric oxide values in static conditions
were significantly lower, 2 ± 0.35 µmol/ml, 24 hr post-seeding and 1.7 ± 0.3 µmol/ml,
48 hr post-seeding (Figure 1).
Figure 1. Nitric oxide measurement (µmol/ml) in culture medium in static versus dynamic conditions,
24 and 48 hr after seeding MC3T3 on the devices, and immediately after stimulation at 1 and 3 Hz.
NO values are significantly higher in the dynamic group.
The NO measurement results of culture under dynamic conditions versus static
conditions, suggest osteoblasts detect and respond in a reproducible way to small dis-
placements and strain levels.
Changes induced by mechanical stimulation on the cytoskeleton were qualitatively
assessed through indirect immunofluorescence. Primary antibodies against actin, lam-
inin and tubulin were used; stains show stronger fluorescence on mechanical stimu-
lated cells, clearer images of the cytoskeleton elements and nucleus delimitation and
prominent cytoplasmatic extensions (Figures 2 to 4).
Figure 2. MC3T3 cells on the active area of the device immediately after mechanical stimulation.
Indirect immunofluorescence using primary antibody against actin (Actin, pan Ab-5, Thermo
Scientific, used at 1:50) and secondary antibody (Chromeo™ 488 conjugated Goat anti-Mouse IgG,
Active Motif 1:500); (400X, microscope Olympus BX41, Olympus Cell A Imaging Software).