Bone Mechanical Stimulation with Piezoelectric Materials - Natural Polymers, Biopolymers, Biomaterials, and Their Composites, Blends, and IPNs - page 284

To estimate the magnitude of stress/strain, finite numerical models were applied
and theoretical data was complemented by optic experimental data. The finite nu-
merical method estimated displacement varying from 6.44 to 77.32 nm in uncoated
films, with strain levels around 2.2 μ strains along the surface. The Electronic Speckle
Pattern Interferometry (ESPI) method showed the displacement in coated films was
lower, and the maximum substrate displacement was 0.6 μm, in the central area of the
coated devices; displacement was minimum in the encastre (clamped) region.
Piezoelectric substrates (standing on culture dishes, TPP) and controls (standard
culture dishes, TPP) were seeded with 16 × 10 4 cells, with a total volume of 100 µl of
cell suspension. Cells were allowed to adhere to the substrate, then the rest of culture
medium added (n = 6); and cells grown in both static and dynamic piezoelectric sub-
strates. The MCT3T3-E1 cells were cultured in standard conditions, using α-MEM
medium (Cambrex), 2 mM L-Glutamine (Cambrex), 10% of bovine fetal serum (Gibco),
0.5% gentamicin and 1% amphotericin B (Gibco).
The statistical analysis was done using software Origin Pro 8 (OriginLab Corpora-
tion, USA).
Normal distribution of the results was verified using the Kolmogorov-Smirnov
test, homogeneity of variance assessed through the Levene test and differences be-
tween groups tested using one-way ANOVA (at a level of 0.05).
Cell viability and metabolic activity was accessed through the resarzurin method,
after stimulation of dynamic group; viable cells reduce resarzurin, producing resoru-
fin, a highly fluorescent product. Previous studies indicated PVDF affects negatively
adherent cell lines' viability (Hung et al., 2006; Tabary et al., 2007).
The assessment of cell viability and proliferation evidenced a material's poorer
performance than control standard culture vessels, in spite of the coating procedure
(Table 1). The results are expressed as percentage of the value of controls (considered
as 100%) ± standard error of the mean and show higher viability values on mechani-
cally stimulated substrates, although the differences are not statistically significant.

table 1. Cell viability 24 hr and 48 hr after seeding and daily stimulation of the dynamic group,
results are expressed in percent related to controls (standard cell culture dish), assumed as 100%.
Means and Error bars show Means ± Standard Error of the Mean.

Proliferation and Viability Static Dynamic

24 hours post-seeding 49.9 ± 5.25 59.7 ± 15.7

48 hours post-seeding 76.4 ± 16.9 83.4 ± 25.1

Nitric oxide (NO) is a messenger molecule produced in response to mechanical
stimulation of osteoblasts and osteocytes, with a large variety of biological functions
(Smalt et al., 1997; Van't Hof, 2001). In this study, culture medium samples were
collected immediately after stimulation and NO measured, using NO Assay Kit
(Biochain), based on the Griess reaction, after sample deproteinization, and according
to the manufacturer's instructions (Figure1) . Culture medium NO measurements in the
samples subjected to mechanical stimulation were of 3.7 ± 0.65 and 3.2 ± 0.54 µmol/ml,