Biomedical Engineering Reference
In-Depth Information
return to their original shape. After the cleaning (ultrasonic cleaning in acetone) and ster-
ilization (4-h UV exposure) processes, the PRP was seeded onto the surfaces of the a-C(Si)
coated substrates placed in a Petri dish. Incubation was carried out at 37°C in 5% CO
2
for
30 min. Afterward, the supernatant was discarded, and the samples were rinsed twice
with Phosphate Buffered Saline (PBS) to remove the proteins and nonadherent platelets.
The cells on the specimens were fixed with 2.5% gluteraldehyde for 30 min, followed by
1% osmium tetraoxide
on ice for 45 min. After the fixation, the samples were washed and
dehydrated in a graded ethanol series of 50%, 70%, 80%, 95%, and 100% (twice) for 10 min
each. Critical point drying can be performed if desired.
A scanning electron microscope can be used to observe the platelets. The samples were
coated with ~15 nm of gold to prevent surface charging. An average platelet count using
the mean of six nonrepeating selected areas was conducted on each sample. Results were
expressed as counts/unit area and standard error. The statistical analysis used was one-
way analysis of variance (ANOVA) on an average of three replicates to compare data
between different periods of culture, and Student's
t
-test was carried out to compare
the culture data between coatings on each count. Statistical significance was considered
at
p
< 0.05. The morphological shape changes were categorized according to Goodman
et al. [42] and are illustrated in Table 2.1. There are five types of platelet morphologies.
Inactivated platelets of Type I are ~2
µ
m in diameter and do not have a pseudopod. With
increasing activation, the platelets develop pseudopodial extensions, which eventually
TABLE 2.1
Five Types of Platelet Morphologies in Relation to Their Activation Level Upon Adhesion to
Foreign Surfaces
Type
MorphologyDescription
I
Round and discoid—No pseudopodia
II
Dendritic
—
Early pseudopodial with no flattening
III
Spread-dentritic—Intermediate pseudopodia with one or more
pseudopodia flattened, no hyaloplasm between pseudopodia
IV
Spreading—Late pseudopodial with hyaloplasm spreading
V
Fully spread—Hyaloplasm fully spread with no distinct pseudopodia
