Biomedical Engineering Reference
In-Depth Information
by the mitochondrial succinate dehydrogenase present in most cells. Thus, MTT assay
is a semiquantitative colorimetric assay for mammalian cell survival and proliferation.
According to Mosmann, the assay detects living, but not dead cells, and the signal gener-
ated is dependent on the degree of activation of the cells. However, according to Page et al.
[94] nonviable cells could reduce the tetrazolium salt. The main advantages of the assay
are rapidity and precision and a lack of radioisotope, thus requiring no washing steps [93].
In an MTT assay conducted on HMEC seeded on a-C:H and a-C:H(Si) films reviews no
significant difference between coated and uncoated control samples (96-well tissue culture
polystyrene) [95]. This shows that the coatings are not cytotoxic to the cells and could be
said to be closely compatible when compared to the tissue culturing material.
Macrophages
Macrophages are white blood cells within tissues, produced by the division of monocytes.
It is a suitable cell for in vitro biocompatibility testing, as it plays a major role in inflamma-
tion and the response to foreign bodies. Macrophages are known to produce a number of
inflammatory mediators, which have an effect on the surrounding tissue [96]. They play
a vital role in the development of atherosclerosis as well as in-stent restenosis. A minor
population of macrophages can proliferate in the atherosclerotic lesions themselves, par-
ticularly in the early stage. Activated macrophages can accumulate cholesterol esters in the
cytoplasm, which leads to foam cell formation in lesion development [97]. It is therefore
desirable for a biomaterial material to have minimum macrophage adhesion and those
that adhere should be viable.
Thomson et al. [98] studied the inflammatory potential of a-C coated tissue culture plates
on a-C coating by measuring the levels of the lysosomal enzyme
N
-acetyl-d-glucosaminidase
in the culture medium following the exposure of macrophages to a-C coatings. Since lyso-
somal enzymes are released from macrophages during inflammation, an increased enzyme
level in the medium means that an inflammatory reaction has been elicited. They found no
significant difference between the enzyme levels from a-C coatings and those from the
control tissue culture plate. This shows that a-C does not induce inflammatory reactions in
the cells any more than the tissue culture plate. Allen et al. [99] have also studied the effects
of a-C-coated polystyrene plates on macrophages. The results showed that macrophages
grew well on a-C-coated plates. Scanning electron microscopic examinations confirmed
the normal behavior of cells and no evidence of extensive cell death, therefore showing
no cytotoxicity was caused by the a-C coating. Lactate dehydrogenase (LDH), an enzyme
released at cell death, has been used as a measure of cell viability [99], LDH assays carried
out provided a measure of cell viability and have shown no evidence of overt cytotoxicity
for a-C:H films [100]. No difference in LDH release between the a-C:H (with a-Si:H bonding
layer) and control specimens has been observed. Furthermore, the absence of abnormal cel-
lular morphology has been demonstrated by optical microscopy. Li and Gu [101] observed a
decrease in the number of adhering macrophages when the sp
3
fraction and hydrophobicity
were higher.
Increasing surface roughness and surface energy can lead to an enhancement of mac-
rophage viability, and the higher the hydrogen content for a-C:H films, the lower the mac-
rophage attachment [102]. The a-C:H films significantly suppressed attachment of viable
macrophages, compared to the uncoated surface (Si), indicating possible lower inflamma-
tory responses. This finding is in agreement with a previous report by Ball et al
.
[103],
where J744 macrophages, cultured on ta-C surfaces, showed reduced hydrogen peroxide
production compared to thermanox, stainless steel, and polyurethane-coated stainless
