Biomedical Engineering Reference
In-Depth Information
a
b
PS
Ti6AI4V
**
140
200
180
160
140
PS
Ti6AI4V
120
**
100
80
120
100
60
80
60
40
20
0
40
20
0
0
0.25
0.5
0
0.25
0.5
H
2
O
2
(mM)
H
2
O
2
(mM)
Fig. 4.5
Measurement of antioxidant enzyme activity in HDMEC grown on PS and Ti6Al4V.
Catalase (
a
) and SOD (
b
) activities were measured in HDMEC 4 h after H
2
O
2
treatment with cor-
responding activity assay and related to protein concentration of the samples. The data are presented
as % of control (untreated HDMEC on PS; means ± SDs; significant difference: **
p
< 0.01)
alloy surface could also lead to the formation of ROS due to the Fenton reac-
tion upon H
2
O
2
exposure.
Cells have several mechanisms to protect themselves against oxidative stress.
SOD, catalase and GSH system are the central defence players in enzymatic ROS
detoxification. The balance between GSH and its oxidised form (GSSG) is critical
for protecting cells against oxidants. Excessive formation of GSSG and glutathione
conjugates with organic molecules during prolonged oxidative stress leads to the
depletion of the GSH pool [
16
]. Thus, the reduced GSH concentrations in endothe-
lial cells grown on Ti6Al4V alloy compared to the cells grown on PS [
87
] could
reflect a state of permanent oxidative stress in cells in contact with Ti6Al4V alloy as
a result of ROS formation on the titanium surface. This is supported by the fact that
the GSH concentration in endothelial cells on Ti6Al4V was reduced by H
2
O
2
treat-
ment, whereas in cells grown on PS the H
2
O
2
treatment induced an elevation of GSH
level. The effects of H
2
O
2
treatment on PS are in agreement with reports of adaptive
increase in GSH concentration after exposure to H
2
O
2
, a fact that can be explained
by the onset of GSH de novo synthesis and H
2
O
2
-induced up-regulation of the
expression of GSH synthesis enzymes (g-glutamylcysteine synthetase and GSH
synthetase) by these temporary oxidative stimuli [
51,
70
] .
Additional evidence for permanent oxidative stress on Ti6Al4V is based on the
analysis of enzymatic activities of catalase and SOD. Cells grown on Ti6Al4V alloy
showed significantly lower SOD activities than cells grown on PS (Fig.
4.5b
). This
could be explained by a persistent ROS formation in cells on the Ti6Al4V surface
resulting in the exhaustion of enzyme activity, e.g. due to protein oxidation by ROS
[
11
]. Catalase activity in endothelial cells grown on Ti6Al4V was only slightly
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