Biology Reference
In-Depth Information
Table 2.1 Calcein uptake in AECs at the 1st, 6th, and 12th passages per days of culture in DM
7gg
14gg
21gg
0.96
a
1st pass.
8.42
8.02
1.28
8.52
0.98
6th pass.
4.45
1.08
5.57
0.84
8.22
1.24
12th pass.
1.72
0.57
2
0.82
9.07
1.26
a
Values expressed as fluorescence units
Immediately after isolation, AECs did not present remarkable signs of global
DNA methylation, while the presence of methyl cytosine was detected in cells at
the 12th passage.
In all cells evaluated, no significant osteogenesis was recorded (increase in both
ALP and calcein, and alizarin positivity) following a culture time of 21 days in
control medium. Conversely, the use of inductive factors resulted in clear signs of
osteogenic differentiation. Indeed, after 7 days of culture in osteoinductive
medium, the quantity of ALP, expressed as absorbance, was 0.25
0.01 in cells
at the 1st passage and 0.17
0.02 for controls (
p
0.02), 0.2
0.01 at the 6th
<
passage vs. 0.1
0.02 in controls (
p
0.05), and 0.2
0.02 at the 12th passage
<
vs. 0.17
-estradiol did not
produce an increase in ALP activity. The values obtained in DMe were similar to
those obtained in DM. Matrix mineralization, detected by calcein uptake after
7 days, was massive in cells at the 1st passage with large matrix droplets of
extracellular mineralization accumulated on the cell monolayer, while calcein
deposition was low in cells derived from the 6th and 12th passages. After
14 days, abundant calcein deposition was recorded in cells from the 1st passage,
evident in the 6th passage, and barely detectable in the 12th passage. After 21 days,
all evaluated cells expressed clear signs of matrix mineralization, with a higher
signal in cells at the 1st passage. The addition of 17
0.03 in controls (
p
0.05). The addition of 17
>
b
-estradiol in culture medium
improved the osteogenic transformation of AECs that remained evident in all cells
types evaluated (calcein uptake in DMe 2-3 times higher than in DM). The
quantification of the osteogenic process is reported in Table
2.1
and shows signifi-
cant differences between control and DM, especially in the 1st passage (
p
b
<
0.01).
In cells cultured in DMe, the intensity of calcein deposition was always signifi-
cantly higher than in DM (
p
0.05). Alizarin staining confirmed the diffuse
process of mineralization in cells of the 1st passage, which was detectable after
14 days of culture and more evident after 21 days. By contrast, this staining
procedure revealed clear signs of mineralization only after 21 days of culture in
cells derived from the 6th and 12th passages but did not reveal any differences
between DM and DMe.
<
2.4 Discussion
The present investigation describes the peculiar properties of AECs, which have a
diffuse expression of adhesion molecules CD29 and CD49 that are likely responsi-
ble for the high plating efficiency of these cells and account for the quick
