Biomedical Engineering Reference
In-Depth Information
A Wst-1 assay
[37]
quantified the metabolic activity of the hUCMSCs cultured for 8 days on
CPC control and CPC-chitosan-fiber scaffold. The cell viability, measured using the absorbance
at 450 nm, was proportional to the amount of dehydrogenase activity in the cells. This absorbance
was measured to be 1.3
0.2 for the CPC control and 1.2
0.1 for the CPC-chitosan-fiber scaffold.
Hence the stronger and tougher CPC-chitosan-fiber scaffold yielded hUCMSC viability that matched
that of the CPC control.
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to
determine osteogenic differentiation of hUCMSCs on CPC. hUCMSCs were seeded on three materi-
als which are CPC control (containing no chitosan or fibers), CPC-chitosan, and CPC-chitosan-fiber
scaffold (20% fibers). CPC control was referred to as the FDA-approved CPC because that material
consisted of the same TTCP-DCPA mixture with no chitosan or fibers
[30]
. A total 150,000 cells
were diluted into 2 ml of osteogenic media and added to each well containing a CPC disk of 2 mm
thickness and 12 mm in diameter. For qRT-PCR (7900HT, Applied Biosystems, Foster City, CA),
cells were incubated for 1, 4, and 8 days. The total cellular RNA on the scaffolds were extracted
with TRIzol reagent (Invitrogen) and reverse transcribed into cDNA. Relative expression level for
each target gene was evaluated using the 2-ΔΔCt method
[53]
. The results are plotted in
Figure 12.8
.
Alkaline phosphatase (ALP) gene expression was minimal at 1 day; it peaked at 4 days, and then
slightly decreased at 8 days. Osteocalcin (OC) gene expression was at highest after 8 days. In both
cases, incorporating chitosan and fibers, which greatly improved the mechanical properties, did not
adversely affect ALP and OC. Instead, the ALP and OC on the CPC-chitosan-fiber scaffold were
higher (
P
0.05) than those on the FDA-approved CPC control.
(A)
(B)
CPC control
CPC control
e
d
CPC-chitosan
CPC-chitosan-fiber
14
CPC-chitosan
CPC-chitosan-fiber
10
12
8
10
d
6
8
c
6
4
c
c
c
4
b
b
2
b
2
a
a
a
a
a
a
a
a
0
0
1 day
4 days
8 days
1 day
4 days
8 days
FIGURE 12.8
hUCMSC osteogenic differentiation. (A) qRT-PCR for hUCMSCs yielded the ALP gene expression. (B) qRT-
PCR yielded OC gene expression. Each value is mean
SD;
n
5. In each plot, bars with dissimilar letters
indicate significantly different values (
P
0.05).
