Biomedical Engineering Reference
In-Depth Information
Fig. 7.2-17 RGD peptides react via the N-terminus with different groups on polymers: (a) carboxyl groups, preactivated with a
carbodiimide and NHS to generate an active ester; (b) amino groups, preactivated with DSC; (c) hydroxyl groups, preactivated as tresilate;
and (d) hydroxyl groups, preactivated as p-nitrophenyl carbonate.
a coupling reaction, a photochemical immobilization
method has been utilized to graft cell-binding peptides.
In order to examine that any cellular responses to the
modified substrates are mediated solely by the immobi-
lized peptides, the experiment is performed under
serum-free conditions.
The modulation is based on biochemical or environ-
mental cues. A central issue in blood vessel culture is to
balance the competing goals of smooth-muscle cell pro-
liferation and ECM deposition (synthetic state or dedif-
ferentiation), and the contractile phenotype associated
with differentiation and maturation. For culture of en-
gineered vessels
de novo,
an increased synthetic state is
required, whereas at the conclusion of vessel culture, a
minimally proliferative, quiescent phenotype is desired.
Since cells would dedifferentiate when seeded into
polymeric structures
in vitro,
in depth investigations are
necessary to find out how dedifferentiation of seeded
cells can be prevented.
To treat traumatic or congenital cartilage defects with
tissue engineering techniques, a relatively small number
of donor cells, either chondrocytes or progenitor cells,
are expanded
in vitro
until sufficient cells are obtained.
However,
in vitro
multiplication of chondrocytes in
monolayer results in dedifferentiation of these cells.
Expansion in high seeding density cultures often fails to
produce sufficient chondrocytes, even after several pas-
sages. Lower seeding densities may increase cell yield,
but bear the risk of decreased redifferentiation capacity.
Differentiation of stem cells to target cells
in vitro
needs
specific culture media.
7.2.6 Cell expansion
and differentiation
For the clinical use of tissue engineering with isolated
cells, a small number of cells are initially isolated from
a small biopsy from the specific body part of patients or
others and then expanded in number in conventional
monolayer culture before they are seeded into scaffolds.
In many cases the number of harvested cells is not large
enough for repairing the lost or damaged tissues, espe-
cially when used clinically in the reconstruction of large
defects. Therefore, it is necessary to multiply the iso-
lated cells for tissue engineering with a sufficient number
of cells. Generally, the smaller the cell amount, the ECM
formation is less. Moreover, if the density of cells seeded
into scaffolds is low
in other words, the distance be-
tween the neighboring cells is long
d
the production of
ECM such as collagen and GAG from the cells will be
poor because of insufficient communication between the
cells. Although the promise of tissue engineering is tre-
mendous, it has only seldom been accomplished in
humans, largely because many cells are needed to gen-
erate even small amounts of tissue. It will be often nec-
essary to generate large amounts of tissue, starting with
very few cells. To circumvent this problem, cell culture is
performed to multiply cells under retention of their
phenotypic characteristics either before or after seeding
them into scaffold.
In cell culture the modulation of cell phenotype be-
tween the synthetic and quiescent states is important.
d
7.2.6.1 Monolayer (2-D) and 3-D culture
Advances in cell culture techniques have culminated in
the field of tissue engineering. The most common
method to increase the number of cells is 2-D monolayer
cell culture on a flat substrate. The 2-D culture is ex-
cellent for cell expansion but sometimes induces the loss
of native functional natures of cells. In monolayer cul-
tures, cells are forced to grow in one plane under space-
limiting conditions. This results in an obviously artificial
growth environment, in contrast to development
in vivo.
