Biomedical Engineering Reference
In-Depth Information
Conventional monolayer cultures of chondrocytes have
the disadvantage of producing matrix that differs from
that produced
in vivo,
losing their typical phenotype
within several days or weeks as they ''dedifferentiate''.
Freshly isolated articular chondrocytes express cartilage-
specific type II collagen and hyaline cartilage markers,
aggrecan, but during prolonged culture and serial sub-
culture these cells lose their spherical shape, begin to
dedifferentiate to a fibroblast-like phenotype, and pro-
duce predominantly unspecific type I collagen. The
phenotypic stability of adult human chondrocytes is lost
quickly on expansion in serial monolayer cultures than
that of cells of juvenile humans. This loss of phenotype in
monolayer culture is reversible if chondrocytes are cul-
tured in 3-D culture systems embedded in solid support
matrices, such as collagen, agarose, or alginate gels.
However, there are disadvantages to these systems,
including the slow rate of proliferation and the sub-
stantial decline of matrix production in suspension cul-
tures. Cardiomyocytes cultured in monolayers also
exhibit properties different from native heart tissue,
because of structural differences between 2-D and native
environments, and because of the effects of cell isolation
and
in vitro
cultivation.
The 3-D culture has often been stated to be preferable
to 2-D culture, because most of the cells in our body are
present in 3-D environments
[12]
. Many cellular pro-
cesses including morphogenesis and organogenesis have
been demonstrated to occur exclusively when cells are
organized in a 3-D fashion. The fundamental process
underlying most tissue engineering methodologies is 3-D
culture at high cell density to enhance cell-cell in-
teractions favorable for ECM production. The 3-D ECM
culture systems have been developed to simulate natural
interactions between cells and the extracellular envi-
ronment. The 3-D culture maintains the cell phenotype
but is poor for cell expansion. Cells in 3-D culture are
surrounded with a substrate not only on one side of the
cells but on many sides of the cells. Generally, the so-
called ''3-D scaffolds'' having distinct pores of sizes much
larger than the cells come in contact with cells only by
their one side, as illustrated in
Fig. 7.2-18
a. Cells in right
3-D culture should be in contact with a substrate from
multiple directions, as shown in
Fig. 7.2.18
b. However,
the 2-D culture may change to 3-D culture once the cells
begin to be surrounded by the matrix produced by the
cells themselves, as shown in
Fig. 7.2.18
d. In culture of
chondrocytes for cartilage repair the isolated cells have
been expanded in monolayer culture, dedifferentiated,
and then redifferentiated in a 3-D cell arrangement
for new cartilage formation. A typical sign of de-
differentiation of chondrocytes is the switch from type II
collagen to type I collagen synthesis. Endothelial cells
grown in 2-D systems vary from 3-D model systems. A
wide variety of cell types exhibit enhanced maintenance
(a)
(b)
(c)
(d)
Fig. 7.2-18 Cells at 2-D and 3-D culture, and seeded on 3-D
porous scaffold: (a) cells on flat dish (2-D culture); (b) cells in gel (3-D
culture); (c) cells adsorbed on 3-D porous scaffold (2-D culture);
and (d) cells entrapped in biosynthesized ECM.
of their differentiated phenotype if cultured in 3-D
systems instead of monolayers, which is attributed to
associated differences in cell shape and/or increases in
intercellular communication. It was shown that neonatal
rat ventricular myocytes in confluent monolayers couple
on average with six cells, whereas the same cells in native
ventricles couple on average with nine cells
[13]
.
One major constraint in the use of 3-D scaffolds has
been the limitation of cell migration and tissue ingrowth
within these structures. Bovine aortic endothelial cells
can survive or proliferate in a 2-Dmodel in the absence of
angiogenic factors; however, they die in a 3-D collagen
lattice in the absence of angiogenic factors
[14]
. Because
cells located in the interior scaffold receive nutrients only
through diffusion from the surrounding media in static
culture, high cell density on the exterior of the scaffold
may deplete nutrient supply before these nutrients can
diffuse to the scaffold interior to support tissue growth.
In addition, diffusive limitations may inhibit the efflux of
cytotoxic degradation products from the scaffold and
metabolic wastes produced in the scaffold interior. Some
examples of possible diffusive limitations of high cell
density 3-D culture are summarized in
Table 7.2-4 [15]
.
In a series of related studies, maximum penetration depth
of osseous tissues was reported to be in the range of 200-
300
m
mwithin porous 3-D PLGA scaffold after 2 months
of static culture. Although several attempts have been
made to alter scaffold geometry to provide adequate
diffusion within 3-D constructs with some success, in-
growth limitations within 3-D scaffolds remain a perva-
sive problem in tissue engineering.
Some of the factors known to influence the phenotype
of cells are growth factors, cell-material
interactions,
