Biomedical Engineering Reference
In-Depth Information
and in snake venoms. About half of the 24 integrins have
been shown to bind to ECM molecules in an RGD-
dependent manner. Since other important adhesive motifs
have been also identified, the RGD sequence is not the
''universal cell recognition motif,'' but it is unique with
respect to its broad distribution and usage. The confor-
mation of the RGD-containing loop and its flanking
amino acids in the respective proteins are mainly re-
sponsible for their different integrin affinity. Other fac-
tors that contribute to integrin-ligand binding affinity
include the activation of integrins by divalent cations and
cytoplasmatic proteins.
The process of integrin-mediated cell adhesion com-
prises a cascade of four different partly overlapping
events: cell attachment, cell spreading, organization of
actin cytoskeleton, and formation of focal adhesions.
First, in the initial attachment step the cell contacts the
surface and some ligand binding occurs that allows the cell
to withstand gentle shear forces. Secondly, the cell body
begins to flatten and its plasma membrane spreads over
the substratum. Thirdly, this leads to actin organization
into microfilament bundles, referred to as stress fibers. In
the fourth step the formation of focal adhesions occurs,
which link the ECM to molecules of the actin cytoskele-
ton. Focal adhesions consist of clustered integrins and
more than 50 other transmembrane-associated and other
cytosolicmolecules. During the four steps of cell adhesion
integrins are employed in physical anchoring processes as
well as in signal transduction through the cell membrane.
It is well established that integrin-mediated cell
spreading and focal adhesion formation trigger survival
and proliferation of anchorage-dependent cells. In this
context the expression of the anti-apoptotic protein Bcl-
2, induced by a5b1 integrins, and the suppression of the
p53 pathway by focal adhesive kinase (FAK) are dis-
cussed. In contrast, loss of attachment causes apoptosis
in many cell types, referred to as ''anoikis'' (a Greek word
meaning homelessness). Anoikis can even be induced in
the presence of immobilized ECM molecules when non-
immobilized soluble ligands like RGD peptides are
added, as illustrated in
Fig. 7.2-16 [11]
. Stable linking
of RGD peptides to a surface is essential to promote
strong cell adhesion, because formation of focal adhe-
sions only occurs if the ligands withstand the cell's con-
tractile forces. These forces are able to redistribute
weakly adsorbed ligands on a surface, which leads only to
weak fibrillar adhesions later on. Furthermore, cells can
remove mobile integrin ligands by internalization.
Surface
Spread cell
Survival, proliferation
Integrin
Integrin ligand
Detached, round cell
Apoptosis
Fig. 7.2-16 Opposite effects of integrin ligands. Immobilized
ligands act as agonists of the ECM, leading to cell adhesion and
cell survival, while non-immobilized ligands act as antagonists,
leading to cell deattachment, a round cell shape, and apoptosis.
these polymers are floating in cell culture medium. When
cells in culturemediumare plated on the top of the porous
scaffold or injected into its interior for seeding, the ma-
jority of its pores remain empty since the scaffold does not
absorb the culture medium. Because it is crucial to obtain
a uniform distribution of initial seeded cells throughout
the scaffold volume for the creation of a tissue with ho-
mogeneous cellularity, some approaches have been
attempted to improve hydrophilicity of polymer scaffolds
and thus to ensure uniform and dense cell seeding. They
include treatments of the scaffolds by prewetting with
ethanol, hydrolysis withNaOH, oxidationwith perchloric
acid solution, oxygen or ammonia plasma discharge
treatment, physical or chemical coating with some hy-
drophilic polymers or cell-adhesive proteins, and blending
with hydrophilic polymers such as Pluronics (ethylene
glycol-propylene glycol block copolymers).
When foreign materials are exposed to a biological en-
vironment, ECM proteins are non-specifically adsorbed
on the surface of nearly all the materials, masking its
specific surface properties, and then cells indirectly in-
teract with the material surface through the adsorbed
ECM proteins
[12]
. A range of proteins from the culture
medium come adsorbed to the substrate surface, re-
flecting the relative abundance of proteins in the culture
medium. However, over time, weakly adsorbed proteins
will leave the surface, while those bound more strongly
will remain. Other protein molecules will come in and
replace those that have been desorbed. The implication
of this series of events is that the final nature of the at-
tached protein layer reflects both the surface chemistry
of the underlying substrate and the composition of the
culture medium. This highlights the importance of op-
timizing the surface chemistry of the substrate to give the
7.2.5.2 Artificial surface in biological
environment
Because common poly(a-hydroxyester)s like PLA and
PGA are hydrophobic, porous scaffolds fabricated with
