Biomedical Engineering Reference
In-Depth Information
differentiation,
7
it is not surprising that an enormous amount of research has
centered on the modification of inorganic and polymer substrates with these
molecules prior to experiments with cells. Most work has focused on laminin,
collagen and fibronectin where either the whole protein complex (or cocktails
thereof) or peptide sequences are 'grafted' onto a substrate. Briefly, the
laminins are a family of glycoproteins that are responsible for structural scaf-
folding in tissue. They are trimeric molecules comprised of a-, b-andg-chains
present in five, four and three genetic variants. Fifteen laminin structures have
been found where variability is defined by the chain composition. The proteins
connect to other ECM macromolecules and membrane components to form
cross-linked scaffolding. Collagen is massively present in connective tissue in
mammals. It is composed of three polypeptide chains with the molecule being
incorporated into a larger fibril structure. The protein is characterized by
possessing high concentrations of glycine, proline and hydroxyproline, which
confer properties with respect to connectivity.
As for the case of polypeptides, the 'grafting' or attachment of ECM protein
or peptides to particular substrates is based conventionally on either non-
covalent or stronger covalent interactions. Hydrophobic force, van der Waals
forces, hydrogen bonding or electrostatic forces are the basis of protein
adsorption by non-covalent interactions. Modification by such binding of
coating materials on the surface is advantageous because of their ease of
application since no chemical modification is required prior to immobilization.
The drawbacks are protein denaturation due to uncontrolled interactions
between the proteins and the surface, and undesired protein desorption during
the study period. A more stable means of protein immobilization is to link a
protein to the surface covalently via a chemical bond.
Widely used peptides derived from ECM proteins that are used for neuron
attachment are GDPGYIGSR, GQAASIKVAV, GRGDS and RHSRN. An
excellent example of this type of chemistry is the use, similar to the hydrogels
mentioned above, of a three-dimensional polymeric matrix specially
synthesized from a methacrylated dextran and aminoethyl methacrylate in the
presence of PEG.
38
This leads to a macroporous structure displaying high
equilibrium water content. The presence of the amino functionality was
assessed semi-quantitatively via a simple ninhydrin color test. Covalent binding
of peptides to the -NH
2
group present on the copolymer was achieved via a
linker. The latter activated the surface group in a somewhat analogous manner
to that employed in biosensor technology discussed in Chapter 1. Peptides
studied were CRGDS or a mixture of CDPGYIGSR and CQAASIKVAV.
Significantly enhanced growth of primary embryonic chick dorsal root ganglia
on peptide-treated polymers was observed (Figure 2.11). Despite reaching the
conclusion that ECM-derived peptides on polymers could provide an ideal
scaffold for neural regeneration, the authors appeared disappointed by the level
of neural attachment and performance obtained in their experiments.
38
Guidance of neurite growth using ECM-based peptides incorporated again
in a three-dimensional polymer hydrogel has been attempted.
39
In this work the
polymer hyduronan was reacted with S-2-nitrobenzyl cysteine using the
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