Scope of tissue engineering - Biomedical Engineering Desk Reference - page 688

best performance of the cells in a particular medium. The
nature of the protein layer that is adsorbed to the un-
derlying substrate has a profound effect on the attach-
ment and subsequent development of the cells in culture.
Not only is the mass of bound protein an issue but, more
importantly, the orientation of the protein with respect
to the surface may be crucial. Complexity of the cell
adhesion process that spans a broad range of time and
length scales no doubt contributes to some of the dif-
ferences among reports of cell interaction with materials.
Adhesion of anchorage-dependent mammalian cells is
traditionally viewed as occurring through at least four
major steps that precede proliferation: protein adsorp-
tion, cell-substratum contact, cell-substratum attach-
ment, and cell adhesion/spreading. Interactions between
cells and polymers in serum are mediated by proteins
that have been either preimmobilized on the material
surface, absorbed from the surrounding medium, or se-
creted by cells during culture. Cell contact and attach-
ment involves gravitation/sedimentation to within 50 nm
or so of a surface whereupon physico-chemical forces
conspire to close the cell-surface distance gap. Attached
cells then spread over the surface, depending on the
compatibility with the surface typically within hours.
Functional scaffolds should be capable of eliciting
specific cellular responses and directing new tissue for-
mation. This is mediated by biomolecular recognition in-
stead of by non-specifically adsorbed proteins, similar to
the native ECM. Extensive studies have been performed
to render scaffolds biomimetic by surface modification, as
this is the simplest way to make biomimetic scaffolds.
Scaffold with an informational function, e.g., with the
RGD sequence which facilitates cell attachment, must be
better than non-informational synthetic polymers. These
biomimetic materials potentially mimic many roles of
ECM in tissues. For example, incorporation of peptide
sequences renders the surface of biomaterials cell adhe-
sive that inherently have been non-adhesive to cells. The
design of biomimetic materials that are able to interact
with surrounding tissues by biomolecular recognition is an
attempt to make the materials such that they are capable
of directing new tissue formation mediated by specific
interactions, which can be manipulated by altering design
parameters instead of by non-specifically adsorbed ECM
proteins. For mimicking the native ECM, either long
chains of ECMproteins such as FN, vitronectin, and LNor
short peptide fragments composed of several amino acids
along the long chain of ECM proteins can be the candi-
dates for surface modification.
The early work on the surface modification of bio-
materials with bioactive molecules has used long chains of
ECM proteins for surface modification. Biomaterials can
also be coated with these proteins, which usually have
promoted cell adhesion and proliferation. Since the finding
of the presence of signaling domains that are composed of

several amino acids along the long chain of ECM proteins
and primarily interact with cell membrane receptors, the
short peptide fragments have been used for surface mod-
ification in numerous studies. Particularly, the use of a short
peptide for surface modification is advantageous over the
use of the long chain of native ECM proteins. The native
ECM protein tends to be randomly folded upon adsorption
to the biomaterial surface such that the receptor binding
domains are not always sterically available. However,
theshortpeptidesequencesarerelativelymorestable
during the modification process than long chain proteins
such that nearly all peptides modified with spacers are
available for cell binding. In addition, short peptide
sequences can be massively synthesized in laboratories
more economically. The biomimetic material modified
with these bioactive molecules can be used as a tissue en-
gineering scaffold that potentially serves as artificial ECM
providing suitable biological cues to guide new tissue
formation.
The most commonly used peptide for surface modifi-
cation is RGD. Additionally, other peptide sequences
such as Tyr-Ile-Gly-Ser-Arg (YIGSR), Arg-Glu-Asp-Val
(REDV), and Ile-Lys-Val-Ala-Val (IKVAV) have been im-
mobilized on various model substrates. In order to pro-
vide stable linking, RGD peptides should be covalently
attached to the polymer via functional groups like hy-
droxyl, amino, or carboxyl groups. Simple adsorption of
small RGD peptides only leads to poor cell attachment.
When a polymer does not have functional groups on its
surface, these have to be introduced by blending, co-
polymerization, or chemical or physical treatments. Dif-
ferent coupling techniques have been employed to ensure
covalent binding of the peptides to the surface of the
materials. In most cases, peptides are linked to polymers
by reacting an activated surface carboxylic acid groupwith
the nucleophilic N-terminus of the peptide, as shown in
Fig. 7.2-17 a [11] . First, the surface carboxyl group is
converted to an active ester, that is less prone to hydro-
lysis, e.g., N -hydroxysuccinimide (NHS) ester, and,
second, this is coupled to the peptide in water. Polymers
that contain surface amino groups can be treated with
succinic anhydride to generate surface carboxyl groups,
which can be reacted with RGD peptides as described
above. Amino groups can directly be converted into
preactivated carboxyl groups by using an excess of bisac-
tivated moieties like N , N 0 -disuccinimidyl carbonate
(DSC), as shown in Fig. 7.2-17 b. Surface containing hy-
droxyl groups can simply be preactivated with, for in-
stance, tresyl chloride ( Fig. 7.2-17 c) or p -nitrophenyl
chlorocarbonate ( Fig. 7.2-17 d).
A bifunctional crosslinker that has a long spacer arm
can be used for the immobilization of peptides to the
surface, which can enable the immobilized peptides to
move flexibly in the biological environment. For polymer
substrates lacking appropriate functional groups for