Surfaces having desirable cell adhesive effects

An absorbed protein-independent cell-adhesive surface is disclosed. The treated surface comprises a chemically derivatized material to which small peptides, having less than 12 amino acid residues and including YIGSR, RGD or REDV amino acid sequence are covalently linked to. The peptides of the present invention include a terminal glycine amino acid. Tresyl chloride activation of surface hydroxyl moieties provides the active surface sites by which a terminal glycine arm of a selected peptide attaches to form covalent bonds between the substrate and peptide. Peptides high with cell adhesive properties are bound in high efficiency. By way of example, surfaces which may be used in conjunction with the present invention include polymer, metal, and ceramic surfaces. The most preferred polymer surfaces include PHEMA and PET polymer surfaces, with the most preferred glass surfaces being glycophase glass. The present methods also include a pretreatment method which provides hydroxyl moieties to surfaces devoid of readily available hydroxyl moieties. The pretreatment, by way of example, comprises immersion of the surface in a mixture of formaldehyde and acetic acid. Methods of preparing the treated surfaces are also included in the present invention. Also included are surface-treated biomedical implant devices and cell culturing devices. The treated surface promotes an enhanced rate and an enhanced amount of cell adhesion to the surface, independent of media serum concentrations or other absorbed proteins. The treated surfaces of the present invention are thermally stable, reusable, peptide efficient (attached to surface only) and resistant to cell proteolysis. The invention further concerns polymeric substrates with a surface having physically interpenetrating water-soluble polymer chains, and methods for production thereof.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The field of the present invention relates generally to the biospecific 
adhesion of cells to a surface. More specifically, the invention relates 
to the chemical modification of a surface and the covalent attachment 
thereto of small peptides to promote cell adhesion. Even more 
particularly, the surface may comprise a ceramic, a metal or a polymer. 
The small peptides include a minimal cell receptor recognition amino acid 
sequence which promotes cell adhesion and is common to a variety of cell 
adhesion molecules. The surfaces and methods of the present invention thus 
relate to cell adhesion techniques which are independent of culture media 
serum composition and adsorbed surface proteins. 
Moreover, the field of the present invention relates to the modification of 
polymeric materials by a solution processing technique to render the 
surfaces extremely nonadhesive to cells. Such surfaces have important 
applications in biomedicine and biotechnology. Furthermore, the field 
relates to the attachment of cell adhesion peptides to these nonadhesive 
surfaces to obtain surfaces that are cell adhesive and have the particular 
advantage of being specifically adhesive for certain cell types but not 
for other cell types. 
2. Description of the Related Art 
The interaction of cells with extracellular matrix in vivo is involved in a 
number of important biological processes, such as the regulation of 
cellular growth, migration, and differentiation. The role of eukaryotic 
cell adhesion in culture largely dictates the success of a particular cell 
culture effort or endeavor. Adhesion, spreading, and contraction on solid 
substances are prerequisites for growth of normal anchorage dependent 
cells in vitro (1, 2). This cellular bioadhesion is affected by several 
factors, including the particular type of cell, the cell culture media 
used, and the particular surface upon which the cells are cultured. 
Many mammalian cells are cultured on polymer surfaces. Nearly all mammalian 
cell adhesion to synthetic polymer surfaces is controlled by adsorbed 
proteins and is receptor mediated. Fibronectin (FN), was the first cell 
adhesion molecule (CAM) that was shown to be involved in the adhesion of 
some avian and mammalian cell types to extracellular substrates (3, 4). FN 
is commonly provided to the in vitro environment through the addition of 
serum, in a form known as cold-insoluble globulin (CIg). For normal 
attachment and spreading of cells to occur, it was found that CIg had to 
be adsorbed to the culture surface (5, 6). 
FN consists of several protease-resistant domains, each of which contain 
specific binding sites for other extra cellular molecules and for the cell 
surface (7). The cell attachment activity has been localized to a 
tripeptide sequence (RGD), located in the cell binding domain of FN as 
well as in several other CAMs (8). Substrate-bound, RGD-containing 
peptide, directly adsorbed to the substrate, or peptide cross-linked to 
adsorbed-albumin or IgG, was found to promote fibroblast attachment and 
spreading. This attachment and spreading activity was found to be readily 
inhibited by the addition of soluble RGD-containing peptides to the medium 
(8). 
Affinity chromatography of cellular extracts on cell attachment-promoting 
FN fragments combined with specific elution utilizing synthetic 
RGD-containing peptides yielded a receptor with two 140 kD subunits (9). 
The mammalian FN receptor and other RGD directed receptors are typically 
heterodimers of two subunits, alpha and beta (10). Families of these 
receptors consist of members with similar beta subunits, whereas the alpha 
subunits are more distinct and restrict the receptor's affinity to one or 
a few CAMs (11). Collectively, these structurally and functionally related 
receptor families are known as the integrin superfamily (12, 13). 
A basic understanding of the molecular mechanisms underlying the process of 
cell adhesion has thus developed regarding the role of the cell culture 
substrates and other surfaces in promoting cell adhesion. Basically, after 
a protein solution is placed on a culture substrate, proteins are 
immediately adsorbed to the surface. If there are receptors for some of 
these adsorbed proteins on the cell surface, and if the conformation of 
the adsorbed protein is not so extensively altered by adsorption as to 
destroy the high ligand-receptor affinity, then cell adhesion to the 
culture substrate and cell spreading can result. 
If the cells are seeded on a substrate in the absence of adsorbed proteins, 
then the proteins on the cell surface may directly adsorb to the surface 
and the cell will, provided favorable conditions, secrete its own proteins 
toward the surface in the form of an extracellular matrix. However, if the 
substrate does not support protein adsorption, or if it supports high 
affinity adsorption of a protein for which there is not a cell-surface 
receptor, then the substrate will not support cell adhesion. In no case 
has the cell in culture been found to actually touch the surface except 
through these intermediate adsorbed proteins. 
Some investigators who study short-term cell adhesion have proposed the use 
of substrate treating systems which promote cell adhesion wherein 
particular peptides are adsorbed to a polymer surface. For example, 
Singer, et al. proposed the adsorption of a 13-mer peptide containing the 
RGD sequence onto a polymer substrate to promote cell adhesion (14). 
However, peptides of this length have been found to be highly susceptible 
to degradation at high temperatures and to the proteolytic action of the 
cultured cells themselves. Additionally, peptides adsorbed to a surface 
are subject to desorption upon repeated use. Thus, surfaces with long 
amino acid residue peptides absorbed thereto have been found to be 
unstable and thus unsuitable in preparing reusable cell culture 
substrates. 
An alternative approach in promoting cell adhesion is through chemical 
modification of the surface to facilitate the adsorption and attachment of 
protein and peptides to the substrate. However, present technology for 
chemical modification of substrates is particularly non-specific and 
empirical. For example, treatment of polymer surfaces with various radio 
frequency plasma discharges, both polymerizing and nonpolymerizing, has 
been proposed. Alternative approaches of surface acid treatment or surface 
incorporation of charged groups have also been described. However, these 
various surface treatments alter only the pattern of protein adsorption on 
the culture surface, which in turn functions to modify the cells' 
characteristic adhesion and spreading behavior. Thus, the protein and 
peptide surface adsorption and desorption problems still remain, limiting 
the reusability of culture plates and other surfaces so treated. 
An alternative to surface adsorption of peptides to promote cell adhesion 
has been to instead chemically attach peptides to a surface. For example, 
the method of polymer surface chemical modification was employed by 
Brandley, et al., (1988) (Analyt. Biochem. 172: 270), who proposed the 
inclusion of a 9-mer peptide in a polymer substrate to promote cell 
adhesion (Id.). While enhanced cell adhesion was attained using the 
Brandley technique, the method required similar concentrations of peptide 
to promote the same level of cell adhesion observed in the adsorbed 
peptide systems. For example, FIG. 1 of Brandley shows surface 
concentrations of peptide on the average of about 6 nanomoles per square 
centimeter (Brandley, at pg. 275). These high peptide concentrations 
suggest the Brandley method does not control for the inclusion of peptide 
at the polymer surface only, but instead permits the incorporation of 
peptide throughout the bulk of the polymer. Given that synthetic peptides 
cost about $5,000 per gram, this method would not facilitate the 
economical preparation of cell culture substrates commercially. 
Thus, a need still exists in the art for an economical system of preparing 
thermally stable, peptide-coated surfaces with cell adhesion promoting 
characteristics which are resistant to the desorptive effects of repeated 
usage and proteolysis by cellular proteases or proteases added to remove 
cells. A more commercially feasible and economical system would be 
substantially more peptide-efficient than those proposed by Brandley and 
others of skill in the art of cell culture and polymer chemistry. 
Currently used biomedical polymers in applications involving blood contact 
have not proved to be sufficiently nonthrombogenic to be useful in small 
diameter vascular grafts. Adhesion of platelets and other blood cells is 
the main cause of low patency of small diameter grafts, and an aspect of 
the present embodiment is to reduce the interactions of blood components 
with biomedical polymers. Because the adhesion of platelets, white blood 
cells, fibroblasts, etc. is mediated by the adsorption of proteins to the 
polymer surface, an approach was adopted which reduced the interaction of 
proteins with these polymers. 
Polyethylene oxide (PEO) surfaces have been observed to resist the 
adsorption of plasma proteins as a result of their strong hydrophilicity, 
chain mobility and lack of ionic charge. Several groups have used PEO or 
PEG (polyethylene glycol) as a modifier in a quest to obtain a 
biocompatible or nonadhesive surface. Different approaches have been used 
to modify polymer surfaces with PEO. Among them are those techniques that 
involve covalent grafting of PEO to a base polymer such a PET, a 
polyurethane, or polyvinyl alcohol, polymerization of a monomer having a 
pendant PEO chain, incorporation of PEO into a base polymer by block 
copolymerization, or direct adsorption of PEO-containing surfactants which 
are typically block copolymers of the AB or ABA type where one of the 
blocks is a PEO. Most of these techniques have utilized PEO of relatively 
low molecular weights (less than 5000 daltons) and only a few have used 
significantly higher molecular weights. 
Although some of the above described techniques work reasonably well in 
reducing cellular interactions at the surfaces of the modified polymers, 
most of them require multiple stages to obtain the necessary surface 
modification. Furthermore, they are limited by the structure and 
availability of labile chemical moieties on the base polymer surface and 
are in many cases, specific for modification of the base polymers. 
The present invention relates to a technique incorporating PEO and other 
water-soluble polymers (WSP) into the surface of a base polymer (BP). 
The following abbreviations are used by Applicants throughout the 
application: 
A=Ala (alanine) 
C=Cys (cysteine) 
D=Asp (aspattic acid) 
E=Glu (glutamic acid) 
F=Phe (phenylalanine) 
G=Gly (glycine) 
I=Ile (isoleucine) 
K=Lys (lysine) 
P=Pro (proline) 
R=Arg (arginine) 
S=Set (serine) 
V=Val (valine) 
Y=Tyr (tyrosine) 
BP=base polymer 
CAM=Cellular adhesion molecule 
CFN=cellular fibronectins 
Cig=cold-insoluble globulin 
DIFW=deionized and filtered water 
FC=focal contacts 
FEP=fluorinated ethylene polymers 
fg=fibrinogen 
FN=fibronectin 
GREDV=glycine, arginine, glutamic acid, aspartic acid, valine or 
Gly-Arg-Glu-Asp-Val 
GRGD=amino acid sequence glycine, arginine, glycine, aspartic acid; or 
Gly-Arg-Gly-Asp 
HFF=human foreskin fibroblast cells 
HVSMC=human vascular smooth muscle cells 
kD=kilodalton 
mer=amino acid residue 
nm=nanomolar 
PAE=porcine aortic endothelial (cells) 
PBS=phosphate buffered saline 
PDMS=poly(dimethyl siloxane) 
PEG=polyethylene glycol 
PELL=polyurethane (pellethane) 
PEO=polyethylene oxide 
PEOX=polyethyloxazoline 
PET=polyethylene terephthalate 
PFN=plasma fibronectins 
PHEMA=poly(hydroxyethyl methacrylate) 
PIPN=physical interpenetrating network 
Plt=human blood platelets 
PMMA=polymethylmethacrylate 
Prestim Plt=human blood platelets prestimulated with 5 .mu.m adenosine 
diphosphate 
PTFE=poly(tetrafluoroethylene) 
PVP=polyvinylpyrrolidone 
REDV=arginine, glutamic acid, aspartic acid, valine or Arg-Glu-Asp-Val 
RGD=amino acid sequence arginine, glycine, aspartic acid, or Arg-Gly-Asp 
SAM=surface (or substrate) adhesion molecule 
TFAA=trifluoroacetic acid 
THF=tetrahydrofuran 
.mu.g=micrograms 
.mu.l=microliter 
WSP water soluble polymer 
YIGSR=tyrosine, isoleucine, glycine serine, arginine, or 
Tyr-Ile-Gly-Ser-Arg 
SUMMARY OF THE INVENTION 
The present invention features a new process by which surfaces may be 
modified to yield proteolytically stable, reusable surfaces which promote 
the amount of and enhance the rate of receptor mediated cell adhesion. The 
specifically directed and controlled chemical processes herein disclosed 
provide for the chemical attachment of peptides at the surface of a flask 
or other device without diffusion of the peptide throughout the bulk of 
the material treated. Thus, the disclosed methods provide a surprisingly 
enhanced cell adhesion promoting surface with the use of only a fraction 
of the peptide required by formerly proposed methods. The peptides are 
chemically attached to a surface, and thus avoid the desorption problems 
which plagued surface peptide-adsorbed systems of the past. These 
advantages are accomplished through the chemical attachment of small 
peptides, for example, those having less than 12 amino acid residues, at 
only the surface of the substrate. 
An additional feature of the present invention lies in that the process 
provides for the use of cell adhesion molecule fragments, rather than 
whole surface adhesion molecule (SAM) proteins. The acronyms SAM and CAM 
are used interchangeably to denote surface, substrate or cell adhesion 
molecules that are proteins which interact with extracellular matrix 
components through a specific binding domain to promote specific 
domain-mediated adhesion of cell receptors (i.e., the cells). SAMs are a 
family of proteins that include fibronectin, vibronectin, thrombospondin, 
laminin, and other proteins. Grafting of small peptide fragments provides 
a further advantage in that surfaces so treated are less subject to 
denaturation and proteolytic degradation than surfaces grafted with whole 
proteins or larger peptides. 
A further feature of the present invention is that it provides a more 
efficient surface modification system. For example, Applicants are able to 
produce a derivatized surface with maximal cell adhesion properties using 
only 1/500th the amount of peptide of formally proposed chemical grafting 
methods. For example, Brandley, et al. employed a method which yielded on 
the average of 6 nanomoles/cm.sup.2 (6,000 picomoles/cm.sup.2) peptide 
surface concentration (Brandley, et al., pg. 274, Table 1). In contrast, 
Applicants have demonstrated enhancement of cell adhesion with a surface 
peptide concentration of as little as 0.001 nanomoles/cm.sup.2 (a six 
million fold improvement). Applicants were able to omit a significant 
amount of the surface peptide in the process of chemically binding a 
peptide to a surface, and were able to achieve such without a loss in cell 
adhesive promoting activity. 
An additional feature of the disclosed surface modification techniques is 
that they provide a novel approach for promoting receptor-mediated cell 
attachment independent of adsorbed serum components. The present invention 
provides a method for the treatment of surfaces which is effective in 
promoting the adhesion of any species and type of cell, including for 
example, porcine, murine and human cells. Types of cells which have 
already been successfully cultured on the treated surfaces include aortic, 
foreskin fibroblast and 3T3 fibroblast cells (ATCC # CRL1658) and vascular 
endothelial cells. 
The present invention also features adaptability of use with a wide variety 
of small peptides. While numerous small peptides may be used in 
conjunction with the present invention, the most preferred small peptides 
include those with less than 12 amino acid residues (12 mer). More 
preferably, these peptides contain 3-9 amino acid residues (3-9 mer). The 
most preferred peptides have either 6 amino acid residues (6 mer) or 4 
amino acid residues (4 mer). The number of amino acid residues in a 
peptide are often denoted herein by such nomenclature (e.g., 6 mer, 4 mer, 
etc.). 
These small peptides are further described as including a minimal 
cell-surface receptor recognition sequence, for example, RGD, YIGSR, or 
REDV. This sequence permits the cell receptor mediated support of cells to 
a treated surface. By way of example, the most preferred peptides which 
include the about minimal cell surface receptor recognition sequences 
include the GRGD (Gly-Arg-Gly-Asp), GYIGSRY (Gly-Tyr-Ile-Gly-Ser-Arg-Tyr), 
GYIGSR (Gly-Tyr-Ile-Gly-Ser-Arg), GRGDY (Gly-Arg-Gly-Asp-Tyr), YIGSR 
(Tyr-Ile-Gly-Ser-Arg), RGD (Gly-Arg-Asp), REDV (Arg-Glu-Asp-Val), GREDV 
(Gly-Arg-Glu-Asp,Val), GREDVY (Gly-Arg-Glu-Asp-Val-Tyr), RGDS 
(Arg-Gly-Asp-Ser), GRGDS (Gly-Arg-Gly-Asp-Ser), RGDF (Arg-Gly-Asp-Phe), 
GRGDF (Gly-Arg-Gly-Asp-Phe), PDSGR (Pro-Asp-Ser-Gly-Arg), GPDSGR 
(Gly-Pro-Asp-Ser-Gly-Arg), GPDSGRY (Gly-Pro-Asp-Ser-Gly-Arg-Tyr), IKVAVC 
(Ile-Lys-Val-Ala-Val-Cys), GIKVAV (Gly-Ile-Lys-Val-Ala-Val), IKVAVY 
(Ile-Lys-Val-Ala-Val-Tyr), GIKVAVY (Gly-Ile-Lys-Val-Ala-Val-Tyr) amino 
acid sequences. These fragments contain either the cell attachment 
sequence of many surface adhesion molecules (RGD) or one of the cell 
attachment sequences of laminin (YIGSR and PDSGR), a particular surface 
adhesion protein, or the cell adhesion molecule fibronectin (REDV). The 
IKVAV peptide from laminin is also useful for particular cells. These most 
preferred peptides may further include a C-terminal Y for radioiodination. 
The N-terminal G is used as a spacer with the particular peptide between 
the adhesive peptide and the surface. The small peptides are used to 
provide cell receptor recognition sites required for cell adhesion on the 
treated surface. 
While a surface concentration of peptides of at least 0.001 
picomole/cm.sup.2 is sufficient to enhance the cell adhesive 
characteristics of a surface, a preferred range of peptide surface 
concentration is between about 0.001 to 100 picomoles/cm.sup.2. A more 
preferred range of peptide surface concentration is 0.5 to 20 
picomoles/cm.sup.2. The most preferred peptide surface concentration of 
the present invention is about 12 picomoles/cm.sup.2. 
Conventional methodologies rely upon direct adsorption of the CAM or the 
peptide to the surface, or adsorption of non-CAM proteins followed by 
cross-linking of the peptides to the adsorbed proteins, thus allowing for 
desorption of these components into the culture media. The present 
invention provides the further advantage of avoiding this problem of 
protein desorption by chemically bonding the peptide through covalent 
bonds to hydroxyl or other reactive moieties of the desired substrate. 
There are many situations in the use of biomedical implants where it is 
desirable that the surrounding cells in the tissues adhere to and spread 
upon (integrate with) the implant surface. The present invention provides 
a method for surface modification to obtain such desired implant 
integration within the host. The present invention further features a 
method for reducing the incidence of infection attendant to the in vivo 
implant of biomedical devices. A major risk associated with implantation 
of biomedical devices has been infection. The lack of a continuous 
protective layer between the device and the biological tissue opportunizes 
the entry of bacteria and other infectious agents into the tissue. With 
the enhanced cell adhesion promoting surface as part of the device, such 
undesirous side effects will be minimized, as a continuous protective cell 
covering is provided, closing the potential entry of infectious agents. 
Furthermore, there are many situations in the use of biomedical implants 
that only specific cells from the surrounding tissues attach to the 
implant surface. For example, in vascular graft technology, it is 
desirable that endothelial cells attach to the implant so that it will 
look like the natural blood vessel wall, which is lined with endothelial 
cells. However, platelets also attach and lead to clotting, and 
fibroblasts and smooth muscle cells also infiltrate from the tissues, 
leading to a very detrimental thickening of the tissue layers within the 
vascular graft. Thus, it would be advantageous to utilize a material to 
which platelets do not attach and to which only endothelial cells attach. 
The present invention provides, as a surprising advantage over adsorbing 
peptides to surfaces, this feature of cell-type specificity. 
Additionally, the long-term stability of the disclosed treated surface 
method makes the system ideal in preparing various biomedical implants for 
extended term body emplacement. For example, in use with nerve growth 
guides and indwelling catheters. The surface modification system of the 
present invention also provides new avenues for mammalian cell bioreactor 
design, since it provides a stable integral surface component which 
supports cell adhesion independent of media CAMs. Such substrates provide 
the further advantage of permitting the use of serum-free media which are 
deficient in cell adhesion molecules. 
The chemistry of the present invention is directly applicable to any 
material with surface hydroxyl moieties or other surface reactive groups 
to which such moieties can be added. Most other surface treatments to 
enhance cell adhesion do so by the enhancement of protein absorption. The 
peptide grafting approach of the present invention eliminates the 
requirement for absorbed proteins completely, as the cell has receptors 
for the surface-coupled synthetic peptides. These covalently bound minimal 
sequences are much more stable to cellular proteolysis and thermal 
degradation than adsorbed cell adhesion proteins or adsorbed proteins 
conjugated with adhesion peptides, since desorption is eliminated and the 
active groups (e.g. RGD, YIGSR, PDSGR, IKVAV or REDV sequence) are not as 
exposed to soluble proteases. 
While a variety of chemical methods exist by which the present surfaces may 
be prepared, the various approaches fall into the general class of surface 
activation, via modification of a nucleophile, such as an amine or 
hydroxyl, followed by coupling to the peptides, via another nucleophile 
such as an amine or hydroxyl or thiol (23). By way of example, the 
activation of surface hydroxyl groups may be accomplished through 
treatment with agents such as tresyl chloride, glutaraldehyde, cyanuric 
chloride, sulfonyl chlorides, cyanogen bromide; surface hydroxyls may be 
added via benzoin with potassium tert-butoxide in dimethyl sulfoxide. 
Treatment with these particular surface activators is followed by a 
procedure by which a peptide is covalently linked to the hydroxyl group. 
Additionally, the present invention may be practiced through the 
production of active carboxyl groups on the surface by using, for example, 
succinic anhydride. The exposed surface is then rinsed and coupled with 
peptide. 
By way of example, biomedical implants which would benefit from the 
inclusion of the present surface peptide treatment include penile, heart, 
vaginal, and hip implants; catheters; artificial veins or arteries, 
artificial tendons and, ligaments; artificial bone screws, bone plates, 
bone fragments and bone joints; artificial skin; nerve growth guides; and 
intraocular lenses and the like. By way of example, materials used as cell 
and tissue culture substrates which profit from the present surface 
peptide treatment include tissue culture flasks, petri dishes, 
microcarrier beads, porous macrocarriers, fibers, hollow fibers, monolith 
supports, and roller bottles. 
The disclosed methods may be used in the derivatization of any surface to 
which enhanced cell adhesion is desired. By way of example and not 
limitation, these surfaces include metal, ceramic or polymer surfaces. A 
preferred embodiment of the invention is directed to the derivatization of 
polymer surfaces. While any polymer surface may be derivatized using the 
proposed methods, particular exemplary polymer surfaces most preferably 
include poly(hydroxyethyl methacrylate) (PHEMA), poly(ethylene 
terephthalate) (PET), poly(tetrafluoroethylene) (PTFE), fluorinated 
ethylene (FEP), poly(dimethyl siloxane) (PDMS) and other silicone rubber 
surfaces. PET, otherwise known as Dacron, is a polyester frequently used 
for biomedical implants. PTFE is otherwise known as Teflon. Most preferred 
polymeric matrix of the present invention comprises poly(hydroxyethyl 
methacrylate) (PHEMA). 
The PHEMA polymeric matrix comprises a gel-like matrix having about a 45% 
water composition, and was, prior to the disclosure of the present 
invention, unable to support cell adhesion. 
Use of peptides in conjunction with other high-water polyacrylamide gel 
matrixes is much less peptide-efficient compared to such use with polymers 
of lower water content. Highly hydrated gels are highly permeable to 
peptides and thus facilitates the substantial and indiscriminant diffusion 
of small peptides into the bulk of the polymer. These highly hydrated 
polymers have been used for protein electrophoresis, demonstrating that 
they are even permeable to whole proteins, which are very large molecules 
(21). Polymer gels comprising polyacrylamide usually include at least 
about 90% water, and, thus would be unsuitable in the practice of the 
present invention. 
Another preferred embodiment of the invention is directed to the 
derivatization of a particular ceramic, glycophase glass (glycerol 
propylsilane bonded glass). By way of example, a preferred metal to be 
used with the described process is titanium. 
The present invention also includes methods of enhancing cell adhesion to a 
surface comprising first activating the surface, coupling a peptide to the 
activated surface, and then plating mammalian cells on the peptide 
derivatized surface, wherein the preferred peptide is smaller than a 12 
met. The process whereby the peptide is coupled to any activated surface 
most preferably comprises exposing an activated surface to a solution 
containing a sufficient amount of the peptides described above (having 
cell-adhesive characteristics). While any concentration of peptide 
solution of at least about 10 ng/ml would produce equal results in the 
present coupling process, solutions between about 10 ng/ml peptide and 100 
ug/ml peptide are also suitable. Most preferably, the process includes a 
peptide solution having a concentration of 10 ng/ml peptide. In one 
preferred embodiment of the invention, the peptide of the peptide solution 
includes the amino acid sequence arginine-glycine-asparagine (RGD). In 
another preferred embodiment of the invention, the peptide of the peptide 
solution includes YIGSR. In still another preferred embodiment of the 
invention, the peptide of the peptide solution includes PDSGR, IKVAV, REDV 
or RGDF. A most preferred peptide to be used in the method of the present 
invention comprises a peptide sequence selected from the group consisting 
of GRGD, RGDY, GRGDY, GYIGSR, GYIGSRY, YIGSR, RGDS, REDV, GREDV, GREDVY, 
RGDF, GRGDF, PDSGR, GPDSGR, GPDSGRY, IKVAV, GIKVAV, IKVAVY, and GIKVAVY. 
These peptides are most preferably used in the derivatization of polymer 
surfaces, such as PET polymer and PHEMA polymer, or glass surfaces, such 
as glycophase glass. However, any peptide which includes an amino acid 
sequence capable of supporting cell receptor recognition may be used in 
conjunction with the present invention. 
Applications of the bioactive cell adhesive peptide grafting approach to 
enhance cell adhesion include the following uses: 
1. For laboratory scale tissue and cell culture of anchorage dependent 
cells and cell lines This approach may be useful in the treatment of 
laboratory glassware and plasticware used as cell culture substrates, such 
as tissue-culture flasks and Petri dishes. It would be useful for animal, 
insect, and plant cells and tissues, as all utilize essentially the same 
molecular biology for adhesion. 
2. For large scale tissue and cell culture. The approach may be useful in 
the treatment of microcarriers, porous macrocarriers, hollow fibers, 
monolith supports, and roller bottles. 
3. For the interior of implantable artificial vascular grafts to promote 
the endothelialization of these surfaces. 
4. For the exterior and anastamotic regions (ends) of vascular grafts to 
promote integration into the tissues. 
5. For other implantable devices where integration with the tissues is 
desirable, such as artificial tendons, ligaments, bone screws and plates, 
bone fragments, joints, and skin. 
6. For the treatment of sutures to promote adhesion with and integration to 
the tissues. 
7. For the promotion of directional growth or migration of cells or 
tissues, this approach may be useful when the peptides are grafted to the 
surface with a gradient of surface concentration. An example where this 
may be useful is in nerve growth guides for peripheral nerve regeneration. 
8. For use in research. The present systems allow for the study of cell 
adhesion in the presence of serum without the confusion of the effects of 
protein adsorption. Thus, background levels in the test system remain low. 
Additionally, the present methods control for the amount of peptide which 
gets coupled to a surface, which is also important in studying cell 
adhesion. 
Surfaces modified to resist cell adhesion prepared according to the present 
invention may be useful for: 
1. Situations in biomedicine where cell attachment is detrimental, such as 
catheters, hemodialysis membranes, blood filters, intraoccular lenses, 
contact lenses, and 
2. Situations in biotechnology where protein adsorption is detrimental, 
such as chromatography support columns.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention is related to surfaces having unique self-adhesive 
properties and methods of preparing treated surfaces which specifically 
enhance cell-surface adhesion thereto independent of separately adsorbed 
peptides and soluble media components. This methodology provides 
techniques for preparing various types of substrates which simplify 
optimization of culture systems and which also control the amount and rate 
of cell bioadhesion to surfaces of various materials. Moreover, materials 
having particular cell nonadhesive properties and methods for preparing 
such surfaces to reduce cell adhesion and protein adsorption are 
disclosed. These materials have important utility as nonadhesive surfaces 
and have particular advantages when modified with adhesion-promoting 
peptides. 
More particularly, the surface modification techniques provide for the 
chemical grafting of peptides to a surface, the peptide comprising at 
least the minimal amino acid sequence included in a cell (surface) 
adhesion molecule, such as RGD in fibronectin, or YIGSR, IKVAV or PDSGR in 
laminin, REDV in some forms of fibronectin, or some other short peptide 
sequence related to or derived from the sequence of a protein involved in 
cell-surface or cell-cell adhesion. The terms "Cell Adhesion Molecule" 
(CAM) and "Substrate Adhesion Molecule" (SAM) are used interchangeably in 
describing the family of proteins and peptides found to facilitate cell 
receptor-mediated adhesion or attachment to a surface. 
The present surface treatment methods comprise chemically activating a 
surface to expose reactive groups, rinsing the actuated surface, and then 
exposing the surface to a solution containing small peptides, for example, 
a peptide which includes the RGD, PDSGR, IKVAV, YIGSR, REDV, or other amino 
acid sequence which facilitates receptor mediated cell attachment having 
less than a total of 12 amino acid residues (12-mer). A covalent chemical 
bond attaches the terminal end of the peptide to a reactive moiety on the 
surface being treated. A variety of chemical methods exist by which a 
surface may be activated to expose reactive groups. One of these methods 
is by way of the tresyl activation. Other alternative chemistries which 
may be used in the practice of the present invention, by way of example 
include: 
(1) Activation of the surface with glutaraldehyde: At temperatures between 
0.degree. C. and 80.degree. C., treat the surface with an aqueous solution 
of 5% to 37% glutaraldehyde for about one hour. The glutaraldehyde will 
couple with any exposed nucleophilic groups, such as amines and hydroxyls. 
Rinse the surface with water, and treat the surface with a solution of 
peptide, between 10 ng/ml and 1 mg/ml. Nucleophilic groups on the peptide, 
such as thiols, amines, and hydroxyls, will be coupled to the 
surface-coupled glutaraldehyde function. 
(2) Activation of the surface with cyanuric chloride: At temperatures 
between 0.degree. C. and 80.degree. C., treat the surface with a 
nonaqueous solution of about 5% cyanuric chloride for about one hour. The 
cyanuric chloride will couple with any exposed nucleophilic groups, such 
as amines and hydroxyls. Rinse the surface with dry solvent, and treat the 
surface with a solution of peptide, between 10 ng/ml and 1 mg/ml in a 
nonaqueous solvent, such as acetonitrile. Any nucleophilic groups on the 
peptide, such as thiols, amines, and hydroxyls, will be coupled to the 
surface-coupled cyanuric chloride function. 
(3) Activation of the surface with other sulfonyl chlorides: Follow the 
procedure outlines in Example 3 using another member of the sulfonyl 
chloride family, such as tosyl chloride. 
(4) Activation of the surface with cyanogen bromide: At temperatures about 
20.degree. C. or below, expose the surface to an aqueous solution of 
cyanogen bromide at pH 10-11 for about one hour. The cyanogen bromide will 
covalently couple and activate surface hydroxyl groups. Rinse the surface 
with water at pH 10-11, and couple with peptide between 10 ng/ml and 1 
mg/ml in this same solution for about 1 hour. The peptide will couple to 
the cyanogen bromide groups via any amine functions on the peptide. 
(5) Activation of the surface to produce active carbonyl-bearing esters, 
e.g. with succinic anhydride: At temperatures of about 20.degree. C., 
expose the surface to a solution of succinic anhydride, whereupon that 
compound will react with surface hydroxyls and amine to produce esters and 
amides, respectively. Rinse the surface and couple with peptide between 10 
ng/ml and 1 mg/ml for about 1 hour. The peptide will react with the 
activated surface via amine or hydroxyl groups. 
(6) Preactivating a polymer by adding hydroxyls with benzoin dimethyl 
sulfoxide: Hydroxyl functions are added to poly(tetrafluoroethylene) 
(PTFE). As described by Costello and McCarthy, Surface-Selective 
Introduction of Specific Functionalities onto Poly(tetrafluoreoethylene), 
Macromolecules 20:2819-2828 (1987)). Benzoin is added to a solution of 
potassium tert-butoxide in dimethyl sulfoxide and placed in contact with 
the PTFE surface. The reaction is allowed to proceed at 50.degree. C. for 
1 hour. The material is removed and rinsed with tetrahydrofuran (THF). 
This intermediate surface is then treated with 1M borane in THF at room 
temperature for 12 hours. The surface is then treated with 1M NaOH 
containing 10% hydrogen peroxide at 0.degree. C. for 3 hours, after which 
it is washed sequentially with dilute NaOH, water, dilute HCl, water, THF, 
and heptane. This produces a surface that is rich in hydroxyls and can 
subsequently be activated by any of the chemistries described above. 
The most preferred method by which peptides are attached to a surface 
comprises surface activation of the surface, by a tresyl immobilization 
method, as described by Nilsson and Mosbach (1981) (Biochem. Biophys. Res. 
Commun., 102: 449-457). Specifically, tresyl activation is a process 
wherein the surface is first immersed in 20 ml. dry ether containing about 
40 .mu.l 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) and about 
2 ml. triethylamine for about 15 minutes at room temperature. These 
activated surfaces were then rinsed with 0.2M sodium bicarbonate pH 10 
buffer. The surfaces were then placed in the same buffer containing about 
60-100 ng/ml of the peptide for about 20 hours at room temperature. Most 
preferably, the concentration of peptide solution is about 10 ng/ml. This 
incubation time allows for the coupling of the peptide to the surface 
hydroxyl groups. This particular embodiment of the invention is preferably 
used with the GRGD peptide. 
The peptides couple to an activated surface most preferably by exposing the 
activated surface to a solution containing an appropriate amount of the 
desired peptide. While any concentration of peptide solution of at least 
about 10 ng/ml would produce equally satisfactory results, solutions 
containing between about 10 ng/ml peptide and 100 ug/ml peptide are 
preferred. Most preferably, a peptide solution of about 10 ng/ml peptide 
is used in the coupling process. 
The treated surfaces produced by the disclosed methods are characterized by 
a peptide surface concentration of at least 0.001 picomole/cm.sup.2. It is 
expected peptide surface concentrations of at least 0.001 
picomole/cm.sup.2 are sufficient to enhance the cell adhesive 
characteristics of a surface. A more preferred range of peptide surface 
concentration is between about 0.5 to 100 picomoles/cm.sup.2. A most 
preferred range of peptide surface concentration is between about 0.5 to 
20 picomoles/cm.sup.2. The most preferred surface peptide concentration is 
about 12 picomoles/cm.sup.2. 
Where no surface hydroxyl moieties exist on the surface to be treated, the 
surface was pretreated. This pretreatment preferably comprised a surface 
hydroxylation procedure wherein an electrophilic aromatic substitution was 
employed to add hydroxyalkyl groups to a surface. A particularly preferred 
method of this pretreatment comprises immersing a surface in an about 
18.5% (v/v) solution of formaldehyde and about 1M acetic acid for about 4 
hours at room temperature. This procedure was used in the 
hydroxymethylation of a particularly preferred embodiment of the invention 
comprising a PET polymer surface. The hydroxylated surface was then tresyl 
activated with the attachment of peptides thereto as described above. Most 
preferably, the pretreatment method disclosed herein is used to prepare 
polymer surfaces, particularly PET polymer surfaces, with GRGD or GYIGSR 
peptides. Various other surface hydroxylations would be equally as useful 
for use in conjunction with other polymers or materials. Various other 
linking characteristics may also be used, where the N-terminal amine or 
another reactive group on the peptide is reacted with a group on the 
surface, perhaps with the use of some linker. 
Any surface may be used in the practice of the present invention. By way of 
example, surfaces particularly suitable for use in the practice of the 
present invention comprise a ceramic, a metal, or a polymer surface. Most 
preferably, the present invention is used in the treatment of polymer 
surfaces and ceramic (glass) surfaces. By way of example, the polymer 
surfaces comprise poly(hydroxyethyl methacrylate) (PHEMA), poly(ethylene 
terephthalate) (PET), poly(tetrafluorethylene) (TPFE), fluorinated 
ethylene (FEP), poly(dimethyl siloxane) (PDMS) and other silicone rubbers. 
While a variety of glass surfaces may be treated with the proposed methods, 
the glass surface most preferred comprises glycerol propylsilane bonded 
glass (glycophase glass). A particularly preferred polymeric surface is 
one with a physical interpenetrating polymeric network of water-soluble 
polymer resisting cell adhesion without peptide substitution. 
A minimal amino acid sequence included in many cell adhesion molecules is 
RGD (arginine-glycine-aspartic acid amino acid sequence), or YIGSR 
(tyrosine-isoleucine-glycine-serine-arginine) or REDV (arginine-glutamic 
acid-aspartic acid-valine). While any peptide containing a minimal amino 
acid sequence active in cell adhesion may be used in the practice of the 
present invention, those sequences most preferred comprise RGD, YIGSR, 
GRGD, GYIGSR, PDSGR, IKVAV, GRGDY, GYIGSRY, RGDY, YIGSRY, REDV, GREDV, 
RGDF and GRGDF. 
In a particularly preferred embodiment of the present invention, the 
peptides GRGDY and GYIGSRY are chemically grafted to a surface of glycerol 
propylsilane bonded glass (glycophase glass) a specific type of ceramic. 
The C-terminal Y of these preferred peptides is included for radio 
iodination, the N-terminal G (glycine) is provided for use as a spacer 
with the particular peptide between the adhesive peptide and the surface. 
The small peptides are used to provide cell receptor recognition sites 
required for cell adhesion on the treated sequence. 
In another particularly preferred embodiment of the present invention, the 
GRGD and GYIGSR peptides are used in the chemical derivatization of 
polymer surfaces. A most preferred embodiment of the present invention 
comprises a PHEMA or PET polymer surface derivatized with GRGD or GYIGSRY 
peptides. Another even more most preferred embodiment of the present 
invention comprises a PHEMA or PET polymer surface derivatized with 
GYIGSRY peptides. 
A surface so treated to include covalently bound peptide provides mammalian 
cell receptor recognition sites which allow the cells to anchor to the 
substrate and grow independent of media serum content and surface adsorbed 
proteins. Thus, the present invention discloses methods which provide for 
receptor-mediated cell adhesion in the absence of any intermediate 
adsorbed protein, i.e., an entirely self sufficient cell adhesion and 
spreading supportive surface. 
While any peptide fragment which includes the minimal amino acid sequence 
of the cell adhesion molecule may be used in the practice of the present 
invention, peptide fragments containing less than 12 amino acid residues 
(mer) are preferred. Peptide fragments having between about 3 and about 9 
amino acid residues are more preferred. The most preferred peptides 
include either 4 or 6 amino acid residues. The length of the peptide 
fragment will affect the susceptibility of the peptide to degradation, and 
therefore, the shorter the fragment, the less peptide surface degradation 
would be expected. 
The present invention further includes surface-treated biomedical implant 
devices and methods for preparing the same. Devices having such surface 
treatments enhance the amount and rate of cell adhesion, and thus the rate 
of tissue integration of the device in vivo. Enhanced cell adhesion and 
tissue integration act to minimize infection, as potential tissue ports of 
entry are "sealed" closed by a protective layer of cells. 
Any device surface to which the described peptides may be chemically 
grafted can be treated with the described methods. By way of example, 
these device surfaces include those of penile, vaginal, heart and hip 
implants or replacements; catheters; artificial skin, veins and arteries; 
artificial tendons and ligaments; artificial bone screws, plates, 
fragments and joints; nerve growth guides; intraocular lenses and the 
like. 
Since polyethylene oxide (PEO) surfaces have been shown to resist 
adsorption of proteins, PEO was incorporated to the surfaces of commonly 
used biomedical polymers such as polyethylene terephthalate (PET), 
pellethane (a commercial polyurethane, PELL) and polymethylmethacrylate 
(PMMA). A novel solution technique was used to incorporate PEO and other 
water-soluble polymers (WSP) such as polyvinylpyrrolidone (PVP) and 
polyethyl oxazoline (PEOX). The presence of PEO, PVP and PEOX on these 
surfaces was verified by using contact angle analysis and ESCA. The 
short-term blood compatibility on the modified polymers was studied in an 
in vitro parallel plate flow system used in conjunction with 
epifluorescence videomicroscopy that could be used to quantify adherent 
platelets on the polymer surfaces. It was found that the PEO modified 
surfaces showed a lower thrombogenicity over the respective control 
surfaces, with the PEO of molecular weight 18500 showing a much lower 
cellular response than the 5000 or the 100000 molecular weight PEO as well 
as the other water soluble polymers. Scanning electron microscopy was 
performed on these surfaces after blood flow and these tests showed 
similar results to the videomicroscopic analysis. Adhesion and spreading 
of human foreskin fibroblasts on the modified surfaces was used to test 
the effectiveness of PEO, PVP and PEOX in preventing cell adhesion. A 
dramatic response was obtained for the PEO 18500 modified surfaces in 
that, over the almost 30 days following the seeding of these cells, an 
extremely low adherence was obtained on the PEO 18500 modified surfaces 
whereas all the others reached confluency within five to ten days. These 
results, along with protein adsorption studies which showed a significant 
reduction in adsorbed protein only on the PEO 18500 modified surfaces, 
suggest that this molecular weight may be suitable in preventing protein 
adsorption and hence cellular interactions at the surface of the modified 
polymer. The PEO 5000 molecule may be too short to perform this function 
effectively and the PEO 100000 may be too long or bulky to be effectively 
incorporated into the base polymer surface. Both PVP and PEOX have amides 
in their repeat unit and this may serve as a potential interaction site 
leading to protein adsorption and cellular attachment. Thus a PEO molecule 
greater than 5000 and smaller than 100000 is usable to preclude protein and 
cellular adhesion to a bare polymer. 
While these cell nonadhesion materials have utility on their own, they are 
also particularly useful when used as a base material to which 
adhesion-promoting peptides are attached. In this situation, proteins do 
not adsorb to the surface, and as such the only adhesion-promoting 
materials on the surface are those which were added chemically in the form 
of the peptide. This allows for cell-type specificity to be built into the 
surface by control of the particular peptide that is incorporated, 
modulation of the linking arm by which the peptide is attached to the 
surface, and modulation of the peptide surface density. Such specificity 
is not possible when the peptide is adsorbed to the surface, as proteins 
would also adsorb, leading to multiple adhesion signals. 
Applicants' evidentiary material presented herein demonstrating the cell 
adhesive properties imparted to a surface treated with disclosed peptides 
and methods of producing and using such treated surfaces. The treated 
surfaces are suitable for the culture of species and type of cell 
including, for example, porcine, murine, insect and human cells. 
Applicants have used a variety of cells in demonstrating the present 
invention, including, by way of example and not limitation, human foreskin 
fibroblast (HFF) cells, porcine aortic endothelial (PAE) cells, embryonic 
and newborn tissue cells. However, the present invention may be used in 
conjunction with any species or tissue source of cell, and is not limited 
to use with any one particular type of cell. The following paragraphs 
outline the certain preferred methods of culturing particular cell types 
used to demonstrate the present invention. 
Cell Culture Procedures 
3T3 CELLS 
NIH/3T3 cells, an immortal cell line of embryonic cells established from 
Swiss mouse embryos, were obtained from the American Type Culture 
Collection cell repository (ATCC # CRL 1658). Cultures were maintained in 
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum 
and incubated at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere. 
Confluent monolayers of cells were harvested by incubation in a solution 
of 0.5% trypsin and 0.53 mM EDTA in phosphate buffered saline (PBS) at 
37.degree. C. for 15 minutes. Cells were resuspended in fresh medium and 
subcultivated by allowing cells to attach and grow to in new culture 
dishes. 
HUMAN FORESKIN FIBROBLASTS 
Human foreskin fibroblasts (HFFs) are primary cell lines that are isolated 
from neonatal foreskin tissue from Seton Hospital, neonatal ward, Austin, 
Texas. About 5-10 newborn foreskins are collected aseptically in sterile 
PBS, minced into 5 mm.sup.2 pieces, and incubated in trypsin-EDTA for 4 
hours. HFFs were collected by centrifugation at 200 g for 5 minutes and 
resuspended in Dulbecco's modification of Eagle's medium (DMEM, Mediatech) 
supplemented with 10% fetal calf serum (GIBCO), 400 u/ml penicillin 
(Irvine) and 400 ug/ml streptomycin (Irvine) and incubated at 37.degree. 
C. in a humidified 5% CO.sub.2 atmosphere. Cells were subcultivated by 
trypsinizing confluent monolayers, suspending cells in fresh medium, and 
seeding cells into new flasks. HFFs were maintained in culture for up to 
20 passages before they are discarded. 
PORCINE AORTIC ENDOTHELIAL CELLS 
Porcine endothelial cells were obtained from sacrificed miniature swine. 
The endothelial cells were isolated by the method of Jaffe, et al. (1973, 
J. Clin. Invest., 52: 2745-2756). Specifically, porcine aorta tissue 
segments from sacrificed miniature swine were rinsed with warm (37.degree. 
C.) PBS to remove blood and placed in 10 cc syringes. The lumen of the 
segments were then filled with a 100 .mu.l/ml solution of Sigma Type II 
collagenase. The tissue was then incubated for 30 min. at 37.degree. C. 
The lumen of the aorta was then washed with PBS, and the cells were 
centrifuged at 200 g for 5 mins. and resuspended in Medium 199 
supplemented with 20% fetal calf serum and antibiotic. The suspension was 
added to culture flasks and the cultures were maintained at 37.degree. C. 
in a humidified, 5% CO.sub.2 atmosphere. 
Human Umbilical Vein Endothelial Cells 
Human umbilical vein endothelial cells (HUVECs) are primary cell lines that 
are isolated from postpartum umbilical cords from Seton Hospital, maternity 
ward, Austin, Tex. by the method of Jaffe et al. (1973, J. Clin. Invest., 
52:2745-2756). Postpartum umbilical cords are clamped in 5-12 inch 
sections, cut from the placental tissue, and collected aseptically in 
sterile PBS. The clamps are removed from the cut cords, clotted blood in 
the umbilical vein is extruded out by applying gentle pressure to the 
cord. Blood is washed from the vein by penetrating one end of the vein 
with a sterile pipet and gently flowing sterile PBS through the lumen of 
the vein. The open end of the vein is then clamped externally with a 
hemostat to seal off the vein and PBS containing 240 U/ml of Type II 
Collagenase (Sigma) was added through the cannula until the vein was 
completely filled with the enzyme solution. The cannula was removed and 
the open end of the vein was clamped so that the cord could be incubated 
with this solution for 30 minutes at 37.degree. C. The hemostats were 
removed after the 30 minute incubation period and the enzyme solution 
containing HUVECs was drained into a sterile centrifugation tube. An 
additional 20 ml of sterile PBS was run through the lumen of the vein and 
collected into the sterile centrifuge tube. The HUVECs in this tube 
collected by centrifugation at 200 g for 5 minutes and resuspended in 
Medium 199 (M199, GIBCO) supplemented with 20% fetal calf serum (GIBCO), 
400 U/ml penicillin, 400 .mu.g/ml streptomycin (GIBCO), 100 .mu.g/ml 
endothelial cell growth supplement (Collaborative Research) and 100 
.mu.g/ml porcine heparin (Sigma) and incubated at 37.degree. C. in a 
humidified 5% CO.sub.2 atmosphere. Cells were subcultivated by 
trypsinizing confluent monolayers, suspending cells in fresh medium, and 
seeding cells into new flasks. HUVECs were maintained in culture for up to 
5 passages before they are discarded. 
Human Vascular Smooth Muscle Cells 
Human vascular smooth muscle cells (HVSMCs) are primary cell lines that are 
isolated from postpartum umbilical cords from Seton Hospital, maternity 
ward, Austin, Tex. by the method of Jaffe et al. (1973, J. Clin. Invest., 
52:2745-2756). Postpartum umbilical cords from which HUVECs were isolated 
were traumatized by repeatedly crushing the cord externally with a 
hemostat. PBS containing 240 U/ml of Type II Collagenase (Sigma) was added 
to the lumen of the vein until the vein was completely filled with the 
enzyme solution. The cord was clamped so that the cord could be incubated 
with this solution for 30 minutes at 37.degree. C. The hemostats were 
removed after the 30 minute incubation period and the enzyme solution 
containing HVSMCs was drained into a sterile centrifugation tube. An 
additional 20 ml of sterile PBS was run through the lumen of the vein and 
collected into the sterile centrifuge tube. The HVSMCs in this tube 
collected by centrifugation at 200 g for 5 minutes and resuspended in 
Medium 199 (M199, GIBCO) supplemented with 20% fetal calf serum (GIBCO), 
400 U/ml penicillin, 400 .mu.g/ml streptomycin (GIBCO), 100 .mu.g/ml 
endothelial cell growth supplement (Collaborative Research) and 100 
.mu.g/ml porcine heparin (Sigma) and incubated at 37.degree. C. in a 
humidified 5% CO.sub.2 atmosphere. Cells were subcultivated by 
trypsinizing confluent monolayers, suspending cells in fresh medium, and 
seeding cells into new flasks. HVSMCs were maintained in culture for up to 
5 passages before they are discarded. 
Human Blood Platelets (Plt) 
Human blood platelets were obtained from heparinized whole blood (4 U/ml) 
which was centrifuged at 250 g for 15 minutes to obtain platelet rich 
plasma (PRP). Blood was collected from healthy male non-smoking donors. 
PRP was removed from the centrifuged blood and used in platelet spreading 
assays. Prestimulated platelets (Prestim. Plt) were treated with 5 .mu.M 
ADP prior to determine platelet spreading. 
The following examples are presented to describe preferred embodiments and 
utilities of the present invention and are not meant to limit the present 
invention unless specifically indicated otherwise in the claims appended 
hereto. 
Example 1 describes the method by which particularly preferred peptide 
fragments were synthesized. 
Example 2 describes the derivatization of a PHEMA polymer surface. 
Example 3 describes the derivatization of a PET polymer surface. 
Example 4 describes the preferred method of preparing a primary cell 
culture from foreskin tissue. 
Example 5 describes the adhesion of cells to a derivatized PHEMA and PET 
polymer surface. 
Examples 6, 7 and 8 describe the derivatization of a glass surface, the 
cell adhesive and supportive characteristics of such a derivatized glass 
surfaces and the comparative cell supportive characteristics provided by 
various peptides to a derivatized verses non-derivatized glass surface. 
Example 9 describes the relative stability of a derivatized PET polymer and 
glycophase glass surface to heat and proteolysis. 
Example 10 presents a comparative study on the cell supportive 
characteristics of a pretreated verses a non-pretreated PET polymer 
surface. 
Examples 11 and 12 demonstrate proposed methods of using the described 
surface derivatization processes with a biomedical implant (Example 
11--indwelling catheter; Example 12--nerve growth guide). 
Example 13 describes HFF cell adhesion and spreading on treated surfaces 
with various levels of peptide substitution. 
Example 14 describes glycophase glass--a model cell nonadhesive substrate 
to which peptides can be coupled. 
Example 15 describes glycophase glass coupled with RGD materials which 
support the adhesion of fibroblasts and endothelial cells but not 
platelets. 
Example 16 describes glycophase glass coupled with YIGSR materials which 
support the adhesion of fibroblasts and endothelial cells but not 
platelets. 
Example 17 describes glycophase glass coupled with PDSGR--materials which 
support the adhesion of fibroblasts and endothelial cells but not 
platelets. 
Example 18 describes glycophase glass coupled with REDV--materials which 
support the adhesion of endothelial cells but not fibroblasts or 
platelets. 
Example 19 describes glycophase glass coupled with IKVAV materials which do 
not support HLFVEC or platelet spreading, but should support neurite 
outgrowth. 
Example 20 describes polyethylene terephthalate to which polyethylene 
glycol has been immobilized--a polymeric cell-nonadhesive substrate to 
which peptides can be attached (abbreviated PET/PEG). 
Example 21 describes PET/PEG coupled with RGD-materials which support the 
adhesion of fibroblasts and endothelial cells but not platelets or 
vascular smooth muscle cells. 
Example 22 describes PET/PEG coupled with REDV-materials which support the 
adhesion of endothelial cells but not fibroblasts or platelets or vascular 
smooth muscle cells. 
Example 23 describes PET/PEG coupled with IKVAV-materials which do not 
support HUVEC or platelet spreading, but supports HFF spreading and should 
support neurite outgrowth. 
Example 24 describes a solution technique to incorporate polyethylene oxide 
and other water soluble polymers into surfaces of polymeric biomaterials. 
EXAMPLE 1--SMALL PEPTIDES 
This example demonstrates particularly preferred methods of synthesizing 
peptides having less than 12 amino acid residues and including at least 
the amino acid sequence arginine-glycine-aspartic acid (RGD). This 
sequence is a particular minimal cell attachment sequence recognizable by 
cells in cell-receptor mediated cell adhesion. 
The peptides used in these studies, including GPDSGRY, GIKVAVY, GREDVY, 
GRGD, GRGE, GYIGSR, GRGDY, GRGEY, and GYIGSRY, were synthesized by known 
procedure at the University of Texas Southwestern Medical School peptide 
synthesis laboratory or obtained from Biosynthesis, Inc., Denton Tex. 
EXAMPLE 2--PREATION OF PHEMA POLYMER SURFACE 
This experiment was designed to describe one particularly preferred method 
of preparing a polymer surface with small peptide fragments covalently 
attached thereto. PHEMA (poly(hydroxyethyl methacrylate)), is a hydrogel 
which has been found unable to support cell adhesion in its untreated 
state. 
The particularly preferred peptides GRGD and GYIGSR were grafted to the 
polymer surface using the PHEMA surface hydroxyl groups and the terminal 
primary amine of the glycine linker arm of the peptide using tresyl 
chloride activation. The G group at the terminal end of each respective 
peptide was used to add distance between the surface-active peptide (GRGD) 
and the polymer surface. 
More particularly, the coupling method utilized activation of PHEMA surface 
hydroxyl moieties by tresyl chloride in an organic solvent for the reaction 
components but a nonsolvent for the polymer. PHEMA films did not require 
pretreatment, since their surfaces are amply supplied with hydroxyethyl 
groups. The cell adhesive promoting activities of this modified surface 
were determined as outlined in Example 4, where in vitro cell adhesion and 
spreading assays were performed. The tresyl leaving group was then 
displaced in an aqueous solvent by the terminal amine of the peptide GRGD 
or GYIGSR. 
The unmodified PHEMA films were then tresyl activated in 20 ml dry ether 
containing 20 .mu.l of 2,2,2-trifluoroethanesulfonyl chloride (tresyl 
chloride) and 2 ml of triethylamine for 15 minutes at room temperature. 
Activated films were then rinsed with 0.2M sodium bicarbonate pH 10 buffer 
and placed in the same buffer containing 80 ng/ml GRGD for 20 hours at room 
temperature to couple the peptide. 
EXAMPLE 3--PREATION OF POLY(ETHYLENE TEREPHTHALATE) (PET) POLYMER 
SURFACE 
This experiment was designed to describe one particularly preferred method 
of preparing a surface devoid of hydroxyl moieties so as to facilitate the 
inclusion of small peptides thereto to promote cell adhesion. 
The PET surface is a polymer which is devoid of hydroxyl moieties, and 
therefore the surface must be pretreated before the surface is thereafter 
modified with a peptide. Specifically, the PET film surface was modified 
via an electrophilic aromatic substitution which added hydroxymethyl 
groups to the surface. The reaction was carried out at room temperature 
and atmospheric pressure. The films were then immersed in 18.5% (v/v) 
formaldehyde and 1M acetic acid for about 4 hours. The pretreated PET 
surface was then modified exactly as described for the PHEMA surface as 
outlined in Example 2. 
The following listing presents the most preferred method of pretreating a 
polymer surface, such as a PET matrix, which does not include hydroxyl 
moieties. The surface modification of hydroxyl moieties and attachment of 
peptides fragments thereto was accomplished with the following protocol: 
1. Addition of surface hydroxymethyl moieties--to the PET polymer surface 
by performing an electrophilic aromatic substitution of surface groups or 
the polymer surface, wherein the PET polymer is immersed in 18.5% (v/v) 
formaldehyde and 1M acetic acid for 4 hours at room temperature. 
2. Tresyl Activation--of surface hydroxyl moieties wherein the pretreated 
PET film is immersed in 20 ml. dry ether containing 40 .mu.l of 
2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) and 2 ml. of 
triethylamine for 15 minutes at room temperature. 
3. Rinse--activated PET film was then rinsed with 0.2M sodium bicarbonate 
pH 10 buffer at room temperature to couple the peptide. 
4. Peptide Coupling to Surface--rinsed PET films were then placed in the 
same buffer containing 80 ng/ml GRGD or GYIGSR peptide for 20 hours. 
EXAMPLE 4--PRIMARY CELL CULTURE FROM HUMAN FORESKIN TISSUE 
Human foreskin fibroblasts (HFFs) are primary cell lines that were isolated 
from neonatal foreskin tissue from Seton Hospital, Neonatal Ward, Austin, 
Tex. The following procedure was used to establish primary cell lines from 
these tissues: 5-10 foreskins were collected aseptically in sterile PBS, 
minced into 5 mm.sup.2 pieces, and incubated in trypsin-EDTA for 4 hours. 
HFFs were collected by centrifugation at 200 g for 5 minutes and 
resuspended in Dulbecco's modification of Eagle's medium supplemented with 
10% fetal calf serum. Cells were subcultivated by trypsinizing confluent 
monolayers, suspending cells in fresh medium, and seeding cells into new 
flasks. HFFs were maintained in culture for up to 20 passages before they 
were discarded. 
EXAMPLE 5--CELL ADHESION STUDIES ON DERIVATIZED PHEMA AND PET POLYMER 
SURFACES 
This experiment was designed to determine if a polymer surface could be 
designed and synthesized which would support receptor-mediated cell 
adhesion in the absence of any intermediate adsorbed proteins, i.e., to 
produce a surface that was entirely self-sufficient in its support of cell 
adhesion and spreading. Immobilization of entire proteins, such as collagen 
or fibronectin, can accomplish this but is associated with the difficulties 
of proteolysis, protein degradation, and protein denaturation. To 
circumvent this, the small, thermally stable peptide region of most CAMs, 
Arg-Gly-Asp (RGD) was covalently coupled to the surface of polymer films 
with a Gly N-terminal linker in the form of Gly-Arg-Gly-Asp (GRGD). This 
produced stable surfaces that were intrinsically bioadhesive, i.e., the 
material surfaces contained groups with a high affinity for cell-surface 
receptors completely independent of adsorbed CAMs from the culture medium. 
This surface modification provided a systematic methodology for developing 
well characterized substrata which simplifies optimization of a culture 
system. Mouse NIH-3T3 (ATCC # CRL 1658) fibroblasts were washed and 
inoculated in serum-free medium on both treated and untreated PHEMA 
substrata. Treated PHEMA substrata was prepared as outlined in Example 2. 
CULTURE METHODS 
NIH/3T3 fibroblasts (ATCC # CRL 1658, Rockville Md.) were cultured in DMEM 
supplemented with 10% calf serum in a humidified 5% carbon dioxide 
atmosphere at 37.degree. C. Porcine aortas were obtained from sacrificed 
miniature swine. Endothelial cells were isolated by the method of Jaffe, 
et al. (1973, J. Clin. Invest., 52: 2745-2756) with a modification to 
facilitate profusion of the lumenal surface of the vessel with 
collagenase. Porcine aortic endothelial cells (PAE) were maintained in 
DMEM supplemented with 10% fetal calf serum with the same incubation 
conditions as above. 
SURFACE MODIFICATION PROCEDURE 
GRGD was grafted on polymer surfaces via the glycyl terminal amine using 
the tresyl chloride immobilization method of Nilsson and Mosbach and as 
described in Examples 3 and 4. Two polymer surfaces were modified, 
poly(hydroxyethyl methacrylate) (abbreviated PHEMA) and poly(ethylene 
terephthalate) (abbreviated PET). The coupling method utilized activation 
of surface hydroxyl moieties by tresyl chloride in an organic solvent for 
the reaction components, which is also a nonsolvent for the polymer. The 
tresyl leaving group was then displaced in aqueous solvent by the terminal 
amine of the peptide. 
Poly(ethylene terephthalate) (PET) has no available hydroxyl groups for 
tresyl chloride activation, therefore a surface hydroxylation procedure 
was developed. An electrophilic aromatic substitution which adds 
hydroxymethyl groups to the PET films was employed (FIG. 1). Specifically, 
the commercially available PET films were immersed in 18.5% (v/v) 
formaldehyde and 1M acetic acid for four hours at room temperature, as 
particularly defined in Example 3. PHEMA films did not require 
pretreatment since their surfaces are amply supplied with hydroxyethyl 
groups. 
The modified PET and unmodified PHEMA films were tresyl activated in 20 ml 
dry ether containing 40 .mu.l of 2,2,2-trifluoroethanesulfonyl chloride 
(tresyl chloride) and 2 ml of triethylamine for 15 minutes at room 
temperature. Activated films were then rinsed with 0.2M sodium bicarbonate 
pH 10 buffer and placed in the same buffer containing about 80 ng/ml GRGD 
for 20 hours at room temperature to couple the peptide. FIG. 2 graphically 
depicts a modified PET film with hydroxymethyl moieties and the subsequent 
steps involved with tresyl activation and GRGD coupling. 
CELL SPREADING ASSAY 
NIH/3T3 and PAE cells were detached from culture flasks by trypsinization 
and suspended in serum-free medium (DMEM with 1 mg/ml bovine serum albumin 
(BSA)). The cells were washed twice by centrifugation in the BSA containing 
medium and seeded at a density of 3,000 cells per cm.sup.2 of film, unless 
otherwise noted, in serum-free medium and incubated in the normal culture 
environment. Spread cells were scored by morphological features such as 
distinct nuclei, pseudopodia, and polygonal shape (FIGS. 3A and 3B). Cells 
were visualized at 200X magnification using Hoffman modulation contrast 
optics on a Leitz Fluovert inverted stage microscope. Cell growth was also 
assessed by determining spread cell counts at various time points, for 
cells cultured in media supplemented with 10% calf serum. 
ACTIN STRESS FIBER VISUALIZATION 
NBD Phallacidin (7-nitrobenz-2-oxa-1,3-diazolylphallacidin) (Molecular 
Probes, Inc. Eugene, Oreg.) was employed to visualize actin stress fibers 
and microfilament bundles in cells attached to the modified surfaces. 
Samples were prepared according to the manufacturer's procedure and 
1000.times. images were viewed utilizing the Fluovert microscope equipped 
with a Leitz E3 excitation filter and UV illumination. 
SOLUBLE PEPTIDE COMPETITION STUDIES 
In the competition studies, 3T3 fibroblasts were preincubated for 30 
minutes in either serum-free medium containing about 90 ug/ml RGDS or no 
peptide. The cells were then inoculated at a density of about 3000 
cells/cm.sup.2 on GRGD derivatized PET and spreading was determined after 
three hours incubation under normal culture conditions. 
The GRGD derivatized substrates were characterized by their ability to 
support active adhesion of cells on their surfaces. The PET pretreatment 
was optimized by coupling GRGD to tresyl chloride activated films that 
were hydroxylated for various time periods. Cell spreading assays using 
NIH/3T3 fibroblasts were performed to determine conditions that supported 
a maximal response. Four hours pretreatment appeared to be optimal for 
maximum cell adhesion and spreading. PHEMA films were derivatized with 
GRGD utilizing low concentrations of peptide (80 ng/ml) which resulted in 
an increase in cellular adhesion by three orders of magnitude (FIG. 4). 
Comparison of GRGD coupled PET films with pretreated untresylated films 
that were incubated with GRGD for the normal coupling time demonstrated 
that either little peptide adsorbed to the latter films or that the 
adsorbed peptide was not available for the receptor-mediated adhesion 
response (FIG. 5). The GRGD modified surfaces supported much better 3T3 
cell adhesion than the untreated PET even in the presence of serum, which 
is indicative of an intrinsic activity on the modified surface (FIG. 6). 
The competition experiment resulted in a 75% reduction of attachment to the 
modified surfaces in the presence of about 90 ug/ml RGDS, which further 
demonstrates the biospecific activity of the substrates (FIG. 7). Gross 
morphology (FIGS. 3A and 3B) of spread 3T3 fibroblasts in serum-free 
medium on the modified films appeared normal (FIGS. 3A and 3B). 
That no cytoskeletal organization is observed on RGDS modified surfaces 
indicates that the complete adhesive response of this cell line and others 
is not obtained on RGDS modified surfaces. This is not a general phenomenon 
however, as some cell types including normal rat kidney fibroblasts and Nil 
8, a normal hamster fibroblast cell line, have been shown to fully respond 
to substrates containing only RGD peptides (14). Growth on the GRGD 
derivatized-PET was serum dependent and was similar to that on unmodified 
PET (FIG. 8), but the initial attachment and spreading was more rapid, as 
indicated by the observation that the GRGD curve leads (open boxes) the 
control curve (solid diamonds) in this figure. 
Attachment and spreading of porcine aortic endothelial cells on the GRGD 
coupled surfaces was also serum independent as expected, since vascular 
endothelial RGD directed receptors have been characterized (15). PAE cell 
spreading in complete medium was much more extensive on the GRGD 
derivatized films than the untreated films at four hours, but both 
surfaces had confluent monolayers of cells at twenty four hours. These 
observations indicate that the kinetics of PAE cell attachment was more 
rapid on the modified surface. 
Applicants' disclosed methods provide a means for obtaining stable, 
chemically defined surfaces for use in studying cellular responses to 
insoluble extracellular matrix signals. It provides a means by which to 
decouple cell adhesion and spreading from protein adsorption. In this 
sense, it may be useful for those who prefer to use serum-free cell 
culture media (i.e., media in which purified proteins are added 
individually rather than introduced from serum) in that cell adhesion 
molecules (CAMs) do not need to be included. Whether these surfaces are 
capable of supporting cell growth at very low serum concentrations remains 
to be determined. 
It should be understood that, in the presence of the media proteins, the 
GRGD surface is rapidly covered by adsorbed proteins. This is not 
problematic, however, as Applicants' studies with albumin in the culture 
media (FIGS. 4, 5, 7) indicated that the high affinity RGD-integrin 
association is capable of competing favorably with the adsorbed proteins. 
Cell detachment may be accomplished by calcium chelation, as the 
RGD-integrin affinity is calcium dependent. 
It should also be noted that cell function is highly dependent upon the 
cell attachment surface (16, 17). This surface may provide a local 
environment that is closer to the one in vivo, and hence stimulate 
stronger adhesion and/or higher productivity. 
EXAMPLE 6--PEPTIDE DERIVATIZATION OF A GLASS SURFACE 
The present example was designed to outline the most preferred method of 
derivatizing a ceramic surface, such as glass, to provide a cell 
receptor-mediated adhesion-promoting substrate. 
Glycophase glass was prepared by the method of Ohlson et al. (18). Glass 
coverslips (18 by 18 mm; Thomas) were soaked in 0.5M sodium hydroxide for 
two hours, rinsed in deionized water, and immersed in an aqueous solution 
(1% pH 5.5) of (3-glycidoxypropyl)-trimethoxysilane (Petrarch Systems, 
Inc.). The preparation was heated and maintained at 90.degree. C. for 2 
hours. The pH was then adjusted to 3.0 followed by heating again for 1 
hour to convert the oxirane moieties on the derivatized glass to glycol 
groups. Dry glycophase glass coverslips were rinsed with dry acetone 
(dried over molecular sieve 4A; Fisher). To about 1 ml of dry acetone, 
about 200 .mu.l of dry pyridine and about 100 .mu.l of dry tresyl chloride 
(Fluka) were added. A minimal volume of this mixture was added to the upper 
surface of each glycophase glass coverslip placed in a glass 
crystallization dish. The reaction was allowed to proceed for about 10 
minutes at room temperature, then the coverslips were rinsed in about 1 mM 
hydrochloric acid and finally rinsed in an about 0.2M sodium bicarbonate 
buffer at pH 9 (coupling buffer). Coupling buffer containing between about 
5-30 ng/ml of peptide, preferably about 10 ng/ml, was added at a minimal 
volume on the coverslips and incubated for about 20 hours at room 
temperature to graft the peptide to the surface. The peptides used in this 
study were synthesized as outlined in Example 1. The peptide containing 
buffer was removed after an about 20 hour incubation period and replaced 
with coupling buffer containing an about 0.8M beta-mercaptoethanol. The 
coverslips were incubated for about 2 hours so that unreacted tresyl 
groups would react with a nonadhesive moiety. 
Measurement of Peptide Surface Concentration 
GRGDY was radiolabeled by adding about 20 ug of peptide to phosphate 
buffered saline, pH 7.4, containing about 5.0 mCi of Na.sup.125 I and 
incubating for 15 about minutes at room temperature with Iodobeads 
(Pierce) according to the manufacturer's instructions. The labeled peptide 
was purified by loading the reaction mixture on a Sep-Pak C.sub.18 sample 
preparation cartridge (Waters) that was washed with methanol-H.sub.2 
O-trifluoroacetic acid (TFA) (80:19:1 v/v) and reequilibrated with PBS. 
The cartridge was washed with 1% v/v TFA to eluate unincorporated iodine. 
The reaction mixture was fractioned using 10% stepwise increases in 
methanol concentration in 1% TFA with the methanol-H.sub.2 O-TFA (80:19:1 
v/v) as the final eluant. The cartridge was washed with each concentration 
until the radioactivity returned to a baseline level. Each fraction was 
analyzed for peptide content by measuring absorbance at 220 nm 
(epsilon=8441 M.sup.-1 cm.sup.-1). Greater than 90% of the eluted peptide 
was in the methanol-H.sub.2 O-TFA (40:59:1 v/v) fraction, which was 
lyophilized and reconstituted in PBS. The specific activity of the peptide 
was then determined after counting a known amount of labeled peptide in an 
automatic gamma counter (Isoflex, ICN Micromedic Systems). The average 
specific activity was 44.0.+-.2.0 mCi/mmol. 
To determine peptide surface concentrations, radiolabeled peptide was added 
to coupling buffer (0.2M sodium bicarbonate, pH 10) at various 
concentrations, and incubated for 20 hours on tresyl activated glass at 
room temperature. Input concentrations were defined as the moles of 
soluble peptide available for reaction per unit area of glass surface. 
Surface concentrations were defined as the moles of peptide coupled per 
unit area of glass and were determined by counting washed glass samples in 
a gamma counter and calculating the values based on the specific activity 
of the labeled peptide. 
The synthetic peptides Gly-Arg-Gly-Asp-Tyr (GRGDY) and 
Gly-Tyr-Ile-Gly-Ser-Arg-Tyr (GYIGSRY), which contain the ligands for two 
important classes of cell adhesion receptors, were covalently coupled to 
the non-adhesive modified glass surface, glycerol propylsilane bounded 
glass (glycophase glass) by the N-terminal Gly. Glycophase glass contains 
a covalently bound organic layer that imbibes water and reduces protein 
adsorption similar to hydrogels without the associated problems of 
swelling and bulk permeation of aqueous solutions. Since glycophase glass 
absorbs proteins poorly, it alone is not suitable for supporting cell 
adhesion, even with serum in the medium. Therefore, GRGDY and GYIGSRY were 
coupled to glycophase glass using the tresyl chloride immobilization method 
of Nilsson, et al. (1987), Methods Enzymol., 135:65-78). The Nilsson, et 
al. article is specifically incorporated herein by reference. 
The N-terminal "G" was used as a spacer between the adhesive peptide and 
the surface, and the C-terminal "Y" was used for radioiodination. Since 
primary amines serve as nucleophiles that react and covalently bind to 
tresyl-chloride-activated supports, the peptides employed linked to the 
glycophase glass exclusively through the primary amine of the N-terminal 
"G". The surface concentration of peptide was measured by .sup.125 I 
radiolabeling and was 12.1 picomoles/cm.sup.2. This derivatization method 
produces chemically stable substrates, which may be useful in studying 
receptor-mediated cell adhesion, as the quantity of peptide available at 
the surface may be precisely measured and controlled. 
EXAMPLE 7--ADHESION AND SPREADING OF CELLS ON A PEPTIDE DERIVATIZED GLASS 
SURFACE 
The present examples was designed to determine the effectiveness of the 
proposed chemical glass surface treatments in promoting the amount and 
rate of cell adhesion to a glass surface. 
SUBSTRATE PREATION 
Glycophase glass substrates were prepared by the method described in 
Example 6. These modified substrates supported the adhesion and spreading 
of cultured human foreskin fibroblasts (HFFs) independently of adsorbed 
proteins. 
AMETERS MEASURED 
The biological activity of both grafted GRGDY and GYIGSRY was assessed by 
measuring the adhesion and spreading of HFF in the presence and absence of 
serum in the medium. Focal contact formation and cytoskeletal organization 
were also observed on these substrates. 
RESULTS 
HFF spreading rates were much slower on grafted YIGSR (GYIGSRY) peptide 
substrates than on the RGD-containing (GRGDY) peptide surfaces. Cells 
formed focal contacts or absence on the RGD-derivatized substrates in the 
presence of serum. Focal contacts formed on the YIGSR-grafted surfaces 
only when serum was present in the medium and had morphologies distinct 
from those observed on the RGD-containing surfaces (FIGS. 10A-10D; FIGS. 
12A-12D). 
Serum influenced microfilament organization and the extent of spreading of 
adherent cells, although adsorption of adhesion proteins was minimal on 
all surfaces. 
EXAMPLE 8--COMATIVE STUDIES OF PEPTIDE FRAGMENTS GRGDY, GYIGSRY AND 
GRGEY ON A DERIVATIZED GLASS SURFACE 
Derivatized glass surfaces were prepared according to the method described 
in Example 6 employing the GRGDY, GRGEY and GYGSRY peptides. HFF cells 
were then plated onto each of the prepared surfaces. Spreading and growth 
rate determinations were then made. Untreated glycophase glass was found 
to support no cell adhesion, even when cells were incubated on this 
substrate in serum-supplemented medium, which is indicative of a low 
protein-binding substrate (Table 1). 
Beta-mercaptoethanol-grafted glass was equally non-supportive of cell 
adhesion and spreading, as indicated in the results of the cell spreading 
studies (FIGS. 2B, D,; 3B, D). Since beta-mercaptoethanol was employed to 
react with any remaining tresyl groups on the surface of the glass, a 
non-adhesive background was established on this surface. Furthermore, 
grafted GRGEY, which does not intrinsically support receptor-mediated cell 
adhesion and adsorbs proteins similarly to grafted GRGDY, did not support 
cell adhesion (Table 1). This result suggests that immobilizing GRGDY on 
glycophase glass does not significantly enhance protein adsorption on this 
substrate. 
Spreading and Growth Rate Determination 
HFF cells were prepared as outlined in Example 4. These cells were 
harvested for experiments and rinsed twice with Ca.sup.2+ - and Mg.sup.2+ 
-free phosphate buffered saline (PBS) and then incubated in 0.05% trypsin 
plus 0.53 mM EDTA in PBS (GIBCO) for 10 minutes. Cells were collected by 
centrifugation and resuspended in serum-supplemented medium or serum-free 
medium which consisted of DMEM with 2 mg/ml of heat-inactivated 
(90.degree. C., 10 min.) albumin (Sigma) and antibiotics. 
Cells suspended in complete or serum-free medium were seeded on the 
substrates at a density of about 10,000 cells/cm.sup.2 and allowed to 
attach and spread at 37.degree. C. in 5% CO.sub.2. An inverted microscope 
(Fluovert, Leitz) equipped with a phase contrast objectives and a high 
resolution video camera (67M series, Dage-MTI) were used to visualize 
spreading cells at various time points. 
Images were digitized with an image processing system (Series 150, Imaging 
Technology Inc.) and the areas of individual cells were determined by 
tracing the perimeter of each cell in the digitized images with a tracing 
pad (Digi-Pad, GTCC) and computing the area enclosed by each trace with an 
integration routine. At least 100 cells were analyzed and cumulative 
histograms were constructed for each time period so that cell spreading 
rates could be determined. 
Cell growth in complete medium on substrates was assayed by visualizing 
cells with 100.times. phase contrast microscopy. At various time points, 
cells were counted in ten fields and the number of cells power unit area 
of growth surface was calculated based on an averaged cell count per area 
of field. 
In the end point cell spreading assays protein synthesis was inhibited by 
treating cells with 20 ug/ml of cycloheximide for about 30 minutes prior 
to inoculating them on substrates. The cells were maintained in medium 
containing cycloheximide throughout the experiment. Cells that were 
treated with soluble peptide were preincubated with medium containing 
peptide for about 30 minutes and were maintained in that medium throughout 
the experiment. Spread cells were scored in 10 fields according to methods 
described by Massia et al. (1989) (Biochem. Engin. VI, Ann. N.Y. Acad. 
Sci., Vol. 589, pp. 261-270, 1990). The percentage of spread cells per 
field was calculated by multiplying the ratio of spread cells to the total 
number of cells per field by 100. 
Morphological Studies 
Cells adherent to peptide-grafted 24.times.50 coverslips (Thomas) were 
mounted in a culture chamber stage and fitted on the Fluorovent inverted 
microscope. A NPL Fluotar 100.times. (Leitz) objective was employed so 
that transmission phase contrast, and interference reflection (IRM) 
microscopy could be performed on the same field without changing 
objectives. Phase contrast and IRM images were acquired from live cells 
immersed in medium and maintained at 37.degree. C. Illumination for phase 
contrast was provided by a 100 W halogen lamp and a model 050260 power 
supply (Leitz) equipped with a heat-reflecting filter. A 100 W mercury arc 
lamp powered by a HBO 100 model 990023 DC source (Leitz) was used for IRM. 
Images were acquired with a high resolution video camera (70 series, 
Dage-MTI) and digitized with the Series 150 image processing system. 
Digitized images were photographed from a high resolution video monitor 
(model PVM 1271Q, Sony) using Illford Pan F film. 
Fluorescence Microscopy 
Cells on peptide-grafted glass coverslips at the end of incubation times 
were rinsed in PBS and fixed for about 20 minutes with 3.7% (v/v) 
formaldehyde in PBS. They were then rinsed in PBS and permeabilized by 
incubation at room temperature for about 1 minute in PBS containing about 
0.2% (v/v) TRITON X-100. Cells were then rinsed in PBS and stained for 
F-actin with a 20 minute incubation at room temperature with about 900 
ng/ml rhodamine-conjugated phalloidin (Molecular Probes, Inc.). The 
coverslips were rinsed thoroughly with PBS and mounted on microscope 
slides in 50% PBS-50% glycerol. These preparations were viewed and 
photographed on the Fluovert microscope equipped with a 100X PL Fluotar 
objective (Leitz). 
RESULTS 
Determination of GRGDY Surface Concentration 
The number of reactive sites and the corresponding peptide concentration on 
the surface were determined by titration with the radiolabeled GRGDY (FIG. 
9). Surface concentration of grafted GRGDY was determined to increase 
linearly with increasing concentrations of peptide available for coupling 
to the surface, reaching a maximum value of 12.1.+-.0.1 picomoles/cm.sup.2 
(FIG. 9). Subsequent increases in input peptide concentrations above 12.0 
picomoles/cm.sup.2 did not further increase the surface concentration of 
the peptide. A maximum surface concentration of 12.1 picomoles/cm.sup.2 
corresponds to a surface coverage of 73,000 molecules per square 
micrometer, or a spacing of approximately 3.3 nm between peptides. 
HFF Spreading Rates on Peptide-Grafted Substrates 
Cells were observed to spread progressively during the 2 hour period on the 
RGD-derivatized glass, both in the presence and absence of serum, with 50% 
of the cells analyzed having areas of 2130 .mu.m.sup.2 or less 2 hours 
after seeding in complete medium (FIG. 10A), and 50% of the cells having 
areas 1210 .mu.m.sup.2 or less 2 hours after seeding in serum-free medium 
(FIG. 10C). The average area of a well-spread cell on tissue culture 
plastic in complete medium was observed to be 2100 .mu.m.sup.2, whereas a 
non-spread cell had an average area of 355 .mu.m.sup.2. The area ranges of 
cells cultured on the nonadhesive surfaces did not vary over time in the 
presence or absence of serum, with 50% of the cells having areas less than 
500 .mu.m.sup.2 (FIGS. 10B, D). These results indicate that no spreading 
occurred on these surfaces, and most of the cells were observed to be 
nonadherent. 
Cell spreading was also observed on YIGSR-derivatizing glass in the 
presence and absence of serum. Spreading rates were much slower on grafted 
YIGSR substrates than on the RGD-containing surfaces, requiring more than 6 
hours for complete spreading in complete (FIG. 10A) or serum-free (FIG. 
10B) medium. After about 9 hours in serum-free medium, 50% of the cells 
analyzed had areas 1400 .mu.m.sup.2 or less (FIG. 10B), which is 
comparable to areas of well-spread HFFs on the RGD surfaces in serum-free 
medium. Serum was observed to enhance cell spreading on the YIGSR 
surfaces; 50% of the cells analyzed had areas of 2333 .mu.m.sup.2 or less 
(FIG. 10A) 9 hours after inoculation. Nonadhesive control surfaces were 
identical to the ones prepared for the RGD-derivatized glass studies and 
cell spreading was not observed on these surfaces; 50% of the cell areas 
never exceeded 600 .mu.m.sup.2 throughout the time frame of the study 
(FIGS. 10C, D). 
Effects of Grafted GRGDY on Cell Growth 
The effect of these RGD-containing substrates on cell growth was checked by 
monitoring growth of HFFs seeded on glass containing coupled GRGDY. No 
difference in growth rate was observed when we compared growth of cells 
cultured on RGD-derivatized glass with that of untreated (not glycophase) 
glass (FIG. 11). 
Characterization of Cellular Responses to the Peptide-Grafted Substrates 
Since serum enhanced cell spreading on the peptide-containing glass, it was 
postulated that the peptides grafted to the surface of the nonadhesive 
glass promoted the adsorption of serum and excreted cellular proteins 
which would augment cell adhesion and spreading on these substrates. To 
check this possible effect, the synthetic peptide GRGEY was coupled to 
glass, and cell spreading on these surfaces was assayed. The substitution 
of glutamic acid (E) for aspartic acid (D) (an addition of one methylene 
group to the carboxylic acid side chain) has been demonstrated to abolish 
the adhesion-promoting activity of the peptide (8) but should have little 
impact on the way the peptide interacts with potentially adsorbing 
proteins. 
No cell spreading was observed on covalently bound GRGEY after about 8 
hours, even when complete medium was used and cellular protein synthesis 
was not inhibited (Table 1). These findings suggest that the 
covalently-bound GRGEY and GRGDY peptides do not significantly increase 
the adsorption of cell adhesion proteins which would promote and enhance 
cell spreading. This is to say that, the cell adhesive behavior of the 
peptide grafted surfaces was due to the peptide's affinity for 
cell-surface receptors and not due to enhanced serum protein adsorption by 
the peptide. 
To determine if protein synthesis played a role in cell spreading on the 
RGD and YIGSR-linked substrates, and if serum significantly increased the 
fraction of cells that spread at a time point where spreading was 
complete, the percentage of spread cells was determined on each surface 
under different conditions after an 8 hour incubation period. It was 
observed that neither protein synthesis nor the presence of serum in the 
medium affected the fraction of cells spread on the RGD-and 
YIGSR-derivatized glass (Table 1). Cell spreading on both the 
peptide-grafted surfaces was completely inhibited, however by the presence 
of soluble peptide in the medium (Table 1), indicating that cellular 
adhesion on these substrates is governed primarily by cell receptor-ligand 
interactions. 
TABLE 1 
______________________________________ 
Peptide Serum in 
grafted medium Cycloheximide 
Peptide Spread 
to (10% in medium in medium 
cells 
surface v/v) (20 ug/ml) (200 ug/ml) 
(%) 
______________________________________ 
GRGEY + + - 0 
+ - - 0 
- + - 0 
- - - 0 
GRGDY + - - 87 .+-. 8 
+ + - 80 .+-. 9 
- - - 91 .+-. 4 
- + - 83 .+-. 10 
- - +(RGDS) 0 
- + +(RGDS) 0 
GYIGSRY + - - 78 .+-. 7 
+ + - 82 .+-. 2 
- - - 81 .+-. 12 
- + - 90 .+-. 2 
- - +(YIGSRY) 
0 
- + +(YIGSRY) 
0 
______________________________________ 
Cell-Substrate Contacts and Cytoskeletal Organization 
Cells were examined live by IRM and phase contrast microscopy at about 4 
hours after seeding on RGD-derivatized substrates and about 8 hours after 
seeding on YIGSR-derivatized substrates. Fixed specimens were stained with 
rhodamine-conjugated phalloidin to evaluate microfilament distribution in 
spread cells. All images were digitized and a high pass filter was 
employed to enhance detail. 
Cells seeded in the absence of serum on RGD-grafted glass, formed small, 
round focal contacts that were observed mainly on the outer margins of 
cells (FIG. 13B). Serum supplemented medium supported formation of large 
elongated focal contacts typical of cells that spread on cell adhesion 
molecule-coated substrates (FIG. 13D). Cells spreading on YIGSR-grafted 
glass did not form focal contacts in the absence of serum (FIG. 14B). 
Focal contacts were well-defined on this substrate when medium was 
supplemented with serum (FIG. 14D), however they were morphologically 
distinct from those of FIG. 13D on the RGD-derivatized substrates. Focal 
contacts on the YIGSR-derivatized substrates were elongated, similar to 
those in FIG. 13D, but were predominantly located in the outer margins of 
cells. Phase contrast images (FIGS. 13A, C; 14A, C) did not reveal any 
obvious morphological differences between spread cells on the various 
substrates, but serve as a corresponding image for the IRM images. 
Rhodamine-phalloidin staining revealed an extensive network of acting 
microfilament bundles in spread cells on RGD-grafted glass, that were 
incubated in serum-free (FIG. 15A) or complete (FIG. 15B) medium. Bundles 
formed by spreading cells incubated in serum supplemented medium stained 
more intensely than those formed by cells incubated in serum-free medium, 
which indicates that thicker fibers formed when serum was present. Cells 
incubated in serum-free medium on the YIGSR-grafted substrates formed very 
few microfilament bundles (FIG. 15C), however thick bundles formed when 
serum was present in the medium (FIG. 15D). 
These studies show that adhesion-promoting synthetic peptides of minimal 
sequences can be covalently grafted to the surface of a nonpermeable, 
nonadhesive material such as glycophase glass to produce biologically 
active, chemically well-defined surfaces that support cell adhesion. These 
substrates may be useful in the study of cell adhesion, as the amount of 
peptide available on the surface may be precisely measured (FIG. 9) and it 
is possible to control the amount of peptide grafted competitively by 
adding controlled amounts of a nonadhesive species, such as glycine, in 
the coupling buffer. This could be an important requirement for model 
substrates of receptor-mediated cell adhesion, since it has been recently 
shown that integrin-mediated cell adhesion to adsorbed RGD-albumin 
conjugates is very sensitive to the density of RGD-containing groups that 
are covalently attached to the native protein. 
EXAMPLE 9--STABILITY OF PEPTIDE-GRAFTED SUBSTRATES 
Radiolabeled GRGDY peptides were covalently grafted to glycophase glass 
substrates in order to determine how stable the immobilized peptides were 
to heat and proteolysis. The percent (%) less in radioactivity was 
interpreted to indicate the percent (%) of immobilized peptide that was 
degraded and thus unavailable for enhancing cell adhesion. 
The data suggests that these peptide-grafted substrates are quite stable to 
autoclaving (steam sterilization at 121.degree. C.), since no loss of 
radioactivity was evident after this treatment (Table 2). Also, no 
radioactivity was lost after culturing cells on these substrates for 1 
week. The data also indicates that the treated substrates are stable to 
cellular proteases. 
TABLE 2 
______________________________________ 
Stability of RGD-derivatized Glycophase Glass 
Environmental Stress 
to the Substrate % loss of radioactivity 
______________________________________ 
Autoclave (121.degree. C., 15 min) 
0 .+-. 0 
Cell Culture (1 week) 
2 .+-. 1 
Exposure to Trypsin 
5 .+-. 4 
______________________________________ 
EXAMPLE 10--PEPTIDE ADSORPTION COMISON OF GRGD COUPLED PET SUBSTRATES 
WITH PRETREATED UNTRESYLATED PET SUBSTRATES 
The GRGD derivatized PET substrates were characterized by their ability to 
support active adhesion of cells on their surfaces. The PET pretreatment 
was optimized by coupling GRGD to tresyl chloride activated films that 
were hydroxylated for various time periods. Cell spreading assays using 
NIH/3T3 fibroblasts were performed to determine conditions that supported 
a maximal response. An about four hour pretreatment appeared to be optimal 
for maximum cell adhesion and spreading (FIG. 17). 
Comparison of GRGD coupled PET films with pretreated untresylated films 
that were incubated with GRGD for the "normal" coupling time (about 20 
hours) demonstrated that either little peptide adsorbed to the film or 
that the adsorbed peptide was not available for the receptor mediated 
adhesion response (FIG. 5). The GRGD modified surfaces supported much 
better 3T3 cell adhesion than the untreated PET, even in the presence of 
serum, which is indicative of an intrinsic activity on the modified 
surface (FIG. 6). The competition experiment resulted in a 75% reduction 
of attachment to the modified surfaces in the presence of about 90 ug/ml 
RGDS, which further demonstrates the biospecific activity of the 
substrates (FIG. 7). 
Gross morphology (FIGS. 3A and 3B) of spread 3T3 fibroblasts in serum-free 
medium on the modified films appeared normal, however, microfilament 
bundle and stress fiber formation could not be detected under these 
conditions. These results indicate, as others have shown with absorbed RGD 
peptides (19, 20, 14), that the complete adhesive response of this cell 
line and others is not obtained on these modified surfaces. This is not a 
general phenomenon however, as some cell types including normal rat kidney 
fibroblasts and Nil 8, a normal hamster fibroblast cell line, have been 
shown to fully respond to substrates containing only RGD peptides (14). 
Growth on the GRGD derivatized PET was serum dependent and was similar to 
that on unmodified PET (FIG. 8), but the initial attachment and spreading 
was more rapid, as indicated by the observation that the GRGD curve leads 
the control curve (FIG. 8). 
Attachment and spreading of porcine aortic endothelial cells on the GRGD 
coupled surfaces was also serum independent (FIG. 16). As expected, since 
vascular endothelial RGD directed receptors have been characterized (15). 
PAE cell spreading in complete medium was much more extensive on the GRGD 
derivatized films than the untreated films at four hours, but both 
surfaces had confluent monolayers of cells at twenty-four hours. These 
observations indicate that the kinetics of PAE cell attachment was more 
rapid on the modified surface. 
EXAMPLE 11--AN INDWELLING CATHETER WITH A BIOADHESIVE DACRON VELOUR CUFF IN 
A RABBIT (PROPHETIC) 
A dacron velour cuff with an inner diameter of about 1 mm and an outer 
diameter of about 3 mm would be placed around a polyethylene catheter at 
the base and glued in place with surgical-grade cement. Prior to placement 
a cuff would be treated in the acetic acid/formaldehyde solution as 
described in Example 3 to hydroxylate the surface. The tetrapeptide GRGD 
would then be covalently attached to the cuff material surface as 
described in Example 3 by tresyl activation and coupling. The fur of the 
animal would be shaved along the abdomen, and the skin opened with a 
lateral cut of about 1 cm length. The catheter would be inserted into a 
vein, e.g. the descending vena cava, and the cuff would be placed just 
beneath the skin. The skin would be sutured together around the protruding 
catheter, such that it covered the cuff. The rates of bacterial infection 
on the catheter would then be measured. Several of the cuffs would be 
removed after a period of time, for example, 1, 2, 3 and 4 weeks for 
histological examination of tissue integration, regrowth, and 
inflammation. Control experiments would utilize unmodified dacron cuffs. 
EXAMPLE 12--A NERVE REGROWTH GUIDE (PROPHETIC) 
A polymer tube with a high permeability to water and oxygen with an inner 
diameter of about 1.5 mm and an outer diameter of about 3 mm would be used 
as a nerve regrowth guide in the rat. An example of a useful material would 
be poly(hydroxylethyl methacrylate). The lumen of the tube would be 
activated and coupled with the peptide GRGD or GYIGSR as described in 
Example 3. A nerve bundle in one leg would be severed and a section 
approximately 1 cm long would be removed. Both ends of the nerve bundles 
would be inserted into the ends of the tubular regrowth guide, and the 
edges of the guide would be tightly sutured to the epiaxonal tissue. The 
wound would be closed, and reinnervation would be measured 
electrophysiologically weekly. Control experiments would utilize 
unmodified poly(hydroxyethyl methacrylate) tubes. 
The following references are cited throughout the Specification, and are 
hereby specifically incorporated in pertinent part by reference herein. 
EXAMPLE 13--COMATIVE STUDIES OF GRGDY GRAFTED AT DIFFERENT DENSITIES ON 
A DERIVATIZED GLASS SURFACE 
Derivatized glass surfaces were prepared according to the method described 
in example 6 employing the GRGDY peptide, except that the amount of 
peptide added to the tresyl activated glass was less than the 12 
picomoles/cm.sup.2. This allowed the formation of substrates with known 
peptide surface densities. The linear region of FIG. 9, referenced in 
Example 6, indicates that the amount of peptide added to the reactive 
glass is equal to the amount of peptide coupled to the glass, so long as 
that amount is less than or equal to 12 picomoles/cm.sup.2. Peptide was 
covalently attached to the derivatized glass as surface concentrations of 
0.001, 0.005, 0.01, 0.05, 0.1, 0.5, and 1.0 picomoles/cm.sup.2. Human 
foreskin fibroblasts were seeded on the material in DMEM containing heat 
inactivated albumin as the only protein and cell spreading was determined 
after a 4 hour incubation. Cells were graded for morphology of spreading 
according to the following scheme: rounded cell, no filopods, stage 1; 
rounded cell, one filopod, stage 2; rounded cell, more than one filopod, 
stage 3; flattened cell with pseudopods, and polygonal-shaped fully spread 
cell, stage 4. The results are shown in FIG. 18 and indicate that levels as 
low as 0.001 picomole/cm.sup.2 are sufficient to promote nearly a full 
spreading response; this is particularly evident from the data on stage 4 
cells, where 0.001 picomoles/cm.sup.2 resulted in 42% of cells in stage 4 
while 1.0 picomoles/cm.sup.2 resulted in 81% of cells in stage 4. Thus the 
cellular response depends upon the peptide surface density, but 0.001 
picomoles/cm.sup.2 is sufficient to give a strong (about half-maximal) 
response to the surface. 
EXAMPLE14--GLYCOPHASE GLASS--A MODEL CELL NONADHESIVE SUBSTRATE TO WHICH 
PEPTIDES CAN BE COUPLED 
This example is included to illustrate that glycophase glass is poorly cell 
adhesive. It, when combined with Examples 15-19, illustrates the 
advantageous cell-type specificity that can be obtained when peptides are 
attached to otherwise cell nonadhesive or poorly cell adhesive substrates. 
Glycophase glass was prepared by the method of Example 6. Cell spreading on 
this surface was measured both in the absence and presence of serum, and 
the results are tabulated below. The cells were human foreskin fibroblasts 
(HFF), human umbilical vein endothelial cells (HUVEC), human blood 
platelets (Plt), and human blood platelets prestimulated with 5 .mu.m 
adenosine diphosphate (Prestim Plt). HFFs and HUVECs were harvested 
nonenzymatically with a PBS solution containing 54 nM EGTA, centrifuged, 
and resuspended in normal culture medium or medium containing 2 mg/ml of 
heat-inactivated serum for serum-free spreading assays. To determine the 
extent of cell spreading, cells were incubated on the substrates for 4 
hours and the percent of spread cells was determined by previously 
described methods. Platelet spreading was determined by adding PRPP to 
each substrate and visualizing platelets with 1000X modulation contrast 
(Hoffman) optics after a 10 minute incubation period. For prestimulated 
platelets, 5 .mu.M ADP was added to PRP prior to incubation on the 
substrates. Results are shown in Table 3. 
TABLE 3 
______________________________________ 
% Spread % Spread without 
Cell type with serum 
serum 
______________________________________ 
HFF 9 1 
HUVEC 8 1 
Plt 0 N.D. 
Prestim Plt 0 N.D. 
______________________________________ 
EXAMPLE 15--GLYCOPHASE GLASS COUPLED WITH RGD MATERIALS WHICH SUPPORT THE 
ADHESION OF FIBROBLASTS AND ENDOTHELIAL CELLS BUT NOT PLATELETS 
The peptide GRGDY was coupled to the glycophase glass surface as described 
in Example 6. Cell spreading assays were performed as described in Example 
14. Cell spreading was measured and is reported in Table 4. This material 
may have advantages for vascular grafts, where the invasion of endothelial 
cells is desirable but attachment of platelets is detrimental. 
TABLE 4 
______________________________________ 
% Spread % Spread without 
Cell type with serum 
serum 
______________________________________ 
HFF 80 81 
HUVEC 62 55 
Plt 0 N.D. 
Prestim Plt 0 N.D. 
______________________________________ 
EXAMPLE 16--GLYCOPHASE GLASS COUPLED WITH YIGSR MATERIALS WHICH SUPPORT THE 
ADHESION OF FIBROBLASTS AND ENDOTHELIAL CELLS BUT NOT PLATELETS 
The peptide GYIGSRY was coupled to a glycophase glass surface. YIGSR is a 
sequence in the CAM laminin which promotes cell adhesion (Graf et al., 
1987, Cell, 48:989-996). Cell spreading was measured by procedures 
described in Example 14. This material may have advantages for vascular 
grafts, where the invasion of endothelial cells is desirable but the 
attachment of platelets is detrimental. The results are shown in Table 5. 
TABLE 5 
______________________________________ 
% Spread % Spread without 
Cell type with serum 
serum 
______________________________________ 
HFF 64 59 
HUVEC 79 73 
Plt 0 N.D. 
Prestim Plt 0 N.D. 
______________________________________ 
EXAMPLE 17--GLYCOPHASE GLASS COUPLED WITH PDSGR--MATERIALS WHICH SUPPORT 
THE ADHESION OF FIBROBLASTS AND ENDOTHELIAL CELLS BUT NOT PLATELETS 
The peptide GPDSGRY was coupled to a glycophase glass surface. PDSGR is a 
sequence discovered in laminin which has cell adhesion-promoting 
activities (Kleinman et al. 1989, Arch. Biochem. Biophys., 272:1:39-45). 
Cell spreading was measured by procedures described in Example 14. The 
results are shown in Table 6. This material may have advantages for 
vascular grafts, where the invasion of endothelial cells is desirable but 
the attachment of platelets is detrimental. 
TABLE 6 
______________________________________ 
% Spread % Spread without 
Cell type with serum 
serum 
______________________________________ 
HFF 66 64 
HUVEC 66 50 
Plt 0 N.D. 
Prestim Plt 0 N.D. 
______________________________________ 
EXAMPLE18--GLYCOPHASE GLASS COUPLED WITH REDV--MATERIALS WHICH SUPPORT THE 
ADHESION OF ENDOTHELIAL CELLS BUT NOT FIBROBLASTS OR PLATELETS 
The peptide GREDVY was coupled to a glycophase glass surface. The sequence 
REDV, which was derived from fibronectin sequence information, was shown 
to promote b16-f10 cell adhesion but not BHK fibroblast adhesion by 
Humphries et al. (1986 J. Cell Bio., 103:6 2637-2647). Cell spreading was 
measured by procedures described in Example 14. The results are shown in 
Table 7. This material may have advantages for vascular grafts, where the 
invasion of endothelial cells is desirable but the invasion of fibroblasts 
and vascular smooth muscle cells and the attachment of platelets is 
detrimental. 
TABLE 7 
______________________________________ 
% Spread % Spread without 
Cell type with serum 
serum 
______________________________________ 
HFF 9 2 
HUVEC 79 69 
Plt 0 N.D. 
Pretim Plt 0 N.D. 
______________________________________ 
EXAMPLE 19--GLYCOPHASE GLASS COUPLED WITH IKVAV MATERIALS WHICH DO NOT 
SUPPORT HUVEC OR PLATELET SPREADING, BUT SHOULD SUPPORT NEURITE OUTGROWTH 
The peptide GIKVAVY was coupled to a glycophase glass surface. The sequence 
IKVAV, which was derived from laminin sequence information, was shown to 
promote cell adhesion but no spreading of several cell types and neurite 
outgrowth of neuronal cell types by Tashiro et al. (1989 J. Bio. Chem., 
264:27 16174-16182). Cell spreading was measured by procedures described 
in Example 14. The results are shown in Table 8. This material may have 
advantages for implants in nervous tissue, where the invasion of neurons 
and the formation of axonal processes (neurites) is desirable. 
TABLE 8 
______________________________________ 
% Spread % Spread without 
Cell type with serum 
serum 
______________________________________ 
HFF 47.4 42.5 
HUVEC 11.2 0 
Plt 0 N.D. 
Prestim Plt 0 N.D. 
______________________________________ 
EXAMPLE 20--POLYETHYLENE TEREPHTHALATE TO WHICH POLYETHYLENE GLYCOL HAS 
BEEN IMMOBILIZED--A POLYMERIC CELL-NONADHESIVE SUBSTRATE TO WHICH PEPTIDES 
CAN BE ATTACHED (PET/PEG) 
Polyethylene glycol (PEG) of molecular weight 18,500 was immobilized to a 
polyethylene terephthalate (PET) film using a solution processing 
technique described later herein. The resulting film (PET/PEG) was much 
more wettable than the unmodified PET and did not support the attachment 
and spreading of cells. The PET/PEG films were extracted in dioxane for 10 
minutes as a control for the solvent used in the peptide grafting 
procedures. Human vascular smooth muscle cells (HVSMC) were also included 
when these PET/PEG-based materials were tested, because this cell type is 
found in vascular tissues and can interact with vascular implants. The 
percentage of spread cells was determined by the procedures described in 
Example 14. The results are shown in Table 9. HFF, HUVEC, and HVSMC 
spreading was determined in the presence of serum-supplemented medium. 
TABLE 9 
______________________________________ 
Cell Type % Spread with serum 
______________________________________ 
HFF 0 
HUVEC 0 
HVSMC 0 
Plt 0 
Prestim Plt 0 
______________________________________ 
EXAMPLE 21--PET/PEG COUPLED WITH RGD-MATERIALS WHICH SUPPORT THE ADHESION 
OF FIBROBLASTS AND ENDOTHELIAL CELLS BUT NOT PLATELETS OR VASCULAR SMOOTH 
MUSCLE CELLS 
The peptide GRGDY was covalently coupled to the PET/PEG surface of Example 
20 using the same chemistry as in Example 15. HFF, HUVEC, and HVSMC 
spreading was determined in the presence of serum-supplemented medium. 
Cell spreading was as shown below in Table 10. This material may have 
advantages for vascular grafts, where the invasion of endothelial cells is 
desirable but the attachment of platelets is detrimental. 
TABLE 10 
______________________________________ 
Cell Type % Spread with serum 
______________________________________ 
HFF 100 
HUVEC 71 
HVSMC 0 
Plt 0 
Prestim Plt 0 
______________________________________ 
EXAMPLE 22--PET/PEG COUPLED WITH REDV-MATERIALS WHICH SUPPORT THE ADHESION 
OF ENDOTHELIAL CELLS BUT NOT FIBROBLASTS OR PLATELETS OR VASCULAR SMOOTH 
MUSCLE CELLS 
The peptide REDV was coupled to the PET/PEG surface of Example 20. HFF, 
HUVEC, and HVSMC spreading was determined in the presence of 
serum-supplemented medium. Cell spreading was as shown below in Table 11. 
This material may have advantages for vascular grafts, where the invasion 
of endothelial cells is desirable but the invasion of fibroblasts and 
vascular smooth muscle cells and the attachment of platelets is 
detrimental. 
TABLE 11 
______________________________________ 
Cell Type % Spread with serum 
______________________________________ 
HFF 0 
HUVEC 78 
HVSMC 0 
Plt 0 
Prestim Plt 0 
______________________________________ 
EXAMPLE 23--PET/PEG COUPLED WITH IKVAV-MATERIALS WHICH DO NOT SUPPORT HUVEC 
OR PLATELET SPREADING, BUT SUPPORTS HFF SPREADING AND SHOULD SUPPORT 
NEURITE OUTGROWTH 
The peptide GIKVAVY was coupled to the PET/PEG surface of Example 20. The 
sequence IKVAV, which was derived from laminin sequence information, was 
shown to promote cell adhesion but no spreading of several cell types and 
neurite outgrowth of neuronal cell types of Tashior et al. (1989 J. Bio. 
Chem., 264:27 16174-16182). Cell spreading was measured by procedures 
described in Example 14 and is shown in Table 12. This material may have 
advantages for implants in nervous tissue, where the invasion of neurons 
and the formation of axonal processes (neurites) is desirable. 
TABLE 12 
______________________________________ 
Cell Type % Spread with serum 
______________________________________ 
HFF 100 
HUVEC 0 
Plt 0 
Prestim Plt 0 
______________________________________ 
EXAMPLE 24--A SOLUTION TECHNIQUE TO INCORPORATE POLYETHYLENE OXIDE AND 
OTHER WATER SOLUBLE POLYMERS INTO SURFACES OF POLYMERIC BIOMATERIALS 
Currently used biomedical polymers in applications involving blood contact 
have not proved to be sufficiently nonthrombogenic to be useful in small 
diameter vascular grafts. Adhesion of platelets and other blood cells is 
the main cause of low patency of small diameter grafts, and an aspect of 
the present embodiment is to reduce the interactions of blood components 
with biomedical polymers. Because the adhesion of platelets, white blood 
cells, fibroblasts, etc. is mediated by the adsorption of proteins to the 
polymer surface, an approach was adopted which reduced the interaction of 
proteins with these polymers. 
Polyethylene oxide (PEO) surfaces have been observed to resist the 
adsorption of plasma proteins as a result of their strong hydrophilicity, 
chain mobility and lack of ionic charge [25]. Several groups have used PEO 
or PEG (polyethylene glycol) as a modifier in a quest to obtain a 
biocompatible or nonadhesive surface. Different approaches have been used 
to modify polymer surfaces with PEO. Among them are those techniques that 
involve covalent grafting of PEO to a base polymer such a PET [26,27], a 
polyurethane [28], or polyvinyl alcohol [29], polymerization of a monomer 
having a pendant PEO chain [30,31], incorporation of PEO into a base 
polymer by block copolymerization [32,33], or direct adsorption of 
PEO-containing surfactants which are typically block copolymers of the AB 
or ABA type where one of the blocks is a PEO [25,34]. Most of these 
techniques have utilized PEO of relatively low molecular weights (less 
than 5000 daltons) and only a few have used significantly higher molecular 
weights [26,27,34]. 
Although some of the above described techniques work reasonably well in 
reducing cellular interactions at the surfaces of the modified polymers, 
most of them require multiple stages to obtain the necessary surface 
modification. Furthermore, they are limited by the structure and 
availability of labile chemical moieties on the base polymer surface and 
are in many cases, specific for modification of the base polymers. 
The present invention relates to a technique incorporating PEO and other 
water-soluble polymers (WSP) into the surface of a base polymer (BP). This 
technique is of general applicability and is limited only by the solution 
properties of the BP in a solvent that also dissolves the WSP to be 
incorporated into its surface. The technique involves immersing the 
polymeric device or material (fabricated from the BP) to be modified into 
a liquid that is a mutual solvent for the BP and the WSP. The interface 
between the BP and the liquid then begins to soften and results in the 
loosening of the polymer network on the surface of the BP. During this 
time the molecules of the WSP are free to diffuse into the semi-dissolved 
interface. After a period of time the system is quenched with water which 
is a nonsolvent for the BP but is mutually soluble with the immersion 
solvent. This results in a rapid collapse of the interface, entrapping the 
chains of the WSP within the BP network. In this way the BP is 
noncovalently but stably modified with the WSP. Alternate quenching 
solvents could also be used rather than water, where the quenching solvent 
is a solvent for the WSP but not for the BP. Examples of alternate 
quenching solvents include but are not limited to methanol, ethanol, 
acetone, dimethyl, formamide, tetrahydrofuran and benzene for the PET 
TIENs; and ethanol and methanol for the PMMA PIPNs. Alternatively a mutual 
nonsolvent for both the WSP and BP could be used to quench and could be 
followed by a rinse by a solvent for the WSP but not the BP. Alternatively 
the system may be dried and then rinsed in quenching solvent to remove the 
excess solvent. A schematic of the proposed entrapment of WSP chains in 
the BP is shown in FIG. 19. The conditions of the entrapment process, such 
as appropriate solvent dilution (to prevent the BP from dissolving 
entirely), concentration of the WSP, and treatment time, of course need to 
be optimized to obtain a suitably modified stable physical interpenetrating 
network (PIPN). It also follows that the molecular weight of the WSP should 
be of some importance because molecules with relatively short chain lengths 
(i.e., low molecular weights) cannot be effectively entrapped by the 
collapsing network on the BP and in due course may succeed in diffusing 
out of the network. By the same token, very large molecules (i.e., high 
molecular weights) have mass transfer limitations and are not incorporated 
into the surface of the BP effectively. 
A technique has been described in the literature [35] which stresses only 
the use of diblock surfactant copolymers where one block is hydrophobic 
and the other hydrophilic. That the technique may have applications in 
biomaterials is mentioned but no data or examples are presented. The 
technique of the present invention is generally applicable to any 
homopolymer to be incorporated into a BP surface and as such is not 
limited to diblock copolymers with surfactant properties. 
MATERIALS AND METHODS 
Preparation of a physical interpenetrating network PIPN on PET. 
Thin films of PET (Mylar, Du Pont) were used for surface modification using 
the PIPN technique to incorporate PEO (of molecular weights 5000, 18500, 
and 100000 g/mol, Polysciences, Inc.), PVP (mol. wt. 24000 g/mol, Aldrich 
Chemical Co.) and PEOX (mol. wt. 439000 g/mol, Dow Chemical Co., # 
XA10874.05). The PET films were extracted in acetone for at least 24 hours 
at room temperature before use. Trifluoroacetic acid (TFAA, Morton Thiokol, 
Inc.) was used as the treatment solvent since it dissolved all the WSPs as 
well as PET. Full strength TFAA was found to dissolve the PET very rapidly 
and hence needed dilution in order that the BP be treated for a sufficient 
length of time. Solutions of the WSPs were made in deionized and filtered 
water (DIFW) at concentrations of 0.4 g/ml except for the PEO 100000 and 
PEOX which were used at 0.2 g/ml due to high viscosity. It was found that 
dilution of TFAA by 18-20% was suitable to prevent dissolution of the PET 
film and retain optical clarity after quenching with water. TFAA was 
diluted to the suitable concentration by adding the aqueous WSP solutions. 
The PET films were then added to the solution of the WSP in diluted TFAA 
and allowed to stand for 30 minutes with periodic swirling at room 
temperature. The mixture was then rapidly quenched with a large excess of 
DIFW to produce the PIPN. The films were then transferred to DIFW in which 
they were stored. The water was changed periodically to remove any WSP that 
may have leached out of the BP surface. A control was also run with the 
same dilution of TFAA with DIFW but without any WSP. 
Preparation of PIPN on Pellethane. 
Pellethane (Dow Chemical Co., # 2363-80AE) was obtained as pellets. A 
solution of pellethane in tetrahydrofuran (THF) at a concentration of 50 
g/l was used to cast films of 50 mil thickness using a casting knife. 
These films were cured in an oven at 60.degree.-70.degree. C. following 
casting to evaporate the solvent. A number of different solvents were 
tried and THF was found to be suitable for generation of the PIPN on 
pellethane. The PIPN was made only using the PEO 18500 WSP for reasons 
explained below. From control runs (i.e., solvent dilutions without any 
WSP) it was found that dilution of THF to 40% with DIFW was necessary to 
retain structural integrity and optical clarity of the film upon 
quenching. The PEO 18500 was insoluble in THF at room temperature but was 
soluble at 60.degree. C., hence the surface modification procedure for 
pellethane was performed at this temperature. The treatment solution 
consisted of 40% THF, 20% of the PEO 18500 solution in water, and the 
remaining 40% DIFW. This mixture was heated to 60.degree. C. until the PEO 
dissolved and the pellethane films immersed in the solution for 15-25 
minutes. The procedure was carried out at 60.degree. C. in an oven. The 
solution was then quenched with an excess of DIFW and the treated films 
transferred to water for storage. A control was also run at the same THF 
dilution without the presence of PEO. 
Preparation of PIPN on PMMA. 
PMMA (medium mol. wt., Aldrich Chemical Co.) was obtained as a powder. It 
was dissolved in acetone at 50 g/l and a film cast similar to the one for 
pellethane. Acetone was used as a modification solvent for PMMA. It was 
found that a dilution of acetone to 60% was suitable for the procedure. 
The treatment solution consisted of 60% acetone, 20% of the aqueous PEO 
18500 solution (40% PEO in water), and the remaining 20% water. The 
treatment was performed at room temperature for 15-25 minutes followed by 
quenching with water. The modified films were once again stored in water 
that was changed periodically. Controls samples were also run. 
Surface Analysis and Characterization. 
Contact angles on the modified surfaces were measured in a custom built 
device. Air contact angles were measured under water to determine any 
change in hydrophilicity following the modification procedure. 
ESCA analysis (VG Instruments, UK) on the polymer surfaces was performed to 
determine the presence of the WSP on the BP surface after the modification 
procedure. 
Measurement of Biological Responses on PIPN Surfaces. 
Protein adsorption studies on the modified surfaces were done using albumin 
(BSA, Sigma). The protein was radiolabelled with I.sup.125 NaI (ICN 
Biomedicals, Inc.) using the iodobead technique [36]. The specific 
activity of the labelled albumin was found to be 53.9 .mu.Ci/mg. Small 
films (0.5.times.0.5 cm.sup.2) were cut from the modified polymers and 
incubated with radiolabelled albumin at a concentration of 0.094 mg/ml in 
phosphate-buffered saline (PBS) for 4 hours at 37.degree. C. Following the 
incubation the films were rinsed with a non-radiolabelled albumin solution 
in PBS of the same concentration as the adsorption solution and the films 
counted in a gamma counter (Isoflex, ICN Micromedic Systems) for adsorbed 
protein. All samples were done in quadruplet. 
Short-term blood compatibility was studied in a parallel-plate flow chamber 
that provided the necessary controlled flow and shear environment needed 
for this study. The flow chamber is shown in FIG. 20. The surface modified 
films were mounted on glass coverslips (24.times.50 mm) which formed the 
base of the flow chamber. All nonrelevant surfaces were coated with 
albumin (4 g/dl, in HEPES buffer) prior to blood contact to avoid 
preactivation of platelets before contacting the test material surface. 
Freshly drawn heparin-anticoagulated (2 units/ml) human whole blood was 
used in these studies. Platelets were labelled with a fluorescent dye, 
mepacrine (10 .mu.M), at the time of venipuncture. Epifluorescence 
microscopy and digital image processing were used to visualize platelet 
adhesion and thrombus formation during flow and to determine adherent 
platelet counts on the tested surfaces. The image acquisition system has 
been described elsewhere [37,38]. Blood flow was maintained for 10 minutes 
at a wall shear rate of 100 sec.sup.-1. 
Following blood flow over the modified polymer surfaces, the films were 
gently rinsed in phosphate buffered saline (PBS) and then fixed in 2.5% 
solution of glutaraldehyde in PBS overnight. The blood contacting surfaces 
were then subjected to scanning electron microscopy (SEM) to evaluate 
platelet adhesion as well as morphology of adhered platelets to assess the 
degree of spreading and activation of the platelets on the different 
surfaces. 
As a further, more rigorous test of the effectiveness of PEO and other WSPs 
in preventing cell adhesion to the modified polymer surfaces, cell culture 
of human foreskin fibroblasts (HFF) was performed on these surfaces. 
Adhesion and spreading assays were done to determine the effectiveness of 
these surfaces in reducing protein adsorption and hence adhesion and 
spreading of these cells on the modified polymers. 
RESULTS AND DISCUSSION 
Measurement of contact angles on the modified surfaces gave an indication 
of the relative hydrophilicity of these surfaces before and after the 
modification procedure. All polymers were extracted in water for at least 
one week before any measurements were made. Table 13 shows the contact 
angle data on the PET modified surfaces. Table 14 shows the contact angle 
data on Pellethane and Table 15, the data for PMMA. 
TABLE 13 
______________________________________ 
Contact angles for modified PET surfaces. 
MODIFICATION CONTACT ANGLE, (.degree.) 
______________________________________ 
PET-untreated 65.3 .+-. 0.9 
PET-control 50.5 .+-. 2.8 
PET-PEO5000 14.9 .+-. 2.8 
PET-PEO18500 19.4 .+-. 3.6 
PET-PEO100000 21.1 .+-. 2.3 
PET-PVP24000 20.6 .+-. 2.8 
PET-PEOX439000 
22.2 .+-. 2.4 
______________________________________ 
TABLE 14 
______________________________________ 
Contact angles for modified Pellethane surfaces. 
MODIFICATION CONTACT ANGLE, (.degree.) 
______________________________________ 
PELL untreated 
47.9 .+-. 3.1 
PELL control 35.2 .+-. 2.7 
PELL-PEO18500 24.6 .+-. 4.3 
______________________________________ 
TABLE 15 
______________________________________ 
Contact angles for modified PMMA surfaces. 
MODIFICATION CONTACT ANGLE, (.degree.) 
______________________________________ 
PMMA untreated 
59.6 .+-. 3.0 
PMMA control 37.0 .+-. 3.3 
PMMA-PEO18500 22.3 .+-. 1.8 
______________________________________ 
Decreasing contact angles indicate increasing hydrophilicity of the polymer 
surfaces. PET-untreated represents the virgin polymer that has only been 
extracted in acetone. The control PET shows a smaller contact angle than 
the untreated polymer indicating that the treatment with TFAA actually 
increases its hydrophilicity to a small extent. The WSP treated films show 
a much increased hydrophilicity indicating the presence of the respective 
WSPs at the surface of the PET. 
Contact angle data on Pellethane (PELL) shows the same trend as for the PET 
surfaces. The PEO 18500 modified pellethane shows a similar contact angle 
to the corresponding PET surface. Once again the same trend is obtained on 
PMMA. Thus all the WSP-modified surfaces have contact angles very close to 
each other indicating similar degrees of hydrophilicity and similar 
interfacial energies. 
An analysis of radiolabeled albumin adsorption on the modified PET surfaces 
gave the results shown in FIG. 21. The data clearly shows a sharp drop in 
albumin adsorption only for the surface modified with PEO 18500. An 
approximately 80% decrease in adsorption over the control is observed for 
this surface. The PVP modified surface shows a decrease of about 20% and 
the PEOX modified surface shows a decrease of approximately 30% over the 
control. The PEO 5000 and 100000 modified surfaces do not show 
significantly different adsorption levels. This data may indicate that the 
PEO 5000 is not of a large enough molecular weight to prevent protein 
adsorption at the polymer surface, and that the PEO 100000 is too bulky to 
be effectively integrated into the polymer surface in a configuration that 
enables it to prevent adsorption of protein. 
An analysis of platelet adhesion to the modified PET surfaces as obtained 
by epifluorescence videomicroscopy during flow of whole blood over the 
polymer surfaces is shown in FIG. 22. The PEO 18500 modified surface shows 
a reduction in platelet adherence to about 5% of the control value. The 
PEOX modified surface also shows a significant reduction to about 10% of 
control levels. The PEO 100000 modified surface shows a reduction to about 
30%, the PEO 5000 and PVP modified surfaces show a reduction to 40-80% of 
control values. This correlates well with the protein adsorption data on 
the PEO 18500 modified PET. The significantly low platelet adherence on 
the PEOX modified PET has not been explained. 
Adhesion and spreading of human foreskin fibroblasts in Dulbecco's 
Modification of Eagle's Medium (DMEM) supplemented with 10% fetal calf 
serum was used as a test of the effectiveness of the WSP modified surfaces 
in preventing protein adsorption. The cells were seeded at a known density 
on the PET, pellethane and PMMA modified surfaces and the number of spread 
cells were counted first at four hours after seeding and periodically 
thereafter until they reached confluency. 
The PET modified films were seeded with 30000 cells/cm.sup.2 after 
extraction in water for 25 days. The most dramatic results were obtained 
here for the PEO 18500 modified surface on PET. As can be seen in FIG. 23, 
all the surfaces except the one modified with PEO 18500 modified PET 
reached confluency within 5-10 days after initial seeding. In spite of 
reseeding the PEO 18500 on day 9 and again on day 15, the cells do not 
adhere to this surface even after more than 30 days in culture. Among the 
surfaces that reach confluency, the PEOX modified surface is the slowest 
to respond but eventually reaches confluency. These results are in strict 
concordance with the data obtained from protein adsorption and platelet 
adhesion experiments. The results for the PEOX surfaces may indicate that 
in a blood contacting application, such a surface may behave suitably in 
the short-term but over longer contact times, eventually may turn 
thrombogenic. 
These results indicate the effectiveness and suitability of PEO 18500 in 
preventing protein adsorption and hence cellular interactions when 
impregnated into a polymer surface in the form of a PIPN. As a consequence 
of these results it was decided to modify the surfaces of Pellethane and 
PMMA only with PEO 18500 and reconfirm the earlier results on PET. Having 
determined the suitable conditions, solvents, etc., these polymers were 
impregnated with PEO 18500 to obtain a PIPN on these surfaces. Cell were 
seeded on these modified surfaces under more rigorous conditions, (i.e., 
2.5 times the PET seeding density). Under these conditions, a reasonably 
adhesive substrate such as the untreated PET would reach confluency in 
less than 2 days. The results obtained for Pellethane are shown in FIG. 24 
and those for PMMA in FIG. 25. Once again, PEO 18500 is successful in 
reducing cell spreading and growth on these substrates. It is seen that as 
the concentration of the treatment solvent is increased, the cell adherence 
decreases indicating a better incorporation of PEO 18500 into the base 
polymer. 
Similar surface hydrophilicities on the different modified surfaces 
resulted in vastly differing cellular responses, indicating that surface 
mobility and a suitable chain length of the incorporated polymer are 
important factors determining the outcome of cellular interactions at a 
polymer surface. 
These results indicate the general applicability of the PIPN technique to 
modification of polymer systems and moreover, the extraordinary ability of 
PEO 18500 to inhibit protein adsorption and hence cellular interactions at 
a biomaterial surface. The potential of this technique in application to 
existing biomedical polymers is enormous. Being a simple solution process, 
prefabricated devices may be modified simply by immersion in a suitable 
solution of the WSP and then transfer to a nonsolvent for the device 
material such as water. 
The PIPN appears to be quite stable over a period of at least several 
weeks. The cell adhesion and protein adsorption experiments with the PEO 
18500 modified surfaces (which showed low cell adherence and protein 
adsorption) were done 25 days and 20 days respectively, after the 
preparation of these PIPNs. During this 20-25 day period they were 
continually extracted in water. 
Potential applications of this process and the water soluble polymer PEO 
(PEG) (preferably of molecular weight 18500) include areas involving 
blood-contacting biomaterials, such as vascular grafts, catheters and 
pacing leads. In addition, the labile PEO end groups on these modified 
polymer surfaces could serve as a site for attachment of suitable 
biospecific peptides (described elsewhere herein) to produce surfaces that 
would adhere only particular cell types. For example, endothelial cells 
might be adhered in case of application as a vascular graft. About half of 
the vascular grafts currently marketed are made from Dacron (PET) which is 
the major focus of this example. Other applications involve materials in 
contact with tissue where cell adhesion is not desired, such as biosensor 
membranes, intraocular lenses (PMMA). Materials may also be produced for 
use with protein affinity chromatography, where non-adsorbing supports are 
preferred for attachment of specifically adsorbing ligands. Non-adsorbing 
tubes and bags might also be useful in other areas of protein and blood 
processing. The technique of the present invention may have application in 
the manufacture or treatment of ultrafiltration membranes for use in 
biotechnology and food processing. Nonbiological applications may also 
exist, such as piping systems for very clean water systems (e.g., as used 
in the microelectronics industry) and perhaps in marine non-fouling 
surfaces. 
Further studies are planned concerning fibrinogen adsorption on the PET 
surfaces, and albumin and fibrinogen adsorption on PMMA and Pellethane 
surfaces; ESCA data showing the presence of PEO or other WSPs on these 
surfaces; blood contact for Pellethane and PMMA surfaces as well as SEM 
analysis following blood contact to assess morphology of platelets on 
these surfaces; and finally further in vivo tests such as subdermal 
implantation in mice. Preliminary data on PET-PEO 18500 showed it to be 
effective in preventing cell adherence. A one week control PET implant 
showed extensive fibrosis and encapsulation while the corresponding PET 
surface modified with PEO 18500 was found to be free floating with minimal 
cellular adherence. 
The following references, as well as those cited elsewhere are incorporated 
in pertinent part by reference herein for the reasons cited. 
BIBLIOGRAPHY 
1. Grinnell, F. (1978), "Cellular Adhesiveness and Extracellular 
Substrata", International Review of Cytology, 53: 67-149. 
2. Couchman, et al., (1982), J. Cell Biol., 93: 402-410. 
3. Pearlstein, E., (1976), Nature, 262: 497-500. 
4. Kleinman, et al., (1976), Biochem. Biophys. Res. Commum., 72: 426-432 
5. Grinnell, F. (1976), Exp. Cell Res., 97: 265-274. 
6. Grinnell, F. (1976), Exp. Cell. Res., 102: 51-62. 
7. Hynes, et al., (1982), J. Cell. Biol., 95: 369-377. 
8. Pierschbacher, et al., (1984), Nature, 309: 30-33. 
9. Pytela, et al., (1985), Cell, 40: 191-198. 
10. Pytela, et al., (1985), Proc. Natl. Acad. Sci. USA, 82: 5766-5770. 
11. Fitzgerald, et al., (1985), J. Biol. Chem., 260: 11366-11374. 
12. Ruoslahti, et al., (1987), Science, 238: 491-497. 
13. Hynes, R. O., (1987), Cell, 48: 549-554. 
14. Singer, et al., (1987), J. Cell. Biol., 104: 573-584. 
15. Cheresh, A. (1987), Proc. Natl. Acad. Sci. USA, 84: 6471-6475. 
16. Variani, et al., (1986), In Vivo, 22: 575-582. 
17. Aubert, et al., (1987), J. Biomed. Mater Res., 21: 585-18. 
18. Ohlson, et al., (1978) FEBS Letters, 93, 5-9. 
19. Woods, et al., (1986), EMBO J., 5: 665-670. 
20. Streeter, et al., (1987), J. Cell. Biol., 105: 507-515. 
21. Paul, et al., (1976), J. Appl. Pol. Sci., 20: 609-625. 
22. Humphries, et al., (1986), J. Cell Biol., 103: 2637-2647. 
23. Mohr and Pommerening, (1985), Affinity Chromatography: practical and 
theoretical aspects, Chapter 4. 
24. Costello and McCarthy (1987), Macromolecules, 20: 2819-2828. 
25. J. H. Lee, J. Kopecek, and J. D. Andrade, J. Biomed. Mater. Res. 23, 
351, 1989. 
26. N. P. Desai and J. A. Hubbell, Proceedings of the ACS Division of 
Polymeric Materials: Science and Engineering 62, 731, 1990. 
27. W. R. Gombotz, W. Guanghui, and A. S. Hoffman, J. Appl. Polym. Sci. 37, 
91, 1989. 
28. N. Chisato, K. D. Park, T. Okano, and S. W. Kim, Trans. Am. Soc. Artif. 
Intern. Organs 35, 357, 1989. 
29. M. V. Sefton, G. LLanos, and W. R. Ip, Proceedngs of the ACS Division 
of Polymeric Materials: Science and Engineering 62, 741, 1990. 
30. S. Nagaoka and A. Nakao, Biomaterials 11, 119, 1990. 
31. Y. Mori, S. Nagaoka, H. Takiuchi, et al., Trans. Am. Soc. Artif. 
Intern. Organs 28, 459, 1982. 
32. E. W. Merrill, E. W. Salzman, S. Wan, et al., Trans. Am. Soc. Artif. 
Intern. Organs 28, 482, 1982. 
33. S. K. Hunter, D. E. Gregonis, D. L. Coleman, et al., Trans. Am. Soc. 
Artif. Intern. Organs 29, 250, 1983. 
34. C. Maechling-Strasser, Ph. Dejardin, J. C. Galin, and A. Schmitt, J. 
Biomed. Mater. Res. 23, 1385, 1989. 
35. J. H. Chen and E. Ruckenstein, J. Colloid Interface Sci. 132, 100, 
1989. 
36. M. K. Markwell, Anal. Biochem. 125, 427, 1982. 
37. J. A. Hubbell and L. V. McIntire, Rev. Sci. Instrum. 57, 892, 1986. 
38. N. P. Desai and J. A. Hubbell, J. Biomaterials Sci., Polym. Ed. 1, 123, 
1989.