(314g) Selective Endothelial Progenitor Cell Adhesion and Growth On Peptide-Linked Scaffolds

Selective Endothelial Progenitor Cell Adhesion and Growth on peptide-linked Scaffolds

Xin Wang¹,Rustin Shenkman¹, Dan
Heath¹, and Stuart Cooper¹

¹The Ohio State University, Dept. Chemical and
BioMolecular Engineering.

Statement of
Purpose: A set of peptides
from a phage display library has recently been found to bind specifically to
adult human blood outgrowth endothelial cells (BOEC) but not HUVEC. We
previously incorporated these peptides into terpolymer methacrylate films,
obtaining materials that selectively adhered BOEC^[1]. However, these polymers lost selectivity in the
presence of serum proteins. In order to prepare improved functional
biomaterials for in vivo applications, we have synthesized a new generation of polymers that incorporate
succinimide acryl-PEG tethers. Candidate
peptides, such as
"TPS" and "GHM" (Table 1), have been incorporated into polymers as well as the non-fouling poly ethylene glycol (PEG) motif. Such templates are being studied for their affinity for Endothelial Progenitor Cells (EPC) with and without shear stress.

Methods: Nonfouling terpolymer is produced by free radical
polymerization of methacrylate monomers, methacrylated PEG, and a succinimide-
or maleimide- methacrylate. EPCs are either procured from adult human peripheral
blood (HBOECs) or directly purchased from Lonza (ECFCs).

EPCs and Human Umbilical Vein Endothelial
cells (HUVECs) are cultured on the polymer to assess adhesion and growth
responses to the peptides. Adhesion is measured by DNA assay and actin staining
for static culture experiments. In dynamic experiments, cell adhesion was
carried out in a radial flow chamber allowing real time quantification of cell
adhesion as a function of shear rate. The radial flow chamber was mounted on a
Nikon inverted microscope, and ImagePro software was used to automatically
select observation fields and count cell number^[2].

Results: Phage display peptides have been
synthesized and incorporated into the methacrylate terpolymer in addition to a
positive control, RGD, and a negative control, RGE. Preliminary data
demonstrate selective Adult
BOEC adhesion from 10% serum to a
scaffold linked with the promising
peptides, especially GHM (Figure 1b).
HUVEC and Lonza ECFC show very low adhesion to surfaces except
RGD and TCPS.

A comparison of HUVEC,
Adult
BOEC and Lonza ECFC attachment
at 10 minutes on RGD linked
films under flow is shown in
Figure 2. For all three cell types, the number of adhered cells increased as
the flow rate decreased. The degree of Lonza ECFCs
adhesion was much higher than that of HUVECs over the entire shear rate range
examined. The cell density of HBOECs
was similar with that of Lonza
ECFCs when the shear rate is low. At high shear rates, adult BOECs are less adhesive compared with cord blood cells but still more
adhesive than HUVECs.

Conclusions: . A novel methacrylate polymer has been
synthesized with both a PEG component to minimize protein adhesion and
nonspecific cell binding as well as a peptide binding component. Cast films of
this polymer incorporating the GHM peptide significantly increased the contact
adhesion of Adult BOEC in the presence of serum
proteins.

The highly proliferative EPCs^[3] also
have higher adhesion propensity than mature EC under flow
and thus may have the potential to contribute to endothelialization of a device. Ongoing work aims to demonstrate selective EPC affinity for
electrospun peptide containing scaffolds under flow conditions. This would permit rapid EPC expansion directly from
blood samples and support vascular graft endothelialization.

Figure 1. (a),(b)&(c) HUVEC
& EPC static adhesion on peptide-linked scaffolds. (d) Fluorescence
images of Adult BOEC showing
cobblestone morphology (Green FITC Ulex lectin and red DIL acetylated-LDL).

Figure 2. Dynamic
adhesion of HUVEC, Adult BOEC and Lonza ECFC to RGD linked polymer films after 10 minutes

Table 1. Peptides from the phage display library

References: 

1. Veleva A.
Biomaterials. 2008; 29:3656-3661.

2. Dickinson RB, et al, AIChE J. 1995; 41:2160-2174

3. Lin Y, et al, J of Clin Invest. 2000; 105:
71-77