(567h) Endothelial Progenitor Cell Adhesion and Growth On Peptide-Linked Scaffolds with and without Flow

Endothelial Progenitor Cell Adhesion and Growth on peptide-linked Scaffolds with and without flow

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

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

Statement of
Purpose: Recent literature
(Hirschi K. Arterioscler Thromb
Vasc Biol. 2008; 28:1584-1595) has
suggested that a key mediator of the endothelium repair mechanism is the bone
marrow-derived mononuclear cell, endothelial progenitor cell (EPC). But EPC are especially rare, difficult to
define by surface antigens, and expand to colonies very slowly in vitro.

A possible clinical
application might be to adhere  EPC and expand them quickly on 3D
scaffolds embedded with growth factors and/or affinity tags specific for EPC.
Affinity tags now exist in the form of peptides, isolated via phage display,
that bind specifically to EPC but not adult endothelial cells (Veleva A.
Biomaterials. 2008; 29:3656-3661). Tentative names are based on peptide
sequences, e.g. "TPS", and "GHM". Candidate peptides have been incorporated
into a novel non-fouling polymer. This polymer may be cast into films or
electrospun into 3D nonwoven meshes. Such templates are being studied for their
specific affinity for 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 (Dickinson
RB, et al, Bioengineering, Food and Natural Products. 1995; 41:2160-2174).

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 EPC adhesion to scaffold linked with the promising peptides,
especially GHM (Figure 1C). HUVEC shows very low adhesion to surfaces except
RGD and TCPS.

A comparison of EDTA-lifted HUVEC and EPC
attachment at 10 minutes on TCPS is shown in Figure 2. The number of adherent
HUVECs is less than the number of EPCs for the range of shear rates examined (5
to 30s-1). At higher shear rates, the adhesion ability of EPCs is higher by a
factor of 3 to 4 compared with
that of HUVECs.

Figure 1. (A)&(B)
Fluorescence images of HUVECs
adherent to RGE and RGD after 2 hours of culture (C)  HUVEC & EPC static adhesion
on peptide-linked scaffolds

Figure 2. Dynamic adhesion of HUVECs and EPCs
to TCPS and base polymer (H20) after 10 minutes

Conclusions: Methacrylate terpolymers are amenable to
processing, and can incorporate virtually any short peptide or combination of
peptides. Phage display peptides with specific affinity for human peripheral
EPC have been synthesized and incorporated into substrates for cell adhesion:
cast films, and electrospun scaffolds. Increased adhesion of cells to the RGD
binding sequence proves that the peptides remain active. Low HUVEC adhesion and
relatively high attachment of EPC on GHM illustrates specific binding ability
of the substrate.

The highly
proliferative EPCs (Lin Y, et
al, J of Clin Invest. 2000; 105: 71-77) also have higher adhesion propensity than 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 scaffolds. This would permit rapid EPC expansion directly from blood
samples and support vascular graft endothelialization.