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.