(318f) Shear-Dependent Endothelial Cell Attachment to Polymeric Biomaterials

Statement of Purpose: This research studies the adhesion of human Umbilical Endothelial Cells (HUVEC) and Endothelial Colony Forming Cells (ECFC) to polymeric biomaterials under shear flow. EPC are present in small quantities in peripheral blood; their relative concentration has been linked to a number of disease states.  Polymeric materials have been designed: 1) to scavenge and maintain these rare circulating cells for fast and accurate assay devices and 2) to enhance the endothelialization of blood-contacting medical implants.

Methods: As a base polymer we use a methacrylic terpolymer (H20 or H90) polymerized from hexyl methacrylate (HMA), methyl methacrylate (MMA) and methacrylic acid (MAA)^[1], Methacrylated polyethyleneglycol (PEG) was substituted for some of the HMA and polymers were produced  by free radical polymerization. Tissue culture polystyrene (TCPS) and glass served as control surfaces. Cell-binding peptides were bound to the base polymer through two mechanisms: 1) by a chain-transfer mechanism to the bulk polymer, and 2) by covalent binding of the peptide to a succinimide- or  maleimide- terminated methacylated polyethylene glycol. Peptides were also bound to glass slides via heterobifunctional crosslinkers. The peptides included RGD and several candidates from phage display ^[2] that bind specifically to EPC.

Cell adhesion was examined in both parallel-plate and radial flow chambers, which allow real-time quantification of cell adhesion as a function of shear rate. The chambers were mounted on a Nikon inverted microscope, and ImagePro® (ver 5.5) software was used to automatically select observation fields and count cell number ^[3].

Results and discusssion: HUVEC and ECFC adhesion studies were carried out on TCPS and H20. At low flow rates, both cell types adhered linearly with time, but at higher shear rates, a steady state was reached at lower cell densities illustrating shear dependent cellular adhesion as observed in Figure 1.

Figure 1. Real-time shear dependent ECFC adhesion to TCPS

A comparison of EDTA-lifted HUVEC and ECFC cell attachment at 10 minutes on TCPS is shown in Figure 2.

Figure 2. Dynamic adhesion of HUVECs and ECFCs to TCPS and H20 after 10 minutes

The number of adherent HUVECs is less than the number  

of ECFCs for the range of shear rates examined (5 to 30s-1) illustrating that these adult circulating stem cells are more  adhesive to these biomaterials under flow. At higher shear rates, the adhesion ability of ECFCs is higher by a factor of 3 to 4 compared with that of HUVECs (Figure 2). Figure 2 also shows that ECFCs are less sensitive to shear rate when adhering to H20. 

A comparison of HUVEC interactions between different polymer surfaces under flow is shown in Figure 3. The attachment of cells on pegylated polymer was significantly lower than on all other surfaces. The differences between H90 and RGD incorporated H90 may indicate that the RGD moieties interact with the receptors on the cell membrane and successfully retain more cells on the polymer surface.

Figure 3. Real time HUVEC adhesion to polymer surfaces at 5 s^-1 shear rate

Conclusions: Our results suggest that the highly proliferative ECFCs^[4] also have higher adhesion propensity than EC under flow and thus may have the potential to contribute to endothelialization of a vascular graft. The presence of RGD also increases HUVEC attachment under flow. Further work on the methacrylate terpolyer biomaterials will involve the incorporation adhesion ligands to promote specific binding of ECFC ^[5].

Reference:

1. Fussel GW, et al., Biomaterials. 2004; 25: 2971-2978

2. Veleva, A., et al., Biotechnol Bioeng. 2007; 98: 307-311 

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

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

5. Anka NV, et al, Biomaterials. 2008