(637d) Polymer Systems Tuned To Human Blood Outgrowth Endothelial Cell Adhesion

Statement of Purpose: The function of many blood contacting biomedical devices is compromised by thrombus development at the biomaterial/blood interface.  No surface except a healthy endothelium is fully blood compatible.  However, a confluent and functioning endothelial cell layer on a biomaterial surface has not yet been successfully achieved in humans.  In this research we attempt to design a surface tuned to the selective adhesion of human blood outgrowth endothelial cells (HBOECs).  When such a surface is exposed to blood it will scavenge these circulating adult stem cells and the cells will spread, divide, differentiate, and eventually lead to a confluent and functioning endothelial cell layer. 

Methods: The polymer system used in this research is a random acrylic terpolymer polymerized from hexyl methacrylate (HMA), methyl methacrylate (MMA), and methacrylic acid (MAA).  Acrylic polymers were chosen for their ease of synthesis and biocompatibility.  By controlling the molar ratio of HMA, MMA, and MAA we can tailor the physical properties of the polymer [1].  Furthermore, the material has been shown to resist hydrolytic and oxidative degradation in vitro [2].  Selective adhesion of the HBOECs is achieved through the covalent incorporation of HBOEC specific ligands found through phage display screening [3].  The ligands were covalently immobilized to the biomaterial through chain transfer chemistry:  a novel approach for biofunctionalization [4].  To maximize the cellular response the polymer material was electrospun into a fibrous structure with high porosity.  The HBOECs used in this study are obtained by collecting endothelial progenitor cells from fresh human whole blood via density centrifugation and expanded for 4 weeks on rat tail collagen. 

Results/Discussion: The first step in this research was a study of HUVEC and HBOEC adhesion to unfunctionalized polymer surfaces (solution cast, random electrospun mats, and aligned electrospun mats).  Tissue culture polystyrene (TCPS) was used as a positive control.  The HBOEC results from this study are shown in Figure 1 and serve as baseline data to which we can compare HBOEC adhesion to the ligand-derivitized material [5]. 

Figure 1:  HBOEC adhesion to unfuctionalized polymer surfaces after 6 hours of incubation.

From the phage display study, three ligands were chosen for further exploration (one is a negative control) and were terminated by a GGGSC spacer group whereby the cysteine residue, containing a mercaptan functional group, enables the novel incorporation method.  All three of the HBOEC ligands have been successfully immobilized to the polymer and electrospun.  An electron micrograph of electrospun functionalized polymer is shown below in Figure 2. 

Figure 2:  Electrospun biofunctional polymer.

Figure 3 compares the ligand concentration in the polymer before and after electrospinning for selected reactions.  The data indicates that the ligands were not degraded during the electrospinning process. 

Figure 3:  Comparison of ligand concentration of functionalized polymer materials before and after electrospinning.

Data on the HBOEC adhesion and proliferation on ligand containing films and electrospun surfaces will be presented. 

Conclusions:  The research presented in this abstract illustrates the cytocompatibility of the methacrylate terpolymer base material.  Furthermore, we have illustrated the ability to covalently incorporate novel peptide ligands into the polymer material and process this material into fibrous constructs via electrospinning. 

References: 

[1] Fussell GW, et al, Biomaterials. 2004; 25: 2971 ? 2978.

[2] Veleva AN, et al, JBMR. 2005; 74A:  117 ? 123.

[3] Veleva AN, et al, Biotechnology and Bioengineering.  In press. 

[4] Fussell GW, et al, JBMR.  2004; 70A:  265 ? 273.

[5] Veleva AN, et al, JBMR.  In preparation.