(10d) Tailoring the Interface of Methacrylic Terpolymer Biomaterials for Endothelialization

Statement of Purpose: The function of many blood contacting biomedical devices is undermined by biomaterial-induced thrombosis.  Many biomaterials scientists believe that the only truly blood compatible surface is a functioning endothelium ? the monolayer of endothelial cells which lines all native vasculature.  The work presented in this abstract discusses the design of a novel methacrylic terpolymer system and the subsequent topographical, chemical, and biological surface modifications use to tailor the interface for the specific adhesion and growth of either human umbilical vein endothelial cells (HUVECs, a population of mature endothelial cells) or human blood outgrowth endothelial cells (HBOECs, a population of multipotent and circulating adults stem cells with high proliferation capacities and rates and the ability to differentiate into cells with endothelial phenotype). 

Materials and Methods:  The biomaterial system is a random copolymer produced through the free radical copolymerization of hexyl methacrylate (HMA), methyl methacrylate (MMA), and methacrylic acid (MAA).  2 mol % of MAA was incorporated into all polymers to enable post-synthesis derivatizations while the ratio of HMA to MMA was used to tune the glass transition ? and thus the mechanical properties ? of the biomaterial system.¹  Polymers were then electrospun into three dimensional scaffolds with either random or aligned fiber architectures.²  The materials were functionalized with either the RGD tripeptide unit to enhance the integrin mediated binding of mature endothelial cells or novel peptide groups to up-regulate the attachment of the HBOEC adult stem cell population.³  In order to minimize the adsorption of proteins and the adhesion of undesired cell types to the biomaterial interface polymer formulations were produced which contained either poly(ethylene oxide) or sulfobetaine chemical groups.⁴ 

Results:  Biomaterials with moduli ranging from 3 ? 500 MPa were produced.  Interestingly, it was found that the stiffness of the terpolymer does not affect the ability of HUVECs to adhere and proliferate on the surface.  However, HUVECs adherent to the tissue culture polystyrene positive control showed a higher degree of cell spreading indicating that improvements can be made to increase the cytocompatibility of the terpolymer.¹ 

Polymers were electrospun into three dimensional scaffolds and it was found that materials which were above their glass transitions at ambient conditions produced scaffolds with fused fibers and low void fractions (18%) while glassy materials produced scaffolds with discrete fibers and high void fractions (85%).  By electrospinning onto a rotating collector scaffolds with aligned fiber architectures were produced.  Scanning electron micrographs of the scaffolds are shown in Figure 1.  It was found that HUVECs seeded onto the low void fraction materials (Figure 1, panel A) responded to the topography favorably as illustrated by increased cell proliferation, enhanced enzymatic activity, and a higher degree of cell spreading.  Cells seeded onto aligned fiber scaffolds did not show increased rates of proliferation; however, they did obtain an elongated morphology more reminiscent of what is seen in vivo. ²

Figure 1:  Electrospun scaffolds:  (A) slightly porous, (B) highly porous, (C) partially aligned, (D) highly aligned. 

Chain transfer chemistry (a novel biofunctionalization technique) was used to incorporate either the RGD-tripeptide unit or novel peptides discovered through phage display to enhance HUVEC and HBOEC adhesion, respectively.  As shown in Figure 2, up-regulated and specific cellular attachment was observed to the peptide-modified materials (HBOEC data shown); however, these results were only obtained in serum free media conditions possibly due to the adsorption of serum proteins to the interface.³ 

Figure 2:  HBOEC adhesion to peptide modified materials.

To create materials resistant to the adsorption of proteins and the attachment of undesired cell types, new formulations of terpolymer were synthesized.  The MAA was removed and instead polymers containing various amounts of either poly(ethylene glycol) or sulfobetaine methacrylate repeat units were polymerized.  In each case materials polymerized from ≥ 15 mol % of the new comonomer exhibited robust resistance to biofouling as probed through fibrinogen adsorption studies and cell adhesion assay as illustrated in Figure 3.⁴ 

Figure 3:  Fibrinogen adsorption to materials containing either sulfobetaine or poly(ethylene oxide) groups. 

Conclusions:  A novel and cytocompatible biomaterial system with tunable mechanical properties was developed.  Fibrous scaffolds were fabricated through electrospinning and HUVECs adherent to the low porosity scaffolds showed increased cellular proliferation, enzymatic activity, and cell spreading.  Peptide ligands were incorporated into the materials through chain transfer chemistry and specific up regulation of either HUVEC or HBOEC adhesion was observed.  However, these results were only obtained in serum free media possibly due to the interfacial adsorption of serum proteins.  Therefore, terpolymer formulations containing either poly(ethylene oxide) or sulfobetaine chemical groups were synthesized and shown to drastically reduce the adsorption of proteins and attachment of undesired cell types (ex. platelets).