Interactions between endothelial cells and electrospun methacrylic terpolymer fibers for engineered vascular replacements

Abstract

A compliant terpolymer made of hexylmethacrylate (HMA), methylmethacrylate (MMA), and methacrylic acid (MAA) intended for use in small diameter vascular graft applications has been developed. The mechanical properties and in vitro biostability of this terpolymer have been previously characterized. The goal of this investigation was to examine the interactions between endothelial cells and the new terpolymer and to evaluate endothelial cell function. Electrospinning was used to produce both oriented and random terpolymer fiber scaffolds. Smooth solution cast films and tissue culture polystyrene were used as negative and positive controls, respectively. Human blood outgrowth endothelial cells and human umbilical vein endothelial cells were incubated with the test and control samples and characterized with respect to initial cell attachment, proliferation, viability, and maintenance of the endothelial cell phenotype. It was found that the terpolymer is cytocompatible allowing endothelial cell growth, with random fibers being more effective in promoting enhanced cellular activities than oriented fibers. In addition, endothelial cells cultured on these substrates appeared to maintain their phenotype. The results from this study demonstrate that electrospun HMA:MMA:MAA terpolymer has the potential to be used successfully in fabricating small diameter blood vessel replacements.

Figure 1

SEM micrographs of electrospun HMA:MMA:MAA (20:78:2) terpolymer fibers from 13 w/v % acetone-ether solution (A) oriented fibers, (B) random fibers, (C) oriented fibers—high magnification, (D) random fibers—high magnification. To achieve fiber orientation, the terpolymer fibers were deposited on a rotating collector.

Figure 2

Diameter distribution of the oriented and random HMA:MMA:MAA terpolymer fibers (top). Pore area distribution of the HMA:MMA:MAA fiber meshes (bottom).

Figure 3

Bar graph depicting cell attachment as a function of plating density for HBOEC (top) and HUVEC (bottom) after 6 h of cell culture on electrospun terpolymer and control surfaces. The §, #, and † symbols indicate statistically significant (p < 0.05) higher cell numbers with respect to solution cast film (§), oriented (#), and random (†) fibers, respectively. For example, a “# †” over the value bar means that condition has statistically higher attachment than either the oriented or the random fiber specimens.

Figure 4

Bar graph depicting cell proliferation of HBOEC (top) and HUVEC (bottom) cultured on solution cast terpolymer films, oriented fibers, random fibers, and control TCPS surfaces. The §, #, and † symbols have the same meaning as before however “*” is now used to indicate a significantly increased cell number with respect to preceding time point. For example, * over TCPS on day 9 in the bottom panel indicates a significantly increased cell number with respect to TCPS on day 6.

Figure 5

Metabolic activity of HBOEC and HUVEC as measured by CCK-8 mitochondrial activity assay after 9 days of culture on electrospun terpolymer and control surfaces. This is a direct comparison between the cells on the different surfaces without considering the varying cell numbers on each surface. The § and # symbols indicate statistically significant (p < 0.05) higher overall metabolic cell activity with respect to solution cast film (§) and oriented fibers (#).

Figure 6

von Willebrand factor expression and localization by HBOEC seeded on (A) solution cast films, (B) oriented fibers, (C) random fibers, (D) TCPS after 9 days of in vitro cell culture; scale bar 50 µm. The double arrowhead indicates the orientation of the fibers. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]