In vitro endothelialization of electrospun terpolymer scaffolds: evaluation of scaffold type and cell source

Abstract

A family of methacrylic terpolymer biomaterials was electrospun into three-dimensional scaffolds. The glass transition temperature of the polymer correlates with the morphology of the resulting scaffold. Glassy materials produce scaffolds with discrete fibers and large pore areas (1531±1365 μm(2)), while rubbery materials produce scaffolds with fused fibers and smaller pore areas (154±110 μm(2)). Three different endothelial-like cell populations were seeded onto these scaffolds under static conditions: human umbilical vein endothelial cells (HUVECs), adult human peripheral blood-derived outgrowth endothelial cells, and umbilical cord blood-derived human blood outgrowth endothelial cells. Cellular behavior depended on both cell type and scaffold topography. Specifically, cord blood-derived outgrowth endothelial cells showed more robust adhesion and growth on all scaffolds in comparison to other cell types as measured by the density of adherent cells, the number of proliferative cells, and the enzymatic activity of the adherent cells. Peripheral blood-derived outgrowth cells exhibited less ability to inhabit the terpolymer interfaces in comparison to their cord blood-derived counterparts. HUVECs also exhibited less of a capacity to colonize the terpolymer interfaces in comparison to the cord blood-derived cells. However, the mature endothelial cells did show scaffold-dependent behavior. Specifically, we observed an increase in their ability to populate the low-porosity scaffolds. All cells maintained an endothelial phenotype after 1 week of culture on the electrospun scaffolds.

FIG. 1.

Schematic of terpolymer molecular composition.

FIG. 2.

Scanning electron micrographs of terpolymer scaffolds of various chemical compositions: (A) H30 low magnification, (B) H30 high magnification, (C) H70 low magnification, (D) H70 high magnification, (E) H80 low magnification, and (F) H80 high magnification. When originally sized low-magnification images are at 100× magnification and high-magnification images are 300× magnification.

FIG. 3.

Scatter plots of (A) fiber diameter and (B) pore area data collected from quantification of scanning electron micrographs. About 150 fiber diameter and pore area data points were collected per biomaterial. Horizontal hash marks indicate the averages of these data.

FIG. 4.

Fluorescence microscopy images of endothelial cell nuclei after 5 days of incubation on terpolymer scaffolds. DAPI (blue) stains all cells within the electrospun scaffold, while EdU (red) indicates cells with active DNA synthesis during the incubation period. DAPI, 4′,6-diamidino-2-phenylindole; EdU, 5-ethynyl-2′-deoxyuridine; HUVECs, human umbilical vein endothelial cells; OECs, outgrowth endothelial cells; TCPS, tissue culture polystyrene. Color images available online at www.liebertpub.com/tea

FIG. 5.

Quantification of adherent endothelial cell density on terpolymer scaffolds after 5 days of incubation. To the right of the bar chart, you will find a table that denotes statistical differences. Means in the chart which are labeled with the same letter are not significantly different. For instance, the density of adult OECs on TCPS and cord OECs on TCPS are not statistically different because they are both labeled with A. pbOECs, peripheral blood-derived outgrowth endothelial cells; cbOECs, cord blood-derived outgrowth endothelial cells.

FIG. 6.

Quantification of endothelial cell proliferation on terpolymer scaffolds after 5 days.

FIG. 7.

Enzymatic activity of adherent endothelial cells after 7 days of culture.

FIG. 8.

Fluorescence microscopy images of lectin-stained cells after 5 days of culture on terpolymer scaffolds.