Structural and cellular characterization of electrospun recombinant human tropoelastin biomaterials

Abstract

An off-the-shelf vascular graft biomaterial for vascular bypass surgeries is an unmet clinical need. The vascular biomaterial must support cell growth, be non-thrombogenic, minimize intimal hyperplasia, match the structural properties of native vessels, and allow for regeneration of arterial tissue. Electrospun recombinant human tropoelastin (rTE) as a medial component of a vascular graft scaffold was investigated in this study by evaluating its structural properties, as well as its ability to support primary smooth muscle cell adhesion and growth. rTE solutions of 9, 15, and 20 wt% were electrospun into sheets with average fiber diameters of 167 ± 32, 522 ± 67, and 735 ± 270 nm, and average pore sizes of 0.4 ± 0.1, 5.8 ± 4.3, and 4.9 ± 2.4 µm, respectively. Electrospun rTE fibers were cross-linked with disuccinimidyl suberate to produce an insoluble fibrous polymeric recombinant tropoelastin (prTE) biomaterial. Smooth muscle cells attached via integrin binding to the rTE coatings and proliferated on prTE biomaterials at a comparable rate to growth on prTE coated glass, glass alone, and tissue culture plastic. Electrospun tropoelastin demonstrated the cell compatibility and design flexibility required of a graft biomaterial for vascular applications.

Figure 1

Comparison of electrospun rTE fibers from 9, 15, and 20 wt% solutions. The rTE fibers were randomly oriented. Fiber diameters were directly proportional to the concentration of the rTE solution. The electrospun rTE fibers from 9 and 15 wt% solutions had only round cross-sections, while isolated electrospun rTE fibers from 20 wt% solutions had flat cross-sections. SEM micrographs at magnifications of 1000X, 5000X, and 10000X.

Figure 2

SEM micrograph of extracted native elastin from a porcine carotid artery. Scale bars indicate 10 μm.

Figure 3

TEM micrograph of substructure of 15 wt% electrospun rTE fibers. Voids are visualized within the fibers. Scale bar indicate 500nm.

Figure 4

SEM micrograph of uncross-linked electrospun rTE (left) and cross-linked electrospun prTE (right) fibers produced from a 15 wt% rTE solution. No change in fiber structure was seen using the DSS cross-linker.

Figure 5

Characterization of the adhesion of SMCs to adsorbed rTE. SMCs were allowed to adhere to rTE, fibronectin (Fn), collagen type I (Col), or tissue culture treated plastic (TCP) in either adhesion media or adhesion media with a blocking reagent: EDTA, Pertussis toxin, or lactose. EDTA significantly reduced SMC adhesion to the rTE and Fn coated wells. *p<0.05, compared to the same coating with adhesion media, ANOVA with a Tukey post-hoc test.

Figure 6

Morphology of SMCs seeded onto electrospun 15 wt% prTE for (A) 24 and (B) 48 hours. Confocal images of prTE fibers (green) with actin cytoskeleton-phalloidin (red) and nuclei (blue) of SMCs. SMC pseudopodia made attachments to individual prTE fibers (arrows in A), SMC concentrations increased between 24 and 48 hours, and SMCs formed actin stress fibers (arrows in B). Scale bar indicates 20 μm.

Figure 7

SMC proliferation on electrospun prTE, coated prTE, lysine D glass, and tissue culture plastic (TCP) over a 7 day time course. Cell metabolic activity was quantified using alamarBlue assay and the data normalized to the fluorescence reading at day 1. Data are average of 3 lots of rTE solutions.