Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering

Abstract

Electrospinning using natural proteins or synthetic polymers is a promising technique for the fabrication of fibrous scaffolds for various tissue engineering applications. However, one limitation of scaffolds electrospun from natural proteins is the need to cross-link with glutaraldehyde for stability, which has been postulated to lead to many complications in vivo including graft failure. In this study, we determined the characteristics of hybrid scaffolds composed of natural proteins including collagen and elastin, as well as gelatin, and the synthetic polymer poly(epsilon-caprolactone) (PCL), so to avoid chemical cross-linking. Fiber size increased proportionally with increasing protein and polymer concentrations, whereas pore size decreased. Electrospun gelatin/PCL scaffolds showed a higher tensile strength when compared to collagen/elastin/PCL constructs. To determine the effects of pore size on cell attachment and migration, both hybrid scaffolds were seeded with adipose-derived stem cells. Scanning electron microscopy and nuclei staining of cell-seeded scaffolds demonstrated the complete cell attachment to the surfaces of both hybrid scaffolds, although cell migration into the scaffold was predominantly seen in the gelatin/PCL hybrid. The combination of natural proteins and synthetic polymers to create electrospun fibrous structures resulted in scaffolds with favorable mechanical and biological properties.

Figure 1

Scanning electron micrographs of electrospun fibers consisting of collagen5%/elastin2.5% (A), collagen10%/elastin5% (B), collagen10%/elastin5% cross-linked in glutaraldehyde vapor for 2h (C), collagen10%/elastin5%/PCL10% (D), gelatin5% (E), gelatin10% (F), gelatin10% cross-linked in glutaraldehyde vapor for 2h (G) and gelatin10%/PCL10% (H). The scale bar shown applies to all images and equals 20 μm.

Figure 2

Simultaneous transmitted light-differential interference-contrast (DIC), multiphoton-induced autofluorescence and SHG imaging demonstrates that the majority of the electrospun fibers are autofluorescent at wavelengths of 760 nm (green: elastic fibers) and 840 nm (red: collagen), revealing that the fabricated fibers are composed of both collagen and elastin. Scale bar equals 50 μm.

Figure 3

DAPI-stained hASCs cultured on collagen10%/elastin5%/PCL10% (A) and gelatin10%/PCL10% (C) for 2 weeks. Scanning electron micrographs confirm the attachment of hASCs on collagen10%/elastin5%/PCL10% (B) and gelatin10%/PCL10% (D).

Figure 4

H&E-stained hASCs cultured on collagen10%/elastin5%-PCL10% (A) and gelatin10%/PCL10% (B) for 2 weeks.

Figure 5

Images show DAPI-stained hASCs that migrated throughout collagen10%/elastin5%/PCL10% (A-E) and gelatin10%/PCL10% (F-J) scaffolds.