Embossed Membranes with Vascular Patterns Guide Vascularization in a 3D Tissue Model

Abstract

The vascularization of three-dimensional (3D) tissue constructs is necessary for transporting nutrients and oxygen to the component cells. In this study, a vacuum forming method was applied to emboss a vascular pattern on an electrospun membrane so that guided vascular structures could develop within the construct. Two- or six-layer constructs of electrospun membranes seeded with endothelial cells and pericytes were stacked and subcutaneously implanted into mice. Blood vessel formation in the implanted constructs with six alternating layers of flat membranes and membranes embossed with a blood vessel pattern was observed after two weeks of implantation. The formation of blood vessels was observed along the embossed blood vessel pattern in the structure of the embossed membrane laminated at four weeks and eight weeks. Vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang-1) were highly expressed in the vascularized structures. Therefore, we demonstrated that a structure capable of producing a desired blood vessel shape with electrospun membranes embossed with a blood vessel pattern can be manufactured, and that a variety of structures can be manufactured using electrospun membranes in the tissue engineering era.

Keywords: 3D tissue; embossed membrane; guided vascularization; tissue-engineered vascularization.

Figure 1

Generation of electrospun membranes with embossed patterns for guided vascularization of seeded cells. (A) Schematic diagram of forming of embossed sheet followed by seeding with human endothelial and mouse fibroblast cells. (B) Cell-seeded embossed sheets were stacked and cultured for 3 days before implantation into subcutaneous pockets in mice. (C) Experimental groups for in vivo experiment included stacks of two flat membranes (group 2× flat), an embossed membrane stacked on a flat membrane (group 2× embossed), six layers of flat membranes (group 6× flat), and six layers of alternating flat and embossed membranes (group 6× embossed).

Figure 2

Embossed polycaprolactone (PCL) membrane. (A) SEM imaging shows embossed pattern of vacuum-formed electrospun PCL membrane. (B,C) Further magnification of formed PCL embossed membrane shows detail of embossing and woven electrospun PCL fibers. (D) Confocal image and (E) orthogonal views of human umbilical vein endothelial cells (HUVECs) on embossed PCL membrane on day three. Scale bars are 200 μm. Green: F-Actin, red: HUVEC, blue: DAPI.

Figure 3

Embossed sheets after subcutaneous implantation in mice at one, two, four, and eight weeks.

Figure 4

Hematoxylin and eosin-(H&E) stained cross-section images of experimental groups at one, two, four, and eight weeks after implantation. Arrowheads show red blood cells indicating blood vessel formation. Dashed lines indicate edge of transplanted embossed sheets.

Figure 5

Immunofluorescence images showing differences in human mitochondria expression in cross-sections of experimental groups at one, two, four, and eight weeks. Dashed lines indicate edge of transplanted embossed sheets. Green: human mitochondria, red: HUVEC, blue: DAPI.

Figure 6

Immunofluorescence images showing differences in VEGF expression in cross-sections of experimental groups at one, two, four, and eight weeks. Dashed lines indicate edge of transplanted embossed sheets. Green: VEGF, red: HUVEC, blue: DAPI. Scale bars are 200 μm.

Figure 7

(A) Immunofluorescence images showing differences in Ang-1 expression in cross-sections of experimental groups at one, two, four, and eight weeks. Dashed lines indicate edge of transplanted embossed sheets. Arrowheads indicate Ang-1 expression in images after staining. Green: Ang-1, red: HUVEC, blue: DAPI. Scale bars are 200 μm. (B) Mean percent area of Ang-1 at four weeks within implanted layered sheet regions of interests (ROIs) in each experimental group. (C) Mean percent area of Ang-1 expression of group 6x embossed at one, two, four, and eight weeks. (n = 4; data represent average ± SD, * p < 0.05).

Figure 7

(A) Immunofluorescence images showing differences in Ang-1 expression in cross-sections of experimental groups at one, two, four, and eight weeks. Dashed lines indicate edge of transplanted embossed sheets. Arrowheads indicate Ang-1 expression in images after staining. Green: Ang-1, red: HUVEC, blue: DAPI. Scale bars are 200 μm. (B) Mean percent area of Ang-1 at four weeks within implanted layered sheet regions of interests (ROIs) in each experimental group. (C) Mean percent area of Ang-1 expression of group 6x embossed at one, two, four, and eight weeks. (n = 4; data represent average ± SD, * p < 0.05).

Figure 8

(A,B) Fluorescent images showing differences in Fluorescein isothiocyanate (FITC)-dextran perfusion, indicating connection between host and implanted sheets (group 6× embossed) in vivo. Dashed lines indicate edge of transplanted embossed sheets. Green: FITC-dextran, red: HUVEC. (C,D) Immunofluorescence images showing α-SMA expression in cross-sections of group 6× embossed at one and four weeks. Green: α-SMA, red: HUVEC, blue: DAPI. Arrowheads indicate α-SMA expression.