Thickness-controllable electrospun fibers promote tubular structure formation by endothelial progenitor cells

Abstract

Controlling the thickness of an electrospun nanofibrous scaffold by altering its pore size has been shown to regulate cell behaviors such as cell infiltration into a three-dimensional (3D) scaffold. This is of great importance when manufacturing tissue-engineering scaffolds using an electrospinning process. In this study, we report the development of a novel process whereby additional aluminum foil layers were applied to the accumulated electrospun fibers of an existing aluminum foil collector, effectively reducing the incidence of charge buildup. Using this process, we fabricated an electrospun scaffold with a large pore (pore size >40 μm) while simultaneously controlling the thickness. We demonstrate that the large pore size triggered rapid infiltration (160 μm in 4 hours of cell culture) of individual endothelial progenitor cells (EPCs) and rapid cell colonization after seeding EPC spheroids. We confirmed that the 3D, but not two-dimensional, scaffold structures regulated tubular structure formation by the EPCs. Thus, incorporation of stem cells into a highly porous 3D scaffold with tunable thickness has implications for the regeneration of vascularized thick tissues and cardiac patch development.

Keywords:

• electrospinning
• nanofibrous scaffold
• tunable thickness
• vascularization
• stem cell

Supplementary material

Flow cytometry analysis

Flow cytometry analysis was performed to characterize the surface markers of the endothelial progenitor cells (EPCs), as previously described.^1,2 EPCs were identified using a fluorescence-activated cell sorter (FACS; BD FACSCanto II; BD Biosciences, San Jose, CA, USA) using labeled endothelial cell (EC) makers, including an anti-human CD31 (BD Biosciences), ti-human VEGFR-2 (BD Biosciences), and anti-human CD144 (BD Biosciences); hematopoietic stem cell (HSC) markers, including anti-human CD34 (BD Biosciences), anti-human CXCR4 (BD Biosciences), and anti-human c-Kit (DakoCytomation, Glostrup, Denmark); and hematopoietic lineage markers, including anti-human CD11b (BD Biosciences), anti-human CD14 (BD Biosciences), and anti-human CD45 (BD Biosciences). Isotype-matched IgG antibodies (BD Biosciences) were used as negative controls.

Figure S1 Characterization of EPCs.

Notes: Flow cytometry analysis was performed to characterize the surface markers of the EPCs. EPCs were labeled with anti-human antibodies to CD31, VEGFR2, CD144, CD34, CXCR4, c-Kit, CD11b, CD14, and CD45. EPCs expressed, (A) EC makers (CD31, VEGFR2, and CD144) and (B) HSC markers (CD34, CXCR-4, and c-Kit), but did not express (C) hematopoietic lineage markers (CD11b, CD14, and CD45). The percentages of cell surface markers expressed on the EPCs are shown.

Abbreviations: CD, cluster of differentiation; CXCR-4, chemokine receptor 4; EC endothelial cell; EPC, endothelial progenitor cell; HSC, hematopoietic stem cell; VEGFR2, vascular endothelial growth factor receptor 2.

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Acknowledgments

This research was supported financially by the National Research Foundation funded by the Korean government (MEST; 2010-0020260, NRF-2012R1A2A2A01045085, and NRF-2014R1A1A2008392) and a grant from the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (A120247).

Disclosure

The authors declare no conflicts of interest in this work.