Electrospinning-Generated Nanofiber Scaffolds Suitable for Integration of Primary Human Circulating Endothelial Progenitor Cells

Abstract

The extracellular matrix is fundamental in order to maintain normal function in many organs such as the blood vessels, heart, liver, or bones. When organs fail or experience injury, tissue engineering and regenerative medicine elicit the production of constructs resembling the native extracellular matrix, supporting organ restoration and function. In this regard, is it possible to optimize structural characteristics of nanofiber scaffolds obtained by the electrospinning technique? This study aimed to produce partially degraded collagen (gelatin) nanofiber scaffolds, using the electrospinning technique, with optimized parameters rendering different morphological characteristics of nanofibers, as well as assessing whether the resulting scaffolds are suitable to integrate primary human endothelial progenitor cells, obtained from peripheral blood with further in vitro cell expansion. After different assay conditions, the best nanofiber morphology was obtained with the following electrospinning parameters: 15 kV, 0.06 mL/h, 1000 rpm and 12 cm needle-to-collector distance, yielding an average nanofiber thickness of 333 ± 130 nm. Nanofiber scaffolds rendered through such electrospinning conditions were suitable for the integration and proliferation of human endothelial progenitor cells.

Keywords: endothelial cells; nanofiber scaffolds; tissue engineering.

Figure 1

Description of the electrospinning system. The parts of the equipment and their location are shown. (1) High-voltage power supply; (2) infusion pump; (3) needle with horizontal movement; (4) manifold with rotating chuck.

Figure 2

Nanofibers’ diameter and electrospinning technique. The figure shows the nanofiber diameters obtained after the variation in the following working parameters: (A) Voltage; (B) Voltage and infusion rate; (C) Voltage and rotating collector speed; and (D) Voltage and collector-to-needle distance. (*) Statistical difference p < 0.0001.

Figure 3

SEM images of nanofibers morphology. (A,B) Particle/aberration within the fibers, observed at condition of 10 kV, 0.06 mL/h, 2000 rpm and 10 cm needle-to-collector distance. (C,D) The panels show partial particle formation at a condition of 10 kV, 0.06 mL/h, 2000 rpm and 12 cm needle-to-collector distance. (E,F) Fibers obtained at spinning conditions of 15 kV, 0.06 mL/h, 1000 rpm and 10 cm needle-to-collector distance. (G,H) Fibers obtained at spinning conditions of 15 kV, 0.06 mL/h, 2000 rpm and 10 cm needle-to-collector distance. Black arrows indicate the presence of aberrations. The scale bar represents 5 microns.

Figure 4

Immunophenotype and proliferation kinetics of EPCs. (A) Immunophenotype of EPCs obtained after 9 days of subculture. Morphology at bright field, DAPI nucleus stain, CD133 (green), CD31 (red) and merge are shown. (B) Proliferation kinetics hEPCs during 7 days of culture, either on TCP (control, growth 0.03 cells∙h^-1 and a doubling time of 23.10 h during the exponential growth phase) or scaffolds (growth 0.0056 cells∙h^-1 and a doubling time of 123 h during the exponential growth phase). Cells showed specificity; the scale bar represents 100 microns.

Figure 5

SEM images of EPCs integrated into scaffolds. Microphotograph showing the integration of EPCs to partially degraded collagen nanofibers (280 ± 193 nm diameter), obtained by electrospinning technique at 15 kV, 0.06 mL/h, 2000 rpm and 10 cm needle-to-collector distance working parameters. Detailed fiber–hEPCs interaction, showing cell adhesion, elongation and alignment along the fiber, are provided. Lower power field is also provided in the left corner. hEP cell integration was evaluated after 6 days of culture on DPC nanofibers. Scale bar represents 50 µm. At the bottom, an analysis of cell orientation according to scaffold is shown.