An Aligned Patterned Biomimetic Elastic Membrane Has a Potential as Vascular Tissue Engineering Material

Abstract

Cardiovascular disease is the leading cause of death worldwide, with an annual mortality incidence predicted to rise to 23.3 million worldwide by 2030. Synthetic vascular grafts as an alternative to autologous vessels have shown satisfactory long-term results for replacement of large- and medium-diameter arteries, but have poor patency rates when applied to small-diameter vessels. Nanoparticles with low toxicity, contrasting agent properties, tailorable characteristics, targeted/stimuli- response delivery potential, and precise control over behavior (via external stimuli such as magnetic fields) have made possible their use for improving engineered tissues. Poly (styrene-block-butadiene-block-styrene) (SBS) is a kind of widely used thermoplastic elastomer with good mechanical properties and biocompatibility. Here, we synthesized anthracene-grafted SBS (SBS-An) by the method for the fabrication of a biomimetic elastic membrane with a switchable Janus structure, and formed the patterns on the surface of SBS-An under ultraviolet (UV) light irradiation. By irradiating the SBS-An film at different times (0, 10, 20, 30, 60, and 120 s), we obtained six well-ordered surface-patterned biomimetic elastic film with SBS-An at different heights in the thickness direction and the same distances of intervals (named sample-0, 10, 20, 30, 60, and 120 s). The structural effects of the SBS-An films on the adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) were studied, and the possible mechanism was explored. When the HUVECs were cultured on the SBS-An films at different heights in the thickness direction, the sample-30 s with approximately 4 μm height significantly promoted adhesion of the HUVECs at the early stage and proliferation during the culture period compared with the samples of the lower (0, 10, and 20 s) and higher (60 and 120 s) heights. Consistent with this, the sample 30 s showed a higher stimulatory effect on the proliferation- and angiogenesis-related genes. These results suggest that SBS-An with appropriate height could efficiently control bioactivities of the biomimetic elastic membrane and might have great potential in vascular tissue engineering application.

Keywords: HUVECs; anthracene-grafted SBS; biocompatibility; nanoparticles; vascular tissue engineering.

FIGURE 1

(A) The chemical structure of the anthracene modified SBS. Laser scanning confocal microscopy (LSCM) images showing patterns written with different geometric configuration controlled with masks. (B) Positive and negative hybrid concentric rings (size width/space: 50/50 μm). (C) Positive and negative hybrid concentric triangles (size width/space: 100/100 μm). (D) Positive and negative hybrid concentric quadrangles (size width/space: 50/50 μm). The thickness of the polymer blend film was ≈200 μm. The intensity and exposure time of 365 nm UV light were ≈50 mW/cm² and 30 s, respectively.

FIGURE 2

(A) 3D images of sample-0, 10, 20 30, 60, and 120 s by LSCM, respectively. (B) 2D images of sample sample-0, 10, 20 30, 60, and 120 s by LSCM, respectively. (C) Height of the sample-0, 10, 20 30, 60, and 120 s, respectively. Scale bar: 100 μm.

FIGURE 3

Adhesion of HUVECs on the biomimetic elastic membrane with different height in the thickness direction (*p < 0.05). (A–G) HUVECs were stained with 0.1% crystals after cultured in normal condition and on the sample-0, 10, 20, 30, 60, and 120 s for 6 h. Scale bar: 100 μm. (H) Quantitation of adhesive cells. The data are shown as the mean ± SD of the number of adhesive cells from three independent experiments. *p < 0.05 represents statistical significance.

FIGURE 4

Proliferation of HUVECs on the biomimetic elastic membrane with different height in the thickness direction. (A) The proliferation of HUVECs were assayed by CCK8 after cultured in normal condition and on the sample-0, 10, 20, 30, 60, and 120 s for 72 h. The mRNA expression of proliferation-related genes in HUVECs shown in each panel were (B) p21, (C) PCNA, (D) CDK2, and (E) cyclin A2, respectively (*p < 0.05).

FIGURE 5

The mRNA expressions of the angiogenesis-related genes in the HUVECs cultured in normal condition and on the sample-0, 10, 20, 30, 60, and 120 s for 72 h. The angiogenesis-related genes expression in HUVECs shown in each panel were (A) KDR, (B) eNOS, and (C) VE-Cad, respectively (*p < 0.05).