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. 2018 Jan 1;10(1):39.
doi: 10.3390/polym10010039.

Vascular Cell Co-Culture on Silk Fibroin Matrix

Fangfang Tu  1 , Yunfei Liu  2 , Helei Li  3 , Pange Shi  4 , Yunxia Hao  5 , Yue Wu  6 , Honggen Yi  7 , Yin Yin  8 , Jiannan Wang  9   10
Affiliations

Vascular Cell Co-Culture on Silk Fibroin Matrix

Fangfang Tu et al. Polymers (Basel). .

Abstract

Silk fibroin (SF), a natural polymer material possessing excellent biocompatibility and biodegradability, and has been widely used in biomedical applications. In order to explore the behavior of vascular cells by co-culturing on regenerated SF matrix for use as artificial blood vessels, human aorta vascular smooth muscle cells (HAVSMCs) were co-cultured with human arterial fibroblasts (HAFs) or human umbilical vein endothelial cells (HUVECs) on SF films and SF tubular scaffolds (SFTSs). Analysis of cell morphology and deoxyribonucleic acid (DNA) content showed that HUVECs, HAVSMCs and HAFs adhered and spread well, and exhibited high proliferative activity whether cultured alone or in co-culture. Immunofluorescence and scanning electron microscopy (SEM) analysis showed that HUVECs and HAFs co-existed well with HAVSMCs on SF films or SFTSs. Cytokine expression determined by reverse transcription-polymerase chain reaction (RT-PCR) indicated that the expression levels of α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) in HAVSMCs were inhibited on SF films or SFTSs, but expression could be obviously promoted by co-culture with HUVECs or HAFs, especially that of SM-MHC. On SF films, the expression of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (CD31) in HUVECs was promoted, and the expression levels of both increased obviously when co-cultured with HAVSMCs, with the expression levels of VEGF increasing with increasing incubation time. The expression levels of VEGF and CD31 in cells co-cultured on SFTSs improved significantly from day 3 compared with the mono-culture group. These results were beneficial to the mechanism analysis on vascular cell colonization and vascular tissue repair after in vivo transplantation of SFTSs.

Keywords: cells interaction; co-culture; proliferative activity; silk fibroin; vascular cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cell morphology and cell proliferation of human aorta vascular smooth muscle cells (HAVSMCs)/human umbilical vein endothelial cells (HUVECs) co-cultured on silk fibroin (SF) films. (A) HAVSMCs on tissue culture plate (TCP); (B) HAVSMCs on film; (C) HUVECs on TCP; (D) HUVECs on film; (E) HAVSMC/HUVEC co-cultures on film and (F) DNA contents, Bar = 200 μm, ** p < 0.01.
Figure 2
Figure 2
Cell morphology and cell proliferation of HAVSMCs/human arterial fibroblasts (HAFs) co-cultured on SF films. (A) HAVSMCs on TCP; (B) HAVSMCs on films; (C) HAFs on TCP; (D)HAFs on films; (E) HAVSMC/HAF co-cultures on films and (F) DNA contents, Bar = 200 μm, ** p < 0.01.
Figure 3
Figure 3
Immunofluorescence of HAVSMCs co-cultured with HUVECs or HAFs on SF films. Green was for α-SMA in HAVSMCs, blue was for cell nucleus and red was for CD31 in HUVECs, (A) HAVSMCs on films; (B) HAVSMCs on TCP; (C) HUVECs on films; (D) HUVECs on TCP; (E) HUVEC/HAVSMC co-cultures on film and (F) HAF/HAVSMC co-cultures on film, Bar = 50 μm.
Figure 3
Figure 3
Immunofluorescence of HAVSMCs co-cultured with HUVECs or HAFs on SF films. Green was for α-SMA in HAVSMCs, blue was for cell nucleus and red was for CD31 in HUVECs, (A) HAVSMCs on films; (B) HAVSMCs on TCP; (C) HUVECs on films; (D) HUVECs on TCP; (E) HUVEC/HAVSMC co-cultures on film and (F) HAF/HAVSMC co-cultures on film, Bar = 50 μm.
Figure 4
Figure 4
SEM photographs of HUVECs and HAVSMCs on SFTSs. Bar = 100 μm.
Figure 5
Figure 5
Specific identification of each primer. (A) Melting curve and (B) DNA agarose gel electrophoresis. Different colors in the figure A represent the different samples.
Figure 6
Figure 6
Cytokine expression of HUVECs/HAVSMCs co-cultured on SF films. (A) α-SMA; (B) SM-MHC; (C) VEGF and (D) CD31, * represents p < 0.05, ** represents p < 0.01.
Figure 7
Figure 7
Cytokine expression of HAVSMCs/HAFs co-cultured on SF films. (A) α-SMA and (B) SM-MHC, * p < 0.05, ** p < 0.01.
Figure 8
Figure 8
Cytokine expression of HUVECs/HAVSMCs co-cultured on SFTSs. (A) α-SMA; (B) SM-MHC; (C) VEGF and (D) CD31, * p < 0.05, ** p < 0.01.
Figure 9
Figure 9
Cytokine expression of HAVSMCs/HAFs co-cultured on SFFSs. (A) α-SMA and (B) SM-MHC, * p < 0.05, ** p < 0.01.
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