Evaluation of remodeling and regeneration of electrospun PCL/fibrin vascular grafts in vivo

Abstract

The success of artificial vascular graft in the host to obtain functional tissue regeneration and remodeling is a great challenge in the field of small diameter tissue engineering blood vessels. In our previous work, poly(ε-caprolactone) (PCL)/fibrin vascular grafts were fabricated by electrospinning. It was proved that the PCL/fibrin vascular graft was a suitable small diameter tissue engineering vascular scaffold with good biomechanical properties and cell compatibility. Here we mainly examined the performance of PCL/fibrin vascular graft in vivo. The graft showed randomly arranged nanofiber structure, excellent mechanical strength, higher compliance and degradation properties. At 9 months after implantation in the rat abdominal aorta, the graft induced the regeneration of neoarteries, and promoted ECM deposition and rapid endothelialization. More importantly, the PCL/fibrin vascular graft showed more microvessels density and fewer calcification areas at 3 months, which was beneficial to improve cell infiltration and proliferation. Moreover, the ratio of M2/M1macrophage in PCL/fibrin graft had a higher expression level and the secretion amount of pro-inflammatory cytokines started to increase, and then decreased to similar to the native artery. Thus, the electrospun PCL/fibrin tubular vascular graft had great potential to become a new type of artificial blood vessel scaffold that can be implanted in vivo for long term.

Keywords: Electrospinning; In vivo; Inflammatory cytokines; PCL/fibrin vascular grafts; Tissue remodeling and regeneration.

Fig. 1

Preparation and observation of PCL/fibrin vascular grafts. The schematic diagram of electrospun PCL /fibrin grafts (A), macroscopic appearances of the PCL/fibrin grafts (B). Morphology of vascular grafts was observed by SEM (C and D). Scale bars represented 10 μm in C and 50 μm in D, respectively.

Fig. 2

Implantation into the abdominal aorta, changes in the mechanical characteristics and morphology of the electrospun graft. Macroscopic pictures of the graft at 1, 3 and 9 months after transplantation (A). The red arrow indicated the anastomotic location of vascular graft. The appearance of the graft at 9 months after treatment with physiological saline solution (B). Gross view of the cross-sections of explanted PCL/fibrin grafts after 1, 3 and 9 months (C). (D), Compliance values of PCL/fibrin and PCL vascular grafts after 1, 3, and 9 months (n = 3). The mechanical properties of the vascular graft (n = 3) indicated the breaking strain (E), elastic modulus (F) and maximum stress (G). Changes of PCL/fibrin vascular grafts at different time periods were observed with SEM (H) and the percentage of weight retention of different grafts were measured at 1, 3 and 9 months respectively (I) (n = 3). Scale bar: (C) 1 mm. *indicated P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Regeneration of graft vascular function after 9 months. Contraction effects of KCl and AD in native artery, PCL/fibrin graft and PCL graft (A) (n = 3); Relaxation effects of Ach and SNP (B) (n = 3). _⁎ referred to statistical significance, ns indicated no statistical significance.

Fig. 4

General situation of endothelialization in different grafts. The general morphology of the inner layer surface of different grafts was observed by SEM at 1 mm (A, B and C), 50 μm (D, E and F), and 10 μm (G, H and I). Red arrows indicated vascular ECs. Immunofluorescence images of PCL grafts (J and M), PCL/fibrin grafts (K and N) and native artery (L and O) stained with vWF (red) and DAPI (blue), respectively. Scale bar: 100 μm. Endothelial coverage of different grafts was calculated (P) (n = 3). _⁎ p<0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Regeneration of vascular media layer within PCL/fibrin grafts at 9 months in vivo. HE stained images of different grafts after 9 months (A–C). Statistical analysis of cell numbers within graft different grafts (D) (n = 3). Distribution of VSMCs infiltration in different grafts under immunofluorescence test (α-SMA, green) (E-G). Fractional area of α-SMA within the graft (H) (n = 3). Immunofluorescence experiments of grafts with contractile VSMCs (SM-MHC, green) (I–K). Fractional area of SM-MHC within the graft (L) (n = 3). Cell nuclei were stained with DAPI (blue). Δ indicated the lumen. _⁎ represented P < 0.05. Scale bar: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Characterization of ECM deposition in 9 months after implantation. Microscopic photos of cross sections stained with Masson's for collagen (blue, A–C), safranin O for glycosaminoglycansin (red, E–G) and Verhoeff's for elastin (black, I–K) on different scaffolds. Total collagen, GAG, and elastin were assessed by corresponding detection kit respectively (D, H, L) (n = 3). Scale bar: 100 μm. _⁎ indicated statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Vascular density distribution and calcification in different grafts transplanted in abdominal aorta. Image of PCL graft and PCL/fibrin graft stained at 3 months with CD34 marker (A and B). Vessels numbers in PCL grafts and PCL/fibrin grafts at 1, 3 and 9 months was illustrated (C) (n = 3). Von Kossa staining of PCL/fibrin graft and PCL graft at 3 months (D–E). Black arrow indicated calcification. Calcification area ratio of grafts at different time points (F) (n = 3). Scale bar: 50 μm (A, B), 100 μm (D, E). _⁎ indicated statistical significance. Δ indicated the graft lumen.

Fig. 8

Distribution of macrophages and inflammatory-related cytokines during PCL/fibrin vascular graft remodeling. CD68, iNOS and CD206 markers were fluorescently stained (green) in PCL/fibrin graft at different time periods and native artery(A). The ratio of M2/M1 macrophages in PCL/fibrin and PCL grafts (n = 3) (B). Evaluation of inflammation related cytokines IL-4 (C), IL-10 (D), TNF-α (E), IL-6 (F) and IL-1β (G) by ELISA experiments (n = 3). P < 0.05 = _⁎. ns represented no statistical significance. Scale bar: 50 μm. Blue indicated the nucleus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)