Extracellular Vesicles Enhance the Remodeling of Cell-Free Silk Vascular Scaffolds in Rat Aortae

Abstract

Vascular tissue engineering is aimed at developing regenerative vascular grafts to restore tissue function by bypassing or replacing defective arterial segments with tubular biodegradable scaffolds. Scaffolds are often combined with stem or progenitor cells to prevent acute thrombosis and initiate scaffold remodeling. However, there are limitations to cell-based technologies regarding safety and clinical translation. Extracellular vesicles (EVs) are nanosized particles released by most cell types, including stem and progenitor cells, that serve to transmit protein and RNA cargo to target cells throughout the body. EVs have been shown to replicate the therapeutic effect of their parent cells; therefore, EVs derived from stem or progenitor cells may serve as a more translatable, cell-free, therapeutic base for vascular scaffolds. Our study aims to determine if EV incorporation provides a positive effect on graft patency and remodeling in vivo. We first assessed the effect of human adipose-derived mesenchymal stem cell (hADMSC) EVs on vascular cells using in vitro bioassays. We then developed an EV-functionalized vascular graft by vacuum-seeding EVs into porous silk-based tubular scaffolds. These constructs were implanted as aortic interposition grafts in Lewis rats, and their remodeling capacity was compared to that observed for hADMSC-seeded and blank (non-seeded) controls. The EV group demonstrated improved patency (100%) compared to the hADMSC (56%) and blank controls (82%) following eight weeks in vivo. The EV group also produced significantly more elastin (126.46%) and collagen (44.59%) compared to the blank group, while the hADMSC group failed to produce significantly more elastin (57.64%) or collagen (11.21%) compared to the blank group. Qualitative staining of the explanted neo-tissue revealed improved endothelium formation, increased smooth muscle cell infiltration, and reduced macrophage numbers in the EV group compared to the controls, which aids in explaining this group's favorable pre-clinical outcomes.

Keywords: aortic graft; exosomes; mesenchymal stem cells; microvesicles; vascular tissue engineering.

Figure 1.

Characterization of extracellular vesicles (EVs) derived from conditioned media (CM) generated by adipose-derived mesenchymal stem cells (hADMSCs). (A) Schematic of EV isolation that was achieved using differential ultracentrifugation and filtration of hADMSC CM. (B) Morphology of EVs as observed using transmission electron microscopy. The left and right panels depict separate areas of the same EV isolate at 60 000 and 80 000× magnification, respectively. Examples of EVs are highlighted with arrows. (C) Size distribution of particles within the EV isolate as determined using dynamic light scattering in terms of intensity and (D) number of readings. Each curve represents the analysis of a separate EV isolate. (E) Total protein content of the EV isolate before and after lysis. (F) Presence of the EV-enriched tetraspanin CD63 as determined using Western blot; * represents p < 0.05.

Figure 2.

(A) Proliferation and (B) migration of smooth muscle cells (SMCs) and endothelial cells (ECs) when exposed to extracellular vesicle (EV)-based treatments; * represents p < 0.05, ** represents p < 0.005, and *** represents p < 0.0001.

Figure 3.

Development of the EV–TEVG system. (A) Schematic depicting the seeding of silk-based vascular scaffolds with hADMSC-derived EVs. (B) The rotational vacuum device used to seed scaffolds with EVs. (C) The total protein content of the EV isolate (intact and lysed) before and after seeding of the scaffolds. (D) Percentage coverage of the scaffold with Cy5 fluorescent dye following seeding of the scaffolds with stained EVs using the different methods. (E) Fluorescent images of blank scaffolds, scaffolds soaked in spun down Cell Mask dye, scaffolds infused with Cell Mask dye, scaffolds soaked in stained EVs, and scaffolds infused with stained EVs. Green represents the FITC auto-fluorescence of the scaffold (left of the diagonal dashed line), and magenta represents Cy5 staining of the EVs in that same scaffold (right of the diagonal dashed line). Dotted lines have been added to the scaffolds with minimal Cy5 staining to indicate the inner and outer boarders. Scale bars depict 500 μm. (F) SEM imaging of scaffolds seeded with unstained EVs vs (G) unseeded blank scaffolds; *** represents p < 0.0001.

Figure 4.

Graft patency following eight-week implantation. (A) Schematic representation of scaffold seeding and implantation as an aortic interposition graft in a rat model. (B) Angioplasty and gross imaging of a representative patent explant and (C) angioplasty and gross imaging of a representative occluded explant. Scale bars depict 500 μm. Red arrows indicate the graft location during angioplasty. (D) Patency rates of each group as well as the manner of graft occlusion.

Figure 5.

Remodeling of implants following eight-week implantation. (A) Elastin and (B) collagen content of each explant as a percentage of the total explant protein. (C) Immuno-histological and immuno-fluorescent images representing explants from each group. Scale bars depict 100 μm; * represents p < 0.05, and ** represents p < 0.005.