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.

Extracellular vesicles for tissue repair and regeneration: Evidence, challenges and opportunities

“…Loading efficiency can be conveniently calculated by determining the quantity of EVs in the EV isolate solution before and after loading onto the scaffold. Cunnane et al identified a reduction in protein content within the EV isolate after infusing scaffolds with EVs [267]. Results indicated that approximately 45% of the EV isolated protein content was retained within a tubular silk fibroin scaffold.…”

“…More detailed analysis methods are frequently employed by relevant studies, including fluorescent imaging and SEM. In the studies that perform fluorescent imaging of EV-biomaterial systems, the EVs are clearly visible and in most cases a good distribution and near complete coverage of EVs within the biomaterial is achieved [33,215,263,265,267,[270][271][272], although artifacts arising from shedding dye from the EVs labelled with lipophilic dyes cannot be excluded. SEM images of appropriate systems display nanoparticles on the material surface with the morphology typically displayed by EVs [33,267,271,272].…”

Glycosaminoglycans: From Vascular Physiology to Tissue Engineering Applications

“…The rate of temperature decrease can influence pore size and pore connectivity. By this approach, scaffolds of silk fibroin loaded with extracellular vesicles isolated from Adipose-derived Mesenchymal Stromal Cells (ADMSC) were fabricated ( Cunnane et al, 2020 ). These grafts presented 100% patency at the early stages of the study (4 weeks), compared to the bare scaffold and scaffold seeded with ADMCS prior to implantation.…”

Extracellular Vesicles Derived from Primary Adipose Stromal Cells Induce Elastin and Collagen Deposition by Smooth Muscle Cells within 3D Fibrin Gel Culture

“…EVs may represent a more translatable cell-free tool to combine with biodegradable scaffolds intended for vascular grafting applications as we demonstrate here that they are capable of encouraging deposition of ECM structural proteins and therefore likely to encourage positive remodeling of an implanted graft. Indeed, our group has already shown that incorporating EVs into vascular grafts results in increased elastin and collagen production compared to blank scaffolds [ 33 ]. The matrix synthesizing effects exhibited by EVs in this study are therefore readily transferable to the production of de novo vascular graft tissue in situ.…”