Combining Extracellular Vesicles with Porous Electrospun Scaffolds for the Development of New Multifunctional Membranes for Tissue Engineering Applications

Extracellular vesicles (EVs) are membrane-enclosed vesicles that are released by cells and mediate cell–cell communication via their protein, lipid, carbohydrate, and nucleic acid (RNA, DNA) cargo. EVs are involved in a multitude of physiological processes including development, cell differentiation and angiogenesis having also been associated with tissue repair. The aim of this project was to optimise the functionalisation of electrospun scaffolds with EVs and to evaluate biological functionality for their future use in tissue engineering applications. Extracellular vesicles were successfully isolated from oral cancer cell line (H357), normal oral fibroblast (NOF) and human dental pulp stem cells (HDPS) cells in vitro using ultracentrifugation (UC) and size exclusion chromatography (SEC) as assessed by NTA and determination of the presence of EV marker proteins (CD9, CD63 and CD81). Electrospun scaffolds were manufactured using an upright in-house electrospinning rig using poly(caprolactone) (PCL). A functionalisation comparative study showed that the use of air plasma treatment (when compared to heparin treatment) increased the number of EVs attached to the scaffolds and provided homogeneous incorporation of EVs within the fibrous network. Over the course of 21 days, NTA showed a slow release of 40% of EVs from the scaffolds. The zetapotential of EVs and scaffolds was measured to provide a better understanding of the mechanism of EVs attachment and release. All EVs exhibited anionic character regardless of the cell type (with a significantly higher anionic character of H357 EVs when compared to primary cells EVs). Plasma-treated scaffolds exhibited a positive zetapotential in comparison to the neutral charge of PCL and highly negative charge of heparin-treated scaffolds which may explain the high attachment of negatively charged EVs on plasma-treated scaffolds. In vitro, no effect of myofibroblast EVs or EVs-modified scaffold was found on the migration of NOF cells. When using the Chick Chorioallantoic Membrane (CAM) assay angiogenesis model, HDPS EVs modified scaffolds were able to induce vascular formation at a level comparable to the positive control, VEGF. We also found that plasma-treated PCL scaffolds promoted significant vascular formation in comparison to untreated PCL. However, an osteogenesis assay showed no significant effect of HDPS EVs on the osteo-differentiation rat bone marrow MSCs. In conclusion, here we provide evidence that electrospun scaffolds can be functionalized with EVs and provide sustained slow release, offering an opportunity to develop novel, cell-free and tuneable approaches to tissue engineering. Furthermore, HDPS EV-modified scaffolds provide a promising way to promote vascularization, which can be used for a range of tissue engineering applications.