Complex tissue engineering of vessels structure using PU-PCL nanoscaffold by engaging endothelial lineage and Smooth muscle cells

Abstract

Introduction Ischemic diseases such as cardiovascular disease and atherosclerosis are the leading cause of death worldwide. Peripheral artery and coronary artery bypass grafting are usually performed using an autologous vascular graft. Deficiencies in autografts, such as donor deficiency, risk of thrombosis, and infection, have led to extensive research in vascular tissue engineering (TEVGs). So far, artificial vessels with a low diameter and sound quality (less than 6 mm) have not been made. Superficial clots and emboli that block blood flow are the biggest problems in making low-diameter artificial vessels. Objective The primary purpose of this thesis was to create a small diameter engineered vascular graft scaffold using polyurethane and polycaprolactone polymers with suitable mechanical conditions. Therefore, the electrospun scaffold with optimized physiological and suitable viscoelastic properties was prepared in vitro, and the laboratory maturity was determined under dynamic conditions using an air-lift bioreactor. The ability of the scaffold to support the differentiation of endothelial and pericyte cells in vitro was then investigated. Material and method The prepared polymeric nanofibers were characterized by mechanical properties, cell viability, proliferation, and differentiation of endothelial and mesenchymal stem cells. The most optimal scaffold was elected in terms of mechanical properties and cell compatibility. Based on the results of static cell culture, a tubular scaffold was prepared using PU-PCL (2: 1) formulation and electrospinning method for culturing cells under dynamic conditions, was placed in a dynamic culture medium containing endothelial cells for six days. In order to enhance blood compatibility and prevent thrombosis, heparin cross-link was used as an anticoagulant to the scaffold surface. MSCs were cultured on the scaffold in the presence of an endothelial or pericytes differentiation medium and then the expression of VE-Cadherin and vascular SMA-specific proteins using immunohistochemistry and the expression of PDGFR-β and Von. Willebrand was examined using Western blotting. Results Test results revealed that PU-PCL (2: 1) scaffolds have better mechanical properties (including stress-strain, water contact angle and swelling rate, biocompatibility, nanofiber diameter, and porosity) than other groups. The culture of endothelial cells under static conditions on these scaffolds did not cause nitrosative stress and cytotoxicity compared to the control group. Scanning Electron Microscopy (SEM) images confirmed the ability of endothelial cells to attach to the lumen of the tubular scaffold in an aligned manner under dynamic culture conditions. Immunofluorescence imaging approved the attachment of cells to the lumen surface. ATR-FTIR spectroscopy and AFM and EDAX complementary cross-link tests validated successful heparin. Immunohistochemistry and Western blotting supported the differentiation of mesenchymal stem cells cultured on heparinized scaffolds into endothelial cells and pericytes. Conclusion The results showed that using PU-PCL (2: 1) substrate can stimulate endothelial and mesenchymal cells' activity, proliferation, and differentiation in static and dynamic conditions.