Development of bi-layer vascular constructs by combining polymer biomaterial and cellular self assembly

摘要

Introduction: Coronary Artery Disease (CAD) is one of the major causes of death and morbidity worldwide. This disease is caused by the clogging of coronary arteries due to calcium and fat deposits (known as plaques), that restrict the blood flow to the heart muscle. To overcome this problem, coronary artery by-pass grafting (CABG) surgery is performed. Though this surgical technique is widely used, it generally has drawbacks, such as unavailability of autologous grafts in case of multiple surgeries and immunogenic response with allogenic grafts. A potential alternative is to use synthetic grafts made of polymer materials as vascular substitutes. However, smaller diameter (＜6mm) synthetic grafts often fail due to poor long term patency rates. A promising solution to this problem is the use of a novel methodology that combines biodegradable tubular polymeric scaffold along with cellular self-assembly for the synthesis of small diameter vascular conduits (Internal Diameter=5mm) that could be used as blood vessel substitutes. Materials and methods: The project involves the use of biodegradable Poly Lactic Acid (PLA) nanofiber tubular matrix generated by air spinning technique which works on the principle of using air pressure to obtain nanofibers from polymer solution. The polymer scaffolds are subjected to different surface treatments followed by vacuum drying and sterilization. Vascular Smooth Muscle Cells (VSMC) from low passage (thawed at passage £6) are seeded on these scaffolds in specialized cell seeding chambers and incubated with the cell culture medium. Meanwhile, using low passage fibroblast cells on tissue culture plates under incubation, cell sheets are generated, in 2 weeks. These fibroblast cell sheets are rolled around the VSMC seeded tubular scaffolds and incubated for 3-5 weeks to provide better resistance and to create tunica media and adventitia similar to a native blood vessel. All human cell based experiments were done with approval from Comite d'ethique de la recherche du CHU de Quebec. Results: Tubular PLA scaffolds up to a length of 8 cm were fabricated through air spinning. The thickness of these scaffolds is calculated to be around 400pm, using digital micrometer. The nanofiber morphology, porosity were characterized using Scanning Electron Microscopy (SEM) and the average pore size was found to be 5 μm. Immunofluorescence microscopy (IF) with Hoechst staining for nuclei reveals that VSMC infiltration of up to 300 μm is observed in PLA scaffold and the cell number is twice as high on surface gelatinization of PLA. The mean ultimate tensile stress value of the fibroblast cell sheet rolled VSMC seeded PLA scaffold is 0.539 MPa while its elastic modulus (EM) is 2.52 MPa which is less than EM value for conduits developed by self-assembly alone proving that our constructs have better elasticity properties. Discussion and conclusion: The novel method of combining our self-assembly technique with biomaterial scaffold for vascular substitute generation helps mimic the architecture of native blood vessel and creates better elastic properties while also minimizing the time involved.