Vascular Regeneration with Functionalised Biodegradable Scaffold

Abstract

Introduction The development of small-diameter vascular grafts remains a key challenge in Tissue Engineering. Over the last two decades, strides have been made on the biofabrication of vessel mimics with similar biophysical characteristics to those of mammalian vessels but with only modest success. These vessels lack remodelling and regenerative capacity. This thesis aims to use induced pluripotent stem cells (iPSCs) and functionalised biomaterials to produce patient-specific bioengineered vessels. Materials and Methods Human iPSCs were first differentiated into mesenchymal stem cell (MSC), designated as iMSCs, with supplement of SB431542, a TGF-β1 pathway inhibitor. The iMSCs were further differentiated into vascular smooth muscle cells (VSMCs) via platelet-derived growth factor (PDGF)-BB and transforming growth factor beta 1 (TGF-β1) supplementation. Human mesenchymal stem cells derived from bone marrow (hMSCs-BM) from a commercial source were used for optimisation of differentiation protocols and quality control of MSCs. Vascular scaffold was produced using electrospinning of poly-L-lactide (PLLA) and coated with silk fibroin. HMSCs-BM and iMSCs were grown on the scaffold to evaluate the biocompatibility of the materials. HMSCs-BM and iMSCs were seeded in silk fibroin coated electrospun PLLA sheet for in-scaffold differentiation to obtain VSMCs. The cell-laden scaffold sheets were wrapped around a 3 mm diameter stainless steel rod and fixed with an alginate sealing to form a tubular shaped vascular graft. Results During the 34-36 days differentiation, the human iPSCs from three different cell lines gradually gained a fibroblast-like morphology, had increased expressions of MSC marker genes CD73, CD90 and CD105 and stained positive for these markers via immunofluorescence. Flow cytometry of the iPSC derived iMSCs also showed an increase in cell populations positive for these markers during differentiation. Thereafter, hBM-MSCs and iMSCs were successfully differentiating into VSMCs during a 9-day culture in PGDF-BB and TGF-β1 supplemented medium. The MSC-VSMCs express VSMC marker genes of α-SMA and CNN1, SM22 and MYH-11, which were confirmed by immunofluorescence staining. The PLLA silk fibroin coated porous scaffolds was compatible for MSCs adhesion and was best at supporting cell growth during a 10-day culture period over the non-silk fibroin coated pristine and porous scaffolds. The engineered tubular constructs derived from cell-laden PLLA silk fibroin coated scaffolds showed to have cell populations positive for VSMC markers α-SMA and CNN1. Furthermore, these constructs displayed mechanical properties comparable to those of autologous vascular grafts used for coronary artery bypass graft (CABG) like saphenous vein and internal mammary artery. Discussion/Conclusion An optimised protocol for iPSC to VSMC differentiation via MSCs was established. Silk fibroin coating on electrospun porous PLLA scaffold demonstrated an enhanced biocompatibility to MSCs. In-scaffold differentiation of VSMCs from iMSCs was successful which further produced a cell-laden small diameter vascular graft. The fabricated blood vessel mimics have native like biological and mechanical features. Future work will concentrate on the conjugating growth factors to the silk fibroin coating and evaluating the optimal release from these scaffolds to promote VSMC differentiation in situ from iMSCs.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Tao Wang (Supervisor) & Yi Li (Supervisor)