Development of cell-sheet constructs for layer-by-layer tissue engineering using the blood vessel as an experimental model

Abstract

Cell sheet tissue engineering approaches have recently emerged as a method to generate stratified tissue and better recapitulate native architecture. However, completely biological cell sheet approaches are limited by poor mechanical strength and appropriate substrate cues for modulating cell responses. The development of microthin polycaprolactone (PCL) films raised the possibility of using these films as scaffolds to complement the cell sheet approach. The aim of the present thesis was to investigate the use of PCL films as scaffolds in layered tissue engineering, using the blood vessel as a model. The scope covers the engineering of microthin PCL films using bi-axial stretching and surface modification, isolation of vascular progenitor cells and in vitro evaluation of the engineered films.

It was found that solvent cast and electrospun microthin PCL films did not compare favourably against biaxially stretched PCL films (?XPCL). The latter was found to possess the highest tensile strength and resistance to chemical degradation as determined by erosion assays. Tubular constructs formed from ?XPCL were found to possess adequate burst pressure and slow degradation (as determined by DSC and SEM studies), and were predicted to retain tensile properties capable of withstanding physiological loads for up to twelve weeks in vivo.

A novel glow-discharge plasma immobilisation technique was used to functionalise the ?XPCL film with carboxyl or amine groups, which allowed the conjugation of a range of biological moieties. Conjugation of common model proteins, including collagen and cell-capture antibodies, was demonstrated, underlining the possibility of generating customised surfaces for layered tissue engineering.

Subsequently, perivascular cells derived from umbilical cord (UCPVC) were found suitable for reconstruction of the perivascular compartment for vascular tissue engineering. Endothelial progenitor cells (EPCs) were found to exist in perinatal haematopoietic tissue, including two novel sources (fetal blood and liver). In particular, fetal blood derived EPC suggest superior vasculogenic capacity in Matrigel and microarray studies. However, umbilical cord blood EPC (UCB-EPC) were deemed the most suitable for vascular tissue engineering due to lineage stability.

The feasibility of microthin PCL films for layered vascular tissue engineering was explored. ?XPCL film surfaces were modified to support UCPVC and UCB-EPC for the perivascular and intimal compartments respectively. In addition, the intimal surface modification was found to improve haemocompatibility. Finally, sequential cell seeding was employed to generate a layered vascular analogue.