Date of Award

2015

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Fergal J. O'Brien

Funder/Sponsor

The Health Research Board of Ireland and the European Research Council.

Keywords

Orthopaedics, Tissue Scaffolds, Tissue Engineering

Abstract

Bone and articular cartilage are incredibly tough tissues with the ability to withstand repetitive stress throughout an individual’s lifetime. Unfortunately, their ability to heal after injury is finite, resulting in impaired function and degeneration. Despite considerable advances in modern surgical management, such as the use of auto- and allografting, the associated limitations with these approaches has motivated the development of alternative therapeutic interventions to replace or repair bone and cartilage, including tissue-engineered (TE) biomaterials. In relation to bone, a major obstacle to the in vivo use of biomaterials is ensuring an adequate blood supply to meet the metabolic demands of seeded o r invading cells following implantation. In the context of producing superior bone graft substitutes, the overall aim of this research, therefore, was to address the problem of vascularising TE biomaterials by improving the blood supply to collagen-based bone graft substitutes using two distinct vascularisation strategies; in vitro pre-vascularisation of scaffolds (Chapter 2) and an endochondral ossification-based vascularisation strategy (Chapters 3 and 4). In addition, the ability of a novel, multi-layered, cell-free collagen scaffold, that combines hydroxyapatite for bone repair and hyaluronic acid for cartilage repair, to enhance osteochondral repair was also investigated (Chapter 5).

Within the Tissue Engineering Research Group in the RCSI, a series of collagenbased scaffolds have been developed for tissue regeneration. The composition and structure of these biocompatible biodegradable biomaterials has been tailored to provide biological, architectural, and mechanical cues to influence cell infiltration, differentiation and matrix synthesis in a variety of tissues. Throughout this thesis, these scaffolds served as templates for tissue formation in conjunction with seeded cells (Chapters 2, 3 and 4) o r as cell-free implants (Chapter 5). Mesenchymal stem cells (MSCs), with their multi-lineage differentiation potential, were used to establish conditions capable of promoting enhanced vascularisation of these scaffolds fo r bone repair using two distinct methods. In Chapter 2, human umbilical vein endothelial cells (HUVECs) alone, o r in combination with human MSCs, were used to create in vitro micro-vascular networks within collagen-chondroitin sulphate (CCS) scaffolds. These pre-vascularised constructs were shown to significantly enhance bone repair in a critical-sized rat calvarial defect, while the MSCs in the coculture group had an immunomodulatory effect on the host response.

As an alternative method to in vitro pre-vascularisation, attention has recently focused on reproducing aspects of embryological skeletal development and fracture healing via endochondral ossification (ECO) to promote blood vessel invasion of TE constructs in vivo. In Chapter 3, it was demonstrated that MSCs seeded onto two distinct collagen-based scaffolds, a collagen-hyaluronic acid scaffold (CHyA) previously optimised for cartilage formation and a collagen-hydroxyapatite scaffold (CHA) previously optimised for bone formation, could be induced to follow an in vitro process similar to ECO. These constructs synthesised cartilage-specific matrix and were driven towards hypertrophy, resulting in the secretion of the proangiogenic growth factor VEGF, as well as mineralisation in vitro. Chapter 4 investigated the ability of this system to promote healing and bone formation in a critical-sized rat calvarial defect. Compared to traditional intramembranous ossification (IMO)-based approaches that aim to induce direct osteogenic differentiation in vitro, it was found that ECO-based constructs were capable of superior bone formation, construct remodelling and vascularisation in vivo.

Despite a host of repair techniques used to treat chondral and osteochondral injuries, including cell-based and cell-free TE strategies, the generation of durable hyaline-like articular cartilage with appropriate functional properties remains to be achieved clinically. Chapter 5 of this thesis evaluated a multi-layered scaffold, based on seamlessly combining the CHyA and CHA scaffolds, which were investigated independently in Chapters 3 and 4, as a cell-free strategy fo r osteochondral defect repair. This biomimetic scaffold was found to facilitate cellular infiltration and region specific differentiation, resulting in the regeneration of tissue with several key features of native osteochondral tissue, thus confirming its potential as an off-theshelf device fo r direct implantation, overcoming several of the drawbacks associated with current treatments used in orthopaedic clinical practice. Collectively,

Collectively, this study emphasises the significant in vivo capacity of both cell-seeded and cell-free collagen-based scaffolds developed in the RCSI Tissue Engineering Research Group to promote bone and cartilage regeneration using established animal models and highlights the clinical potential of these TE constructs fo r orthopaedic tissue repair applications.

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Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2015.

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