Date of Award


Document type


Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Fergal J O'Brien

Second Supervisor

Dr Garry Duffy


Grant support from the Health Research Board in Ireland (PhD/2007/11), the European Research Council and the EMBO Short Term Travel Fellowship.


Co-Culture, AFSCs, Chondrogenesis, Collagen-Based Scaffolds, Hypoxia


Amniotic fluid-derived stem cells (AFSCs) are a unique stem cell source that demonstrate great potential for use in bone and cartilage tissue engineering (TE) due to their pluripotentiality. One of the major problems in TE is graft failure in vivo due to core degradation and avascular necrosis in constructs designed to regenerate thick tissues such as bone. The engraftment of these constructs post-implantation relies on the rapid formation of stable and functional vasculature, as cell-seeded constructs lack an inherent vascular network. One approach to overcoming this vascularisation problem involves engineering a nascent vasculature in vitro prior to implantation in vivo. This pre-vascularisation approach reduces reliance on vessel invasion from the host, improving the possibility of successful implant engraftment. AFSCs have also previously demonstrated angiogenic potential and as a result, may offer a potential cell source for use in pre-vascularisation.

The primary aim of the research presented in this thesis was to investigate the angiogenic potential of AFSCs in order to develop a novel co-culture system to engineer, in vitro, an inherent network of vessel-like structures within highly porous collagen-composite scaffolds. The application of this co-culture to different scaffolds could allow for the creation of a host of vascularised TE constructs with potential for use in a variety of clinical applications. Hypoxia is a well-known physiological stimulus of angiogenesis so, in order to potentially enhance the formation of vessel-like structures, the effect of hypoxic culture on this co-culture system was also investigated. The final part of the research presented in this thesis aimed to investigate the chondrogenic potential of AFSCs with the aim of creating a novel construct for use in cartilage repair, another major area of orthopaedic TE.

Firstly, Chapter 2 of this thesis investigated the ability of AFSCs to differentiate down an endothelial lineage in order to create an endothelial-like cell for use in pre-vascularisation. This study demonstrated that AFSCs subjected to endothelial stimuli display an endothelial gene expression profile and functional endothelial cell characteristics indicative of early endothelial differentiation. Culture in continuous hypoxia enhanced endothelial gene expression but did not enhance functional endothelial cell characteristics. However, AFSCs were ultimately unable to adopt the mature endothelial cell phenotype necessary for the formation of vessel-like structures required for pre-vascularisation. In Chapter 3, the suitability of AFSCs for use in the role of pericyte in a co-culture with human umbilical vein endothelial cells (HUVECs) was investigated. It was demonstrated that AFSCs were capable of functioning as pericytes and that an AFSC-HUVEC co-culture was capable of successfully pre-vascularising a collagen-chondroitin sulphate (CCS) scaffold via the formation of a robust network of vessel-like structures. In addition, it was found that intermittent and continuous hypoxia reduced vessel-like structure formation and that of the three O2 conditions studied, normoxia promoted the highest level of vessel-like structure formation within CCS scaffolds.

In Chapter 4, this AFSC-HUVEC co-culture was demonstrated to be capable of pre-vascularising two other collagen-based scaffolds from our lab that are the closest to clinical translation: a collagen-hydroxyapatite (CHA) and a collagen-hyaluronic (CHyA) scaffold, indicating the adaptability of this co-culture for use in pre-vascularisation. It was demonstrated that both scaffold types were capable of facilitating similar levels of cell density and vessel-like structure formation, making this approach suitable for use in a variety of TE applications. In Chapter 5, by utilising the knowledge gained in the earlier studies, the focus switched to cartilage repair in order to develop a novel construct which combined AFSCs, the CHyA scaffold and hypoxic culture. It was demonstrated that AFSCs were capable of undergoing chondrogenic differentiation as evidenced by production of cartilage-like matrix. Hypoxic pre-culture accelerated early stage chondrogenic differentiation and inhibited late stage differentiation towards hypertrophy, indicating that hypoxia may enhance the chondrogenic potential of AFSCs. Collectively, this thesis has demonstrated the potential and adaptability of AFSCs for use in both bone and cartilage TE. The research presented in this thesis has led to the development of three pre-vascularised collagen-based scaffolds that have potential for use in a wide range of TE applications. Furthermore, a novel construct composed of AFSCs seeded on a CHyA scaffold has been developed that may have significant implications in the development of advanced TE strategies for cartilage defect repair.

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A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2014.