Date of Award

2014

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Fergal J O' Brien

Funder/Sponsor

European Research Council (ERC grant agreement nº 239685-CollRegen-ERC-2009- STG) under the EU Seventh Framework Programme (FP7).

Keywords

Scaffold, Drug Release, Growth Factor, Bone Repair, Bioglass, Cobalt

Abstract

Although bone has an intrinsic capacity for self repair, the healing of large bone defects that typically present in humans often involves complications which can result in failure to heal, leading to delayed union or non-union of the defect. Due to limitations of current therapeutic approaches of autografting and allografting, the use of tissue engineered scaffolds has emerged. Despite some success with this approach, a major limitation is creating functionally vascularised constructs. In addition, scaffolds often require an additional stimulus such as a growth factor (GF) to fully heal large fractures. However, current GF factor delivery approaches are often associated with limited success due to uncontrolled protein release. These issues arise due to the high concentrations of protein required to elicit healing which, in turn, is due to the result of the short half-life of proteins in vivo. These shortcomings have led to the emergence of scaffolds integrated with polymeric carriers which encapsulate, protect and control the release of GFs.

In the context of producing superior bone graft substitutes, this thesis focused on fabricating a new generation of scaffolds by functionally enhancing collagen-hydroxyapatite (CHA) and collagen-glycosaminoglycan (CG) scaffolds, optimised specifically for bone repair in our laboratory, in order to make them capable of coupling both angio- and osteogenesis for promoting enhanced regeneration. Specifically, this research aimed to investigate the potential for controlled release of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) encapsulated in alginate and poly (lactic-co-glycolic acid) (PLGA) microparticles (MPs) and subsequently, to investigate whether a series of functionalised scaffolds incorporating this system could promote enhanced bone repair in a critical size defect in vivo model. As a GF-free alternative approach, a final aim of this research was to develop and characterise a collagen-based scaffold incorporating pro-angiogenic and pro-osteogenic cobalt-bioactive glass (BG) and to assess the in vitro ability of the material to promote these processes.

In the study shown in Chapter 2 of this thesis, feasible preparation methods for GF-eluting MPs were established. It was shown that PLGA MPs fabricated by double emulsion and spray dried alginate MPs were capable of not only controlling and prolonging the release of two of the main GFs pertinent to bone repair but also retaining their bioactivity. Additionally, it was demonstrated that the release kinetics of GFs can be tailored by using different polymers for encapsulation. In Chapter 3 it was shown that with an optimised fabrication process, it was possible to develop GF-releasing CHA scaffolds containing MPs without interfering with the structural properties of the scaffold previously optimised for bone repair. Additionally, sustained release of bioactive GFs from the optimised scaffolds was demonstrated with kinetics resembling the in vivo condition: the early expression of VEGF from alginate MPs and sustained release of BMP-2 from PLGA MPs. Hence, functionalised scaffolds were capable of eliciting a pro-angiogenic and pro-osteogenic response in vitro. Having demonstrated the functionality of these materials in vitro, VEGF and/or BMP-2-eluting scaffolds were implanted in a rat calvarial defect model where they enhanced healing compared to non-eluting scaffolds as well as non-treated animals. Ultimately, VEGF-releasing CHA scaffolds accelerated healing to the greatest extent offering an ideal platform to promote both vasculogenesis and bone repair. In Chapter 5 a GF-free biomaterial alternative, a novel scaffold was successfully fabricated by the incorporation of cobalt-eluting BG particles into a CG scaffold. This material was capable of stimulating angiogenesis in vitro via the release of cobalt, a known hypoxia mimic, as well as osteogenesis via the dissolution of osteoinductive BG particles. This study indicated that an angiogenic and osteogenic response may be achievable exclusively through a GF-free biomaterials based approach.

Collectively, the research presented in this thesis has led to the development of a new generation of functionally enhanced collagen-based scaffolds. Specifically, polymeric MPs were developed that were capable of controlling the release of bioactive GFs pertinent to bone repair. Functionalised scaffolds containing MPs were capable of the sustained release of bioactive concentrations of VEGF and BMP-2 from MPs within the matrix. The alginate-VEGF scaffold emerged as the GF-eluting scaffold which promoted the most accelerated neovascularisation and bone repair in vivo. Furthermore this thesis has shown that novel cobalt-BG containing CG scaffolds enhanced cell-mediated osteogenesis and angiogenesis representing a desirable, economical, alternative to GF-based delivery for the regeneration of bone tissue. These novel functionalised scaffolds offer the advantage of off-the-shelf availability and lack the need to for in vitro cell culture. In addition, the delivery systems developed in this thesis have enormous potential in regenerative medicine, as they could be tuned in terms of the composition of the collagen-based scaffold and released therapeutic to promote healing of a diverse range of tissues and organs in addition to bone.

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File Size

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Comments

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

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