The influence of structure and surface biochemistry on cell function and angiogenesis in collagen scaffolds

A major challenge in regenerative medicine is the development of grafts that can be vascularised successfully. Capillary networks facilitate processes that are essential for tissue survival including the supply of nutrients to cells and waste exchange. Therefore, in order to prevent core degradation of tissue-engineered grafts, it is important to provide appropriate physico-chemical conditions to encourage angiogenesis in the period immediately following implantation. Ice-templated collagen scaffolds have been used in tissue engineering due to their bioactivity, tunable biodegradability and the ability to produce architectures with tailored porosity, but until now very little work has focused on vascularisation of these structures. The aim of the work described in this thesis was to develop in vitro cell culture models, to study the effects of structural features and surface chemistry properties on the formation of microvessels. Firstly, the influence of mould design on architectural features of collagen scaffolds fabricated using lyophilisation was assessed using micro-computed tomography (Micro-CT) and scanning electron microscopy (SEM). A range of mould designs was investigated and those made of tissue culture polystyrene, and perspex with a stainless steel base mould were found to produce the most suitable isotropic and anisotropic structures, respectively. Both moulds allowed control and optimisation of the scaffold porosity through freezing protocol modification, enabling analysis of cell behaviour in response to scaffold structure. Secondly, the mechanical and biological functionality of the substrates were investigated with the aim of establishing and improving vascularisation in collagen scaffolds. A co-culture of primary human osteoblasts (hOBs) and human dermal microvascular endothelial cells (HDMECs) was chosen to achieve microvessel formation in vitro. Initially, mono-culture studies were carried out to gain an in-depth understanding of the behaviour of the individual cell types involved in co-culture on substrates with varying mechanical properties and architectural features. Mechanical stability of 2D collagen films and 3D scaffolds was adjusted using 1-ethyl- 3-(3-dimethylaminopropyl)-carbodiimide hydrochloride cross-linking in the presence of N-hydroxysuccinimide (EDC/NHS) at concentrations ranging from 0 to 100% of standard conditions (SC) reported widely in the literature. Cell behaviour was quantified using cell metabolic activity, cytotoxicity, proliferation, adhesion and migration assays. A cross-linking treatment at 30% SC provided the most appropriate combination of mechanical stiffness (Young's modulus: 6 kPa) and specific cell attachment for both cell types. Analysis of cell behaviour in response to structural properties revealed that, at early time points, an increase in percolation diameter had the strongest influence on cell invasion, while the effect of pore size became more prominent at later stages (18 days of incubation). Both hOBs and HDMECs exhibited the highest invasion efficiency on scaffolds with the lowest degree of anisotropy and the highest mean pore size and percolation diameter (approximately 175 and 130 μm, respectively). Films and scaffolds cross-linked at 30% SC were taken forward for structural analysis in co-culture. For in vitro vessel formation, an equilibrium is required between the cell types involved in co-culture. To obtain the optimal co-culture conditions on both collagen films and scaffolds, a range of seeding densities and cell ratios were investigated in relation to their influence on cell- metabolic activity and proliferation behaviour. A systematic study using various hOB:HDMEC cell ratios showed that 70:30 and 50:50 provided the optimal conditions for microvessel formation on 2D films and 3D scaffolds, respectively. Nevertheless, vascularisation of these collagen substrates remained limited. Further assessment in 2D revealed that osteoblast detachment was the main factor restricting vessel formation. Pre-coating of the substrates with a 5 μg/mL fibronectin solution provided the appropriate conditions for osteoblast stabilisation, resulting in abundant vessel formation on collagen substrates. Furthermore, the protein coating was found to counteract the negative influence of high cross-linking levels on specific cell attachment. Overall, fibronectin-coated collagen scaffolds seeded with a ratio of 50:50 hOB:HDMEC resulted in abundant microvascular network formation with multiple branching points and lumen formation. Lastly, the influence of scaffold architecture on vessel formation and invasion was examined using the established co-culture system. On all samples, long term survival of microvessels was achieved up to approximately 31 days of incubation. Structural alignment of the scaffold architecture was found to have the strongest effect on vessel density and ingrowth, with a higher mean pore size and percolation diameter as secondary factors. Anisotropic scaffolds with a mean pore size and percolation diameter of approximately 118 and 108 μm, respectively, exhibited the highest vessel density and invasion depth. The work described in this thesis offers new insights into the factors that underpin microvessel formation and demonstrates that by careful control of scaffold architecture and surface chemistry it has been possible to develop a successful in vitro co-culture model.