Development of angiogenic models to investigate neovascularisation for tissue engineering applications

The aim of this project was to develop in vitro models for angiogenesis that have the capability of combining pro-angiogenic cells with an extracellular matrix (ECM) component that can be monitored under flow conditions to learn more about the ‘rules’ of angiogenesis. Significant advancement has been made in the field of tissue engineering in recent years, however one of the current obstacles limiting progression is the production of thick, complex tissues due to the lack of rapid neovascularisation of the constructs upon implantation. Blood vessel formation is tightly regulated and relies on the chronologically precise adjustment of vessel growth, maturation and suppression of endothelial cell growth - all of which are controlled by a large number of factors which influence each other. To induce vascularisation within tissue-engineered (TE) substitutes the same processes need to occur. A number of different vascularisation strategies have been investigated in an attempt to overcome this issue but as yet there is no unified solution to this problem. The most promising attempts have used scaffolds with vascular architectures, perfusion conditions and relevant cell types. Although it is recognised that perfusion conditions, the cell type and scaffold architecture are important with regards to vascularisation strategies many of the techniques fail to consider them in combination. It is therefore important to take a step back and understand how these factors work together to i promote angiogenesis in order to advance this crucial area. This lack of understanding is further compounded by the deficiencies of current angiogenesis models. Current in vitro models fail to combine the use of supporting cells, the extracellular matrix and fluid flow in 3D. Although this complexity exists within in vivo models such assays are primarily limited by the species used, organ sites available and complicated analysis techniques. In this project two in vitro angiogenesis models were developed. The first was derived from the decellularisation of a rat jejunum. Characterisation showed the retention of key extracellular matrix (ECM) components and the removal of almost all cellular material. Re-endothelialisation with human dermal endothelial cells (HDMECs) of the patent vascular network showed enhanced results when co-cultured with human dermal fibroblasts (HDFs). In an attempt to induce angiogenesis, vascular endothelial growth factor (VEGF) loaded gels were placed on top of the scaffold whilst being continuously perfused with media. Placing VEGF loaded gels onto the recellularised jejunum led to the expression of the Notch ligand Delta- like-4 (DLL4) by HDMECs indicating their transformation into tip cells which are synonymous with sprouting angiogenesis. The second was produced through the combination of robocasting and electrospinning. Nanofibrous poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) scaffolds with hollow channels capable of perfusion were produced that could be re-endothlialised with HDMECs. Again the addition of HDFs enhanced cellular distribution in the channels. Placing VEGF loaded gels onto the surface of the scaffolds led to the outgrowth of HDMECs into the gel, forming perfusable tubules. Overall these two models overcome limitations of current in vitro models since they offer the capability of combining pro-angiogenic cells with ECM components that can be monitored under flow conditions. With further development they could provide more sophisticated platforms upon which to investigate the angiogenic process.