Understanding how to engineer Oxygen-dependent angiogenesis in 3D tissue models

Tissue engineering (TE) provides a valuable tool for the surgical repair of damaged body tissue, but designing TE constructs in which cells can survive to aid repair depends heavily on ensuring that these cells receive an adequate supply of O2 and nutrients. The cells residing in any piece of human tissue determine its architecture and function. In the core of tissues, O2 levels are limiting, therefore cells signal to surrounding vessels to infiltrate thereby increasing supply of crucial factors, a process called angiogenesis. This process is difficult to understand as the tissues of the body are complex, so I propose a model to correlate exposure of cells to O2 to help identify important cell specific signals crucial in the process of angiogenesis. By removing cells from tissue and growing them in well defined 3D collagen scaffolds, cell behaviour corresponding to specific positions within a scaffold, with information on the O2 levels triggering this signalling, can be identified. It is crucial to understand cell responses in a 3D environment, as this is the native environment in which cells reside. There are currently few tools at the disposal of researchers that enable them to examine how cells respond to the changing O2 environment during construction and implantation of repair devices. Cell embedded dense collagen sheets, will be spiralled to form 3D constructs. Over time periods of up to 1 month, I will measure O2 levels in the core and other specific regions of the 3D construct using O2 probes, and when these 3D spiralled constructs are unrolled, areas corresponding to known O2 levels will be dissected and examined, resulting in the identification of critical cell responses in relation to their position in a 3D construct, creating a 'map' of cell response dependent upon O2 exposure. These responses can then be manipulated, by controlling exposure of cells to certain O2 levels known to result in upregulation of markers which will aid survival of a construct in the body following implantation. Different biomaterials and composites will be then tested, for different tissue constructs. This model will then test whether specialised Endothelial Cells are attracted by core cell signalling and whether they form vessels, crucial for vascularisation. There is a critical balance to achieve to manipulate cell response: the correct level of O2 exposure to exploit natural cell behaviours. My hypothesis is that cells in the core of 3D TE collagen scaffolds, exposed to low-level O2, will produce important angiogenic signalling molecules, which will attract host vessels into the 3D construct core when implanted in vivo, therefore ensuring survival of a TE construct. The success of this approach will culminate in in vivo studies testing the angiogenic potential of such cellular TE devices. This new and innovative approach will help our understanding of how cells respond in their native 3D environment to different O2 exposure; how this translates to different signalling to induce angiogenesis; and how this information can be manipulated to help survival and maturation of TE constructs for future implantation to replace diseased and injured tissues. Without an understanding of how successful integration and survival of 3D TE constructs can be achieved in vivo, making replacement tissues cannot be a realistic goal.

Current concepts based primarily on tumour research are that cells do not survive >2mm in cell dense organs without vascularisation. The main objective of this study is to challenge conventional views on cell survival in the core of tissue engineered (TE) constructs, which suffer from poor perfusion of O2 and nutrients resulting in cell death. An innovative approach is to use native collagen type I scaffolds, where O2 levels in the core are low but limited cell death occurs. By manipulating material properties of TE constructs, O2 levels can be controlled to exploit the natural cell response to increase angiogenic signalling, to result in TE devices that attract angiogenesis in vivo, aiding survival. I will develop tissue models for investigating the control and generation of angiogenic signals, resulting in successful TE constructs with predictable angiogenic outcome. I will quantitatively define a 3D model to test the interplay of cell/matrix density and diffusion distance on O2 levels and correlate this to cell manufactured angiogenic signals, utilising a cross-disciplinary approach. This will be done using luminescent O2 gradient probes being developed by Oxford OptronixTM and the host institution. Over a 1 month period O2 levels in the core and distinct spatial positions in such constructs will be measured, and when constructs are unfurled and dissected, quantitative measurements of different angiogenic markers can be mapped alongside O2 measurements. The hypothesis being that 'deeper-lying' cells in the core, exposed to low-level O2, will up-regulate angiogenic markers, with a gradient up till the surface. Control experiments will see constructs cultured at physiological O2 levels of tissues in vivo, by utilising gas-mixing chambers. The regulation of angiogenic markers under these conditions compared to standard cultures will indicate responses expected in vivo. Validation of the angiogenic potential of 3D cell/matrix constructs will be conducted in vivo.