Alpha-helical peptide hydrogels as instructive scaffolds for 3D cell culture and tissue engineering

Our research is concerned with understanding how biology builds functional structures using molecular building blocks. We apply this understanding to make new structures from molecules accessible in the lab. In particular, for this proposal, we are interested in making fibrous structures at the scale of billionths to millionths of a metre. With such 'nanofibres' in hand, we wish to construct 'hydrogels'-that is, entangled networks of fibres that are >99% water. These could be used to capture useful molecules (such as growth factors and nutrients), and then to support cell and tissue growth in the laboratory. These new biomaterials would have long-term uses in the area of tissue engineering. Our inspiration comes from biology, which uses fibrous materials to make structures with a wide variety of functions both within and outside cells; for instance, to give shape and stability to cells; to provide molecular highways within cells; and to act as the glue that hold cells together o form tissues, the so-called extracellular matrix (ECM). In our proposed research, we aim to make simpler, or stripped-down and well-understood materials that capture the key properties and biological functions of the ECM. It is early in the development of our understanding of the principles upon which biological components--proteins, cells, tissues etc--are built; and we are only just beginning to tap the potential of this knowledge. Endeavours to reduce biological complexity to principles and manageable building blocks, and then piece these together to form new materials and systems are known as 'synthetic biology'. This is a very new and exciting science. There's a catch, however: we're not very good at it at the moment. A key feature of Nature is that it uses 'self-assembly' to piece its components together--i.e., the biomolecules are somehow programmed to interact and cooperate in precise ways--which is very different from how our everyday technologies are currently built. We are interested in one type of protein that directs and cements interactions between protein chains. This is called the coiled coil. Amongst other things, it is responsible for making structures like porcupine quills. Our interests are down a few orders of magnitude at the scale of billionths to millionths of a metre. We have succeeded in making fibrous structures, like the quills, in the lab on this scale. Recently, we have learnt how to make these fibres more flexible, and, as result, they interact and entangle to make the gels. Our next steps, as proposed here, are: to make the fibres and gels more reliably and cheaply; to alter their physical properties; to decorate them with other functional molecules; and, ultimately, to test how cells grow on, and respond to them. The aim of this proposal is bring together the necessary expertise and create the tools to make these steps. Why do this? The famous physicist Richard Feynman once remarked that what he could not build, he did not understand. This is the principle that we have adopted for our research: we plan to look at natural biological systems, learn from them, and then test our understanding by designing and attempting to construct new simplified systems. This will not be easy and there is a risk of failure. However, the potential rewards are high: we stand to learn how some of biology's components assemble at the very least; and this understanding can then be applied by us and by others to create new biomaterials that might eventually find applications in other fundamental science and medicine.

To achieve our overall aims of (a) producing a reliable recombinant system for the production and development of fibrous peptide hydrogels, and (b) delivering these to end users in cell biology and tissue engineering, requires a multi-disciplinary project. We will combine state-of-the-art peptide chemistry and design; gene design, synthesis and expression; biophysics, including imaging and rheology; and cell biology. Therefore, this proposal brings together local, national and international experts in peptide and materials design and characterization, and cell culture and tissue engineering. Our primary experimental objectives are: 1. To design and synthesize genes for the peptide components of the hydrogels, and to test these in various constructs and expression systems; 2. To engineer the existing, 1st-generation designs to improve and explore physical properties of the gels such as stiffness, strength and longevity; 3. To add function to the hydrogels by introducing modified peptides that are compatible with fibre assembly and hydrogel formation, and then derivatize these with active peptides through click chemistry; 4. To test the recombinant and redesigned functional hydrogels to support cell growth and differentiation for a broad band of basic and more-specific cell and tissue types; 5. To develop next-generation designed biomaterials, in which new physical properties and biological functions can be incorporated and engineered orthogonally. That is, gel strength or stability can be introduced and altered without compromising biological function and vice versa. To do this, we request two PDRAs in molecular and cell biology, and in peptide design and biophysical characterisation, respectively; and propose collaborations with expert cell biologists, tissue engineers and biophysicists from the UK and Canada.

Who will benefit from this research? Likely beneficiaries of this research include: In the immediate term, the employed post-doctoral researchers, and then the wider UK and international academic communities, public and private education and healthcare sectors, and industry, in which they will be employed. Potentially, in the longer-term, and if the hydrogel technology sees it through to commercialisation, the Universities of Bristol and Sussex and University College, London and the UK economy. Finally, through public engagement, the UK public. How will they benefit from this research? These three group will benefit as follows: As the research is at the interface between the physical, biological and medical sciences, it offers training in multi-disciplinary research to post-doctoral researchers, which will equip them with new skills and give them essential experience for research or related jobs in academia, education, healthcare, or industry. As our long-term objective is to generate biomaterials that will find applications in 3D cell culture and tissue engineering, there are potential impacts in new biomedical products, public health and wealth creation to benefit UK industry and the economy. DNW in particular will continue to engage the public at events such as Science Cafés, public dialogues and related events, to explain emerging areas such as nanotechnology, synthetic biology and tissue engineering. What will be done to ensure that they benefit from this research? We will deliver our Impact Plan by: Providing high-quality training to the post-doctoral researchers at the interface between the physical, biological and medical sciences. Working with the University of Bristol Research & Enterprise Development team, and through a recent Enterprise Development Award to explore the potential market for a hSAF hydrogel technology. Continuing to participate and be pro-active in public engagement events and public dialogues, such as Science Cafés.