The Cellular Control of Corneal Development and Transparency and Generation of Biomimetic Corneal Tissue.

The cornea is the clear tissue at the front of the eye, which transmits light and focuses it sharply onto the retina. Accordingly, it is absolutely essential for vision. Composed mostly of collagen, the cornea is not unlike other collagen-rich tissues, such as tendons, cartilages, intervertebral discs, or even the sclera; the "white of the eye". But what makes the cornea optically clear is the exquisite way in which the collagen is structured.

Collagen in the human body exists predominantly in the form of long fibres, which are very strong along their axes, and can be thought of as having mechanical properties rather like ropes on sailing ships or steel cables on suspension bridges. The organisation of collagen fibres in different tissues is contingent on the tissue's function, and the predominant requirement is usually mechanical. The cornea is unique in this regard because as well as the need to fulfil mechanical requirements, it needs to be transparent.

The bulk of the cornea is made up of cells, known as keratocytes, and collagen. Most collagen exists in the form of very long and very thin fibres, or fibrils as they are called in the cornea because they are so thin. Remarkably, and unlike all the other collagen-rich tissues in the body, collagen fibrils in cornea are all of exactly the same diameter and are arranged into a near-perfect, hexagonal-type lattice. It is this precise structural arrangement of collagen which gives the cornea its transparency. But, how does it arise?

Keratocytes in the cornea synthesise and deposit collagen. So, presumably these cells must be at least partly responsible for the way in which the collagen fibrils are laid down and arranged. We will use pioneering microscopic approaches across a wide range of magnifications, using laser and electron imaging technologies and working with scientists in the University of California, to study corneas from developing chicks in fertilised hen eggs. Our aim is to understand precisely how cells in the developing cornea interact with each other to make such a beautifully structured tissue as a transparent cornea (the basis of corneal transparency in the chick is the same as that in humans).

Recently, we pioneered the use of three-dimensional volume electron microscopy for the study of cornea, and discovered that cells in the developing chick cornea all have highly extended, but thin, cell processes. This showed that the cells themselves occupy a volume of the cornea which is much larger than previously believed. Based on this discovery, our hypothesis is that cells in the developing cornea form an extended network in which they communicate with each other, and that, as a group, they have the innate ability to synthesise incredibly thin collagen fibrils and deposit them into a precise lattice-like arrangement to meet the needs of transparency. To test this hypothesis we will study corneas from the very earliest stages of development, which have never before been examined in three dimensions at such high magnifications. New methodologies for the mathematical modelling of corneal light transmission will be applied to this data to ascertain the key structural requirements for corneal transparency. We will also investigate the cellular contribution to corneal transparency, both by mathematical modelling and by direct measurement of light scattering.

Finally, we will interfere with cell-communication pathways in corneal keratocytes, extracted from developing chick corneas and grown in the lab, to pinpoint what molecular mechanisms they use to communicate. These experiments and analyses, using a new way of growing cells in a three-dimensional environment, which we successfully developed to encourage tendon cells to synthesise aligned collagen fibres, will provide great insights for the field of corneal tissue engineering to help inform the intelligent design of the next generation of bioengineered corneal constructs.

Corneal transparency is provided for by the unique arrangement of exquisitely organised, thin-diameter collagen fibrils which make up the corneal extracellular matrix, but how this comes into being is not understood.

The current research will apply pioneering correlative approaches in laser and electron-based imaging modalities - i.e. non-linear optical second harmonic generation (NLO-SHG), high-resolution macroscopy (HRMac), and serial block face scanning electron microscopy (SBF-SEM) - to investigate the fundamental mechanisms of cell-directed development of a functioning, transparent cornea. Importantly, these technologies are complementary across a wide range of structural hierarchies and spatial resolutions, and will provide new and detailed information in 3D about the formation, from the earliest stages of embryonic development, of the cornea's complex, tissue-specific, architecture.

This knowledge will be correlated with our new mathematical approaches, which will model corneal light transmission using Maxwell's equations based on the scattering of incident radiation by corneal components. This will be combined with the first ever direct measurement of the refractive index of corneal keratocytes to interrogate their contribution to corneal light scattering and transparency, and how this changes as development proceeds.

We will also apply scaffoldless 3D cultivation technologies, which we recently developed for the investigation of tendon cells and their in vitro deposition of "neo-tendons", to study corneal cells to ascertain how they communicate and coordinate the formation of the long-range, highly organised collagen-rich matrix. These studies will determine key fundamental mechanisms of corneal development, and will allow us to probe the potential of cell-based approaches for corneal tissue engineering.

The research described in this proposal is predicted to make a demonstrable contribution to enhancing the knowledge economy in the UK and will lead to significant advances in our understanding of corneal development, and of tissue engineering to combat corneal blindness. Advances will be communicated to and debated with the public. The research also has a high potential for future economic impact via the development of a novel scaffold-free 3D tissue culture system for corneal tissue engineering and the subsequent production of biomimetic constructs for surgical use.

Impact Theme 1: Public Engagement.
Collagen is the most abundant protein in the body. Over time, its structural organisation has evolved to meet the requirements of the particular tissue. Collagen fibres in tendons and ligaments, for example, are arranged mostly parallel, consistent with heavy tensile loading; the intervertebral disc on the other hand is layered to resist the compressive, bending and twisting forces in the spine, while collagen in the skin exists as a meshwork. The cornea is perhaps the most spectacular example of the link between tissue form and function, and collagen fibrils are thin and regularly spaced to scatter light in a special way to allow the front of the eye to be transparent. We will engage with the general public via school visits and Science Café events to debate the significance of collagen in health and wellbeing, and how it can be affected by various aspects of lifestyle, such as UV exposure, the intake of foodstuffs and medications such as sugar and aspirin, and, indeed, smoking.
Our Public Engagement activities will also inform the general public about the value of tissue engineering, and will seek their opinions as to the need for this technology and the way in which it is likely to develop over the coming decades. The discussion, using cornea as an example, will cover concepts such as cell-based production of biomimetic tissue (autologous and as an allograft), the use of synthetic biomaterials, and the possible future impact of stem cells, IPS technology and 3D printing. The outcome of these discussions with the public will be communicated to the Chief Optometric Advisor for The Wales Government, Dr Barbara Ryan, to provide some insight into the views of the local public on ocular tissue engineering.
To engage with the community more widely will also create a dedicated website, on which we will summarise our discoveries. This will be written, updated and monitored by Dr Knupp in consultation with the other applicants, and will targeted at the general public and healthcare professionals.

Impact Theme 2: Potential Commercialisation of New Technologies and Tissue Engineered Constructs.
There is a very strong current interest in corneal tissue engineering. Recently, a consortium of academic and industry researchers in the USA, Sweden, and Canada, writing in Science Translational Medicine [2010;2:46ra61], presented data on collagen gels as possible corneal replacements, and highlighted the pressing need for the development of artificial corneal tissue. We will take advantage of the fact that cells in connective tissues make their own collagenous matrices, and will utilise a 3D culture system which we designed for tendon cells (Wreede R, Ralphs JR. Tissue Eng Part A 2009;15:2707-2715). Corneal cells will be grown in a scaffoldless environment to provide essential information about matrix bioassembly processes which lead to the establishment of a strong and transparent cornea. The work will help validate the suitability and optimisation of our cultivation procedures for corneal tissue engineering. Beneficiaries will be medical technology industries with interests in tissue engineering of corneal constructs.