Bioprosthetic cornea: using polymeric templates for directed stem cell growth

The cornea is our window to the world, once compromised by wounding, disease or age, a loss of vision results. By improving our understanding of corneal structure and providing new methods of corneal transplantation the sight of many more patients can be restored. Currently, corneal transplantation requires a continuous supply of healthy donor corneas. However worldwide demand has grown and taken together with an aging population and the rapid rise in laser eye surgery (which can negatively affect the donor tissue suitability for transplantation) the search for an effective engineered replacement is essential if current levels of corneal transplantation are to be maintained. This investigation stems from our previous work in understanding the molecular structure underpinning corneal transparency, the development of novel corneal biomaterials and the limitations of the current corneal stem cell transplantation techniques, specifically the materials used to grow and convey the stem cells to the patient. Previously, we have quantified the nanostructure of the cornea and related this structure to the preservation of corneal transparency; furthermore we have applied these measurements to the design of new corneal biomaterials capable of supporting corneal epithelial stem cell differentiation and growth. Therefore, we will draw on our knowledge of corneal structure, corneal stem cell isolation and cultivation and novel biomaterials to engineer a tissue suitable for corneal transplantation. To do this we will develop a template made from tractable regularly spaced aligned polymers (reflecting the natural state of corneal nanostructure). The template will contain protein fragments recognisable by the human corneal stem cells enabling them to attach in a highly ordered and controlled manner. Once attached the cells will be chemically induced to differentiate and produce collagen fibres. The alignment of these fibres will be guided by the cells orientation. The template will then slowly lose its integrity and detach from the cells by way of enzymes released by the corneal cells, thus releasing the aligned collagen as a tissue engineered collagen mat. These mats will then be stacked and compressed to produce a robust biomaterial made solely from human proteins (mostly collagen), the polymer template having been removed during processing. The biomaterials mechanical strength and ability to support corneal epithelial growth upon its surface will then be tested. We have already shown that compressed mats of rat tail collagen are both mechanically robust as well as excellent substrates for corneal epithelial cell growth. This work represents a significant step forward in the development of biomaterials. Instead of designing and using bio-compatible polymers to represent tissue for transplantation our approach is to use the polymers merely as a template allowing the cells to produce the actual biomaterial. Furthermore, since the template is easily discarded the cell based biomaterial represents the ultimate in biocompatibility as it is comprised of human proteins possibly even derived from cells taken from the patient's own body. The beneficiaries of this work would be those working in the fields of polymer chemistry as the development of aligned polymers containing protein fragments that are both recognised and degraded by cells is not trivial. Scientists in the blossoming field of biomaterials would be given a new direction in the development of truly bio-compatible materials (i.e. ones derived from stem cells). Tissue engineers would be given a new tool with which they could create similar stem cell based materials for bone, skin etc. repair and finally ocular regenerative medicine would benefit from the development of a replacement human donor corneal tissue.

To produce a bioprosthetic cornea, an orientable hydrogel template using novel RGD-based self-assembling peptide systems containing MMP cleavage sites will be fabricated. The hydrogel will be composed of Fmoc-tripeptides aligned by shear flow. Alignment will be quantified by electron /atomic microscopy and small angle x-ray scatter. Measurement of mechanical properties will be achieved through dynamic shear rheometry. The type and number of MMP cleavage sites will be optimised so that the cultured stromal stem cells (once differentiated) cause disruption of the polymer substrate (via MMP expression) in a practicable time i.e. a period of time that is consistent with the production of an aligned collagen mat. The possible presence of residual Fmoc-tripeptides within the collagen mat will be detected by spectroscopic methods. Within the collagen mat the ECM proteins and markers of stromal cell phenotype will be measured by Western blotting, zymography, immunocytochemistry, QPR and cupromeronic blue staining. Arrangement of the newly formed collagen within the mat will be quantified by electron microscopy and small angle x-ray scatter; its mechanical properties will be assessed by measurements of shear modulus and tensile strength. Once characterised the collagen mats will be stacked followed by a process of plastic compression to create an engineered stroma. In this way alternating layers of arranged collagen fibre will be achieved (mimicking the natural corneal stroma). Plastic compression is a cell friendly procedure allowing the cells to remain viable within the compressed matrix. The engineered stroma's ability to support corneal epithelial cell growth and stratification will be shown by the expansion of limbal stem cells upon the surface of the tissue engineered stroma. The capability to support a normal corneal epithelium will be assessed by specific gene and protein levels by QPCR, Western blotting and immunocytochemistry and by cell morphology by microscopy.

The biotechnology industry is rapidly expanding and the emerging field of tissue engineering is projected to have a high commercial impact in the near future. Within this field the orientation of fibres has recently been recognised as one of the important features of a perfect tissue scaffold. Following recent discussions with The Automation Partnership and L'Oreal the long term commercial benefits of aligned biomaterials, as a platform technology, are impressive; for example next generation ocular toxicity assays or artificial corneas. We already have in place NDA's with both these companies. Timescales, which would include finding further funding to properly commercialise the technology are likely to be 6-8 years. Within the health care sector there are significant long term benefits if a biomaterial which can replace the corneal stroma can be manufactured. The consequences would be an improvement in patient care as the transplants could be offered at the exact time they are needed, an increase in availability (i.e. not limited to donor numbers) and a significant saving in the costs associated with eye bank management. These benefits can be realised 5 years on from completion of the current project and would expected to be financed by the MRC or NHS. We have recently established a link with the Micro and Nanotechnology Centre, Science and Technology Facilities Council, RAL. This Publically funded laboratory has strategic ambitions in healthcare futures programmes. The current proposal, and its expected results, would lend intellectual weight to our discussions in using electrospinning for corneal stem cell substrates. Outputs would be in the form of publications and patents leading to licensing agreements (supported by our Tech Transfer Office). Our biomaterial has been identified as a potential battle dressing with obvious benefits to the MOD. An aligned biomaterial (which is light, cheap and stable) that can direct and orientate cell growth could be applied directly to the wounded cornea. By controlling the orientation of collagen regrowth, within the wound, scarring can be reduced reducing the need for corneal transplantation. Using funding from the Centre for Defence Enterprise the time scale for a prototype would be short, 1-2 years. Further meetings are planned with our established contacts at L'Oreal (Dr Jean-Roch Meunier, Director of Safety Research), The Automation Partnership (Rosemary Drake, Chief Scientific Officer), Royal Berkshire Hospital (Mr Martin Leyland, Consultant Ophthalmologist), Micro and Nanotechnology Centre, STFC (Dr Bob Stevens) and Dept of Biomedical Sciences, Defence Science and Technology Laboratory, Porton Down (Dr Chris Green and Dr Leah Scott).