Novel in vitro dynamic corneal model with online mechanical characterisation for pharmaceutical screening and tissue engineering applications

The rabbit eye is frequently used to test new drugs or new therapies for corneal disease. One example of such as disease is keratoconus where the mechanical properties change over time. Concerns over the use of animals for eye research has been growing but efforts to replace these experiments with adequate tissue models have been lacking. To reduce or eliminate the use of animal models for understanding the biology and treatment of corneal disease, more physiologically relevant in vitro models must be developed. One feature of the cornea which is directly linked to some disease conditions is the mechanical integrity of the corneal stroma or tissue. However, to date, none of the 3D tissue models are able to assess or utilise the mechanical properties of the cornea as an outcome measure of drug testing in vitro. In this proposal, we propose to develop a novel on-line characterisation tool for mechanical properties, which can monitor viable tissue construction over time in a non-destructive and on-line manner under sterile conditions. This set up is part of new developments involving the establishment of a bioreactor for corneal tissue models which facilitates growth over a prolonged culture period. This novel methodology enables the mechanical property of the in vitro tissue to be used as a biomarker. After successfully establishing the methodologies, we shall use this in vitro model for the screening of pharmaceutical drugs and new therapies for keratoconus. Efficacy of drugs in our 3D models will be compared with existing data using animal models. Efficacy will be defined as maintained or improved mechanical integrity.

Animal models are currently used extensively for investigations into diseases of the cornea. This is mainly due to a lack ofadequate in vitro human tissue models which can be used for therapy evaluation by the pharmaceutical industry. Although some corneal models have been developed which go some way to allowing us to understand the interactions between the cells and their matrix, the mechanical integrity and properties cannot be assessed as outcome measures for screening. In this proposal, we propose two novel approaches to expand and improve our current in vitro models for use by industry. Firstly, based on our published proof of concept, we will develop an indentation characterisation tool specific to corneal tissue. A key feature of this corneal equivalent is the ability to make on-line and non-destructive mechanical measurements throughout the screening process. Following the proposed modifications, the indentation system will be capable of detection of the force and displacement resolution better than 10 microN and 10 microM respectively. Using a well-developed marker, e.g. graded silicon carbide spheres combined with Finite Element Analyses, will allow us to extend our capability of analysing multi-axis and local strain distribution. Secondly, a novel bioreactor which combines mechanical stimulation and characterisation in one set-up will provide further improvements to our in vitro model. The success of the new model will be tested following application of drugs using mechanical properties such as biomarkers, e.g classes of drugs will include steroids, anti-inflammatory drugs, and growth factors for keratoconus. In addition, we will test a new therapy for corneal ulcer treatments which combines photosensitive chemical riboflavin with UV irradiation which results in an increase in the degree of crosslinking - our system should be able to detect these changes during treatment. Generating new models will ultimately lead to a reduction in animal experimentatio