Development of conjunctival and retinal models for improved eye disease treatments

Abstract

Eye diseases such as glaucoma, macular degeneration and retinitis pigmentosa can lead to significant sight problems and blindness if left untreated. Current treatments are invasive, have a low success rate and lack the ability to regenerate damaged ocular tissue. The requirement for more effective treatments is therefore high. The development of tissue engineering strategies to treat ocular disease has advanced significantly over recent years with promising results. Therefore, the establishment of improved conjunctival and retinal tissue models for investigation of ocular disease mechanisms and testing of new treatments is required. The overall aim of this project was to develop and apply improved conjunctival and retinal models using tissue engineering approaches for eye disease treatments.
2D and 3D Tenon’s capsule and conjunctival tissue (TCCT) models were constructed by seeding porcine TCCT fibroblasts into collagen hydrogel. These were used to investigate key stimulatory factors (growth factors, cytokines, aqueous humour and shear stress) believed to influence tissue fibrosis response after glaucoma surgery. In addition to cellular metabolic assessment, a new non-destructive assay using added azido-L-proline to quantify neocollagen synthesis in response to these factors, was applied for up to 14 days of culture. Results found that 3D TCCT fibroblast metabolism, actin expression and neocollagen synthesis increased by up to 60% by day 7 of culture with the addition of TGF-ß, VEGF, TNF-a (50 ng/mL) or 50% aqueous humour. Furthermore, shear stress-induced mechanotransduction was found to promote metabolic activity across in vitro experimental conditions, with shear stress in combination with aqueous humour triggering the strongest fibrotic response, followed by TGF-ß, TNF-a and VEGF. Shear stress therefore appeared to enhance the influence of growth factors and further promoted fibrotic responses within the TCCT model. These novel findings offer a useful contribution to the understanding of wound healing response triggered by aqueous fluid outflow after glaucoma surgery.
The established TCCT models were also used for evaluation of glaucoma treatment efficacy and in vitro medical device assessment. Key growth factor inhibitors, decorin proteoglycan and the novel application of calcium signalling channel blocker nifedipine were each tested. Furthermore, the Xen Gel Stent glaucoma medical device was tested in vitro for the first time using the established 3D TCCT model. Key results found that TCCT fibroblast metabolism decreased by up to 281.9% by day 3 of culture with the addition of TNF-a and TGF-ß inhibitors (10µM) or nifedipine (100µM). Furthermore, fibroblast activity surrounding the stent lumen was evident when implanting the Xen Gel Stent glaucoma medical device into the 3D TCCT tissue model, altogether offering convenient new methods to assess new glaucoma treatment drugs/devices.
In the final results chapter, a 3D in vitro organotypic retinal tissue model was developed for improved retinal disease study and treatment. A compressed collagen gel was tested for the first time as an (artificial vitreous) substrate for the culture of organotypic retinal explants. Non-destructive imaging modality, OCT, was applied to monitor the full culture period, with cross-validation by conventional histological examinations. Findings confirmed that compressed collagen was beneficial in providing the biochemicals and mechanical strength required to preserve retinal tissue in vitro in the absence of intraocular pressure, whilst still being biocompatible. All layers of porcine retinal tissue were preserved intact and with minimal distortion for up to 2 weeks. The study also demonstrated the novel use of OCT to monitor live in vitro retinal explants during culture. Following establishment of a laser injury method for the retinal tissue, added stem cells were found to aggregate towards the retinal injury site, with differentiation evident.
Overall, the ocular models developed in this thesis offer useful tools for the study of ocular disease mechanisms and testing of potential drug interventions, new medical devices and stem cell therapies, whilst also reducing the requirement for live animal experiments.