Search

Corneal endothelial damage and diseases are two of the major contributors to blindness or severe visual impairment worldwide (1). The corneal endothelium is the innermost cell layer of the cornea and consists of a cellular monolayer that maintains the stroma in a dehydrated state through a “pump-and-leak” mechanism. A critical loss of cells due to damage, disease or aging leads to corneal edema which in turn results in opacification of the cornea (2). Currently, the only treatment consists of a corneal transplantation from healthy cadaveric donor tissue. Unfortunately, only 1 donor is available for every 70 cases (3). To tackle this shortage, the present work focusses on the development of transparent (>90%, 380-700nm), ultra-thin (≤5µm), multilayered sheets that are permeable towards glucose (Papp >2.36*10-3cm/s). These sheets constitute a poly(D,L-lactide) (PDLLA) layer to provide structural rigidity (E=4.95 ± 0.81MPa) and a crosslinkable gelatin-based hydrogel (Gel-MA-AEMA (4)) as an extracellular matrix (ECM) mimic. These sheets provide a substrate for corneal endothelial cells (CECs) (primary human CECs and B4G12 immortalized CECs) to enable subsequent ocular implantation with the aim to restore the damaged endothelium and the patient’s vision (5). Successful membrane production and coating were verified using X-ray photoelectron spectroscopy (XPS) and static contact angle measurements. Cells seeded on the membranes showed their characteristic hexagonal morphology, the presence of Na+/K+ ATPase pumps and tight junctions (ZO-1) as evidenced using immunocytochemical staining. Additionally, research has been devoted towards upscaling of the membranes while ensuring straightforward tunability of the thickness, through doctor blading. References 1. WHO (2017). 2. J. Zhang and D. V. Patel, Exp. Eye Res., 130, 97–105 (2015). 3. P. Gain et al., JAMA Ophthalmol., 134, 167 (2016). 4. J. Van Hoorick et al., Biomacromolecules, acs.biomac.7b00905 (2017). 5. J. Van Hoorick et al., Adv. Healthc. Mater., 2000760 (2020).

The corneal endothelium is the most inner cell layer of the human cornea, which is the transparent window of the eye. It functions as a leaky barrier to facilitate to exchange of nutrients and water to the avascular cornea. It is a monolayer of hexagonal cells with the foremost task to maintain the cornea’s specific hydration status and thus concomitant transparency. However, they do not possess the proper machinery for tissue regeneration. That is why the absolute number of cells only decreases throughout life. When the process of inevitable cells loss is aggravated by disease or (surgical) trauma, the cornea will inadvertently swell and lose its transparency, which leads to visual impairment and blindness. If left untreated, this results in the formation of epithelial bullae, blisters on the front side of the cornea, which cause excruciating pain for the patient on top of visual impairment. At this moment, such patients can only be helped through the transplantation of a donor endothelium from a cadaveric cornea. Despite good clinical outcomes, the amount of available donor corneas does not meet the tissue demand. That is why I explored the cultivation of human corneal endothelial cells on different substrates to develop an ex vivo grown corneal endothelium. In this way, the global donor shortage could be alleviated by providing a therapy to multiple patients with one donor cornea. In this thesis, three different substrates were tested for their propensity to act as a cell scaffold for corneal endothelial culture and transplantation. The first two, the human anterior lens capsule and a collagen fish scale derived scaffold, were biologically derived, but were not appropriate for the proposed application. On the one side, the anterior lens capsule displayed good cytocompatibility, but had a rather limited diameter and was still dependent on a donor supply. On the other side, the fish scale scaffold displayed an extensive surface topography which hampered monolayer formation. That is why we have investigated a third scaffold, namely a synthetic ultrathin scaffold that was developed in collaboration with UGent, composed of a poly-(D,L)-lactic acid base and a gelatin coating. Initial experiments show optimal physicochemical properties and a biological proof-of-concept to grow immortalized and primary corneal endothelial cells. In the future, this novel composite scaffold will further be investigated in an ex vivo model of corneal endothelial transplantation before being tested in a rabbit model of corneal endothelial keratoplasty.