Engineered Basement Membranes for Regeneration of the Corneal Endothelium

Research Description

Regenerating the Corneal Endothelium

Protein Nanofabric Fabrication

Bioengineering the Corneal Endothelium

The corneal endothelium is responsible for maintaining the clarity of the cornea and loss of corneal endothelial cells (CECs) leads to impaired vision and the need for corneal transplantation.  Descemet’s Membrane Endothelial Keratoplasty (DMEK) and related techniques are successful at restoring the pumping function of the endothelium, however donor corneas are limited worldwide and CEC loss can recur due to damage incurred during transplantation.

A bioengineered corneal endothelium could be implanted using existing DMEK surgical techniques, but faces two primary challenges; (i) CECs are non-proliferative in vivo with minimal proliferation in vitro, making expansion of these cells for therapeutic application difficult and (ii) cell-sheet engineering techniques are unable to recreate a basement membrane similar to Descemet’s membrane (DM) for attachment to the anterior side of the stroma.

The nucleus (blue) ZO-1 (red) and F-actin (green) of cells cultured on our biomimetic substrate.

Our initial work toward solving these two major challenges and making a bioengineered corneal endothelium a therapeutic reality involves 1) trying to further expand the CECs and 2) engineering a DM mimic for the culture and transplantation of CECs. In vitro expansion of CECs is limited to a few passages and the CECs rapidly de-differentiate into fibroblast-like cells that lose CEC phenotypic markers. This makes it difficult to expand enough CECs in vitro for therapeutic applications such as bioengineering a corneal endothelium.

We hypothesized that mimicking the extracellular matrix (ECM) and mechanical properties of the DM would promote maintenance of phenotype while enabling cell expansion. Compared to standard tissue culture polystyrene, we have engineered a biomimetic substrate with a unique ECM protein coating and low stiffness that enables bovine CECs to be passaged up to 8-times while maintaining a hexagonal morphology and expression of zona occludens 1 (ZO-1). Preliminary estimates indicate a ~3000-fold expansion of CECs, suggesting that a similar strategy may be successful for expanding human CECs.

Second, DMEK is a clinically successful procedure, suggesting that a bioengineered cornea with similar handling characteristics and pumping function would be a viable option for corneal repair. We hypothesized that engineering a basement membrane with chemical and mechanical properties similar to the DM would be an ideal scaffold. We have used surface-initiated assembly to engineer ECM protein nanofabrics that match the laminin and collagen type IV composition of the DM and are working towards engineering a functional endothelium using these materials.

In summary, we have developed a number of biomimetic technologies for tissue engineering a corneal endothelium. The in vitro expansion of bovine CECs was significantly enhanced by engineering cell culture substrates that match the chemical and mechanical properties of the DM. Using a similar strategy, we have engineered ECM scaffolds from laminin and collagen type IV and are actively developing methods to use these to form bioengineered endothelium.

Future work includes transitioning from bovine to human CECs, establishing in vitro assays to validate pump function of the bioengineered endothelium and in vivo evaluation in an animal DMEK model.