The eggshell membrane: A potential biomaterial for corneal wound healing

The eggshell membrane (ESM) is an abundant resource with innate complex structure and composition provided by nature. With at least 60 million tonnes of hen eggs produced globally per annum, utilisation of this waste resource is highly attractive in positively impacting sustainability worldwide. Given the morphology and mechanical properties of this membrane, it has great potential as a biomaterials for wound dressing. However, to date, no studies have demonstrated nor reported this application. As such, the objective of this investigation was to identify and optimise a reproducible extraction protocol of the ESM and to assess the physical, chemical, mechanical and biological properties of the substrate with a view to use as a wound dressing. ESM samples were isolated by either manual peeling (ESM-strip) or via extraction using acetic acid [ESM-A0.5] or ethylenediaminetetraacetic acid, EDTA [ESM-E0.9]. Energy dispersive X-ray spectroscopy (EDS) confirmed that there were no traces of calcium residues from the extraction process. Fourier transform infrared (FTIR) spectroscopy revealed that the extraction method (acetic acid and EDTA) did not alter the chemical structures of the ESM and also clarified the composition of the fibrous proteins of the ESM. Scanning electron microscopy (SEM) analyses revealed a three-layer composite structure of the ESM: an inner layer as continuous, dense and non-fibrous (limiting membrane), a middle layer with a network of fibres (inner shell membrane) and the outer layer (outer shell membrane) of larger fibres. Material properties including optical transparency, porosity, fluid absorption/uptake, thermal stability, mechanical profiling of the ESM samples were performed and demonstrated suitable profiles for translational applications. Biological in vitro studies using SV40 immortalised corneal epithelial cells (ihCEC) and corneal mesenchymal stromal cells (C-MSC) demonstrated excellent biocompatibility. Taken together, these results document the development of a novel sustainable biomaterial that may be used for ophthalmic wounds and/or other biomedical therapies.