The worldwide limited availability of suitable corneal donor tissue has led to the development of alternatives, including keratoprostheses (Kpros) and tissue engineered (TE) constructs. Despite advances in bioscaffold design, there is yet to be a corneal equivalent that effectively mimics both the native tissue ultrastructure and biomechanical properties. Human decellularized corneas (DCs) could offer a safe, sustainable source of corneal tissue, increasing the donor pool and potentially reducing the risk of immune rejection after corneal graft surgery. Appropriate, human-specific, decellularization techniques and high-resolution, non-destructive analysis systems are required to ensure reproducible outputs can be achieved. If robust treatment and characterization processes can be developed, DCs could offer a supplement to the donor corneal pool, alongside superior cell culture systems for pharmacology, toxicology and drug discovery studies.

Background: There is a clinical need for biomimetic corneas that are as effective, preferably superior, to cadaveric donor tissue. Decellularized tissues are advantageous compared to synthetic or semi-synthetic engineered tissues in that the native matrix ultrastructure and intrinsic biological cues including growth factors, cytokines and glycosaminoglycans may be retained. However, there is currently no reliable, standardized human corneal decellularization protocol. Methods: Corneal eye-bank tissue unsuitable for transplantation was utilized to systematically compare commonly used decellularization protocols. Hypertonic sodium chloride; an ionic reagent, sodium dodecyl sulphate; a non-ionic detergent, tert-octylphenol polyoxyethylene (Triton-X); enzymatic disaggregation using Dispase; mechanical agitation; and the use of nucleases were investigated. Decellularization efficacy, specifically for human corneal tissue, was extensively evaluated. Removal of detectable cellular material was evidenced by histological, immunofluorescence and biochemical assays. Preservation of macroscopic tissue transparency and light transmittance was evaluated. Retention of corneal architecture, collagen and glycosaminoglycans was assessed via histological, immunofluorescence and quantitative analysis. Biocompatibility of the resulting scaffolds was assessed using cell proliferation assays. Results: None of the decellularization protocols investigated successfully removed 100% of cellular components. The techniques with the least residual cellular material were most structurally compromised. Biochemical analysis of glycosaminoglycans demonstrated the stripping effects of the decellularization procedures. Conclusion: The ability to utilize, reprocess and regenerate tissues deemed “unsuitable” for transplantation allows us to salvage valuable tissue. Reprocessing the tissue has the potential to have a considerable impact on addressing the problems associated with cadaveric donor shortage. Patients would directly benefit by accessing greater numbers of corneal grafts and health authorities would fulfill their responsibility for the delivery of effective corneal reconstruction to alleviate corneal blindness. However, in order to progress, we may need to take a step back to establish a “decellularization” criterion; which should balance effective removal of immune reactive material with maintenance of tissue functionality.

Control and maintenance of the keratocyte phenotype is vital to developing in vitro tissue engineered strategies for corneal repair. In this study the influence of topographical and chemical cues on the mechanical, phenotypical and genotypical behaviour of adult human derived corneal stromal (AHDCS) cells in three dimensional (3D) multi‐layered organised constructs is examined. Topographical cues are provided via multiple aligned electrospun nanofiber meshes, which are arranged orthogonally throughout the constructs and are capable of aligning individual cells and permitting cell migration between the layers. The influence of chemical cues is examined using different supplements in culture media. A non‐destructive indentation technique and optical coherence tomography are used to determine the matrix elasiticity (elastic modulus) and dimensional changes, respectively. These measurements were indicative of changes in cell phenotype from contractile fibroblasts to quiescent keratocytes over the duration of the experiment and corroborated by qPCR. Constructs containing nanofibers have a higher initial modulus, reduced contraction and organised cell orientation compared to those without nanofibers. Cell‐seeded constructs cultured in serum‐containing media increased in modulus throughout the culture period and underwent significantly more contraction than constructs cultured in serum‐free and insulin‐containing media. This implies that the growth factors present in serum promote a fibroblast‐like phenotype; qPCR data further validates these observations. These results indicate that the synergistic effect of nanofibers and serum‐free media plus insulin supplementation provide the most suitable topographical and chemical environment for reverting corneal fibroblasts to a keratocyte phenotype in a 3D construct.