Ultrastructural analysis of the decellularized cornea after interlamellar keratoplasty and microkeratome-assisted anterior lamellar keratoplasty in a rabbit model

A worldwide shortage of donated human corneas for transplantation is a serious problem, with increasing need of approximately 1–2 million individuals annually1,2,3. To date, numerous studies have reported various artificial cornea, including keratoprostheses and tissue-engineered (TE) constructs as alternatives to donor corneas4,5,6,7,8. Nevertheless, most of these artificial corneas only partially satisfy the requirements, including biocompatibility, transparency, flexibility, and sufficient suturability strength. Ideal artificial corneas should be equivalent, and preferably superior to human cadaver corneas9. Recently, decellularized corneas have received a great deal of attention as artificial corneal replacements, because they are usually completely free of cellular components and other immunogens from the corneal tissue through the use of chemical, biological, and physical methods, or combinations of these methods, thereby facilitating the suppression of the immune response to the tissue. However, these corneas still maintain the integrity of the extracellular matrix (ECM)9,10,11. We have previously described the preparation of decellularized corneas using the high hydrostatic pressure (HHP) method12,13. The results of our in vitro studies comparing the efficacy of detergent versus HHP decellularization methods showed that the HHP method maintained corneal characteristics, such as the mechanical properties and glycosaminoglycan (GAG) content that are particularly important for the preservation of corneal structure, but not for transparency. Interestingly, subsequent in vivo studies, using interlamellar keratoplasty and deep anterior lamellar keratoplasty (DALK) in a xenotransplantation model, demonstrated that the decellularized cornea was opaque immediately after transplantation, but eventually became transparent within a follow-up period14. In general, corneal transparency is regulated by the specific architecture of the corneal stroma, which is a highly oriented, stacked collagen layer aligned in the vertical and horizontal directions15,16. With such a complex structure, the corneal stroma is very sensitive, and if it is irreversibly damaged by HHP decellularization, the transparency of the decellularized cornea may not recover. Therefore, we hypothesize that the properties of decellularized cornea contribute to the preservation of corneal ultrastructure even after the HHP decellularization. However, the ultrastructure of the decellularized cornea has yet to be demonstrated. In this study, we investigated the ultrastructural alterations of decellularized corneas transplanted into the rabbit stroma with different surgical procedures, using transmission electron microscopy (TEM).