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

Abstract

The decellularized cornea has received considerable attention for use as an artificial cornea. The decellularized cornea is free from cellular components and other immunogens, but maintains the integrity of the extracellular matrix. However, the ultrastructure of the decellularized cornea has yet to be demonstrated in detail. We investigated the influence of high hydrostatic pressure (HHP) on the decellularization of the corneal ultrastructure and its involvement in transparency, and assessed the in vivo behaviour of the decellularized cornea using two animal transplantation models, in relation to remodelling of collagen fibrils. Decellularized corneas were prepared by the HHP method. The decellularized corneas were executed by haematoxylin and eosin and Masson’s trichrome staining to demonstrate the complete removal of corneal cells. Transmission electron microscopy revealed that the ultrastructure of the decellularized cornea prepared by the HHP method was better maintained than that of the decellularized cornea prepared by the detergent method. The decellularized cornea after interlamellar keratoplasty and microkeratome-assisted anterior lamellar keratoplasty using a rabbit model was stable and remained transparent without ultrastructural alterations. We conclude that the superior properties of the decellularized cornea prepared by the HHP method were attributed to the preservation of the corneal ultrastructure.

Decellularization methods for developing porcine corneal xenografts and future perspectives

“…In this method, a pressure of 980 MPa or 10.000 kPa is applied to the cornea for 10 minutes at 10°C or 30°C. In order to achieve sufficient decellularization and maintain corneal histo‐architecture, corneas must then be washed with PBS or EGM‐2 medium containing dextran and DNase I . Applying the HHP method at 10°C instead of 30°C maintained collagen fibrils and interactions to a greater extent .…”

“…“Complete” decellularization was not achieved by 3‐((3‐cholamidopropyl) dimethylammonio)‐1‐propanesulfonate (CHAPS, a zwitterionic detergent). In certain conditions, it resulted in the destruction of the corneal histo‐architecture . Benzalkonium chloride is a cationic surfactant that was unable to “completely” decellularize corneal tissue.…”

“…Triton X-100 is a non-ionic detergent and the second most commonly used detergent in corneal decellularization experiments. [17][18][19] Its decellularization activity is based on the disruption of lipid-to-lipid and lipid-to-protein interactions, but not protein-to-protein interactions. 33 The efficacy of decellularization agents greatly fluctuates with the tissue being treated.…”

“…Decellularization of a three‐dimensional matrix scaffold (ie, organ or tissue, cornea) enables the removal of cellular and immunogenic components from an organ/tissue while preserving ECM with its native micro‐ and macroscopic anatomy . Many different protocols for decellularizing human and porcine corneal tissues have been reported using biological, physical, and chemical compounds . However, there is still no standardized protocol.…”

“…16 Many different protocols for decellularizing human and porcine corneal tissues have been reported using biological, physical, and chemical compounds. [17][18][19][20] However, there is still no standardized protocol.…”