Abstract
Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.
Highlights
Decellularized by various concentrations of sodium deoxycholate (SD) with deoxyribonuclease I in comparison to the normal human cornea (NHC) using hematoxylin and eosin (H&E) staining
Human DM− corneas decellularized with different concentrations of standard deviation (SD) were initially screened by hematoxylin and eosin (H&E) staining, immunofluorescent staining for human leukocyte antigen (HLA)-ABC was used to assess the presence of residual cellular/membranous material, and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) (Fig. 1A)
The use of 0.5% SD did not result in complete removal of cellular debris as apparent in H&E (Fig. 1A, higher magnification, arrowheads) and HLA-ABC staining’s (Fig. 1A, arrows)
Summary
Decellularized by various concentrations of sodium deoxycholate (SD) with deoxyribonuclease I in comparison to the normal human cornea (NHC) using hematoxylin and eosin (H&E) staining. The higher magnification images of H&E staining showing the stromal cells in NHC (arrowheads) and cellular debris in 0.5% SD DAPI staining reveals nuclei in NHC without remaining nuclei/nuclear debris in DHC.
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