Endothelial Wound Repair of the Organ-Cultured Porcine Corneas

ABSTRACT

Purpose: To assess whether injured porcine endothelium of small and large corneoscleral disc differ in its reparative/regenerative capacity under various conditions of organ culture storage.

Material and methods: 166 paired porcine corneas were trephined to obtain tissues with diameter 12.0 mm and 17.5 mm (with area neighboring endothelial periphery). In tested discs, central endothelium was mechanically wounded. Density of live endothelial cells (LECD), percentage of dead cells (%DC), coefficient of variation and cell hexagonality were assessed in central and paracentral endothelium following 5- or 9-day incubation in medium with 2% or 10% fetal bovine serum. The parameters were assessed also in fresh and intact cultured discs. Dead endothelial cells (EC) were visualized by trypan blue, cell borders by Alizarin Red S dye. Endothelial imprints were immunoassayed for the proliferation marker Ki-67 and the nucleolar marker fibrillarin.

Results: In fresh corneas, the LECD/mm² (mean ± standard deviation) were 3998.0 ± 215.4 (central area) and 3888.2 ± 363.1 (paracentral area). Only the length of storage had significant effect on wound repair. Lesion was repaired partially after 5-day and fully after 9-day cultivation. After 9-day storage in medium with 10% serum, the mean LECD detected in small discs were 2409.4 ± 881.8 (central area) and 3949.5 ± 275.5 (paracentral area) and in large discs the mean LECD were 2555.0 ± 347.0 (central area) and 4007.5 ± 261.2 (paracentral area). Ki-67 showed cell proliferation associated with healing of EC of both large and small corneas.

Conclusions: The lesions were completely repaired within 9 days of storage. Presence of the area, where stem cells appear to be located, contributes to stimulation of endothelial reparation less than serum concentration and time of culture. Both cell migration and proliferation contribute to the wound repair.

KEYWORDS:

• Porcine corneal endothelium
• endothelial cell density
• endothelial wound repair
• organ culture
• endothelial cell proliferation

Acknowledgments

We would like to thank Dr. Dusan Cmarko and Dr. Evgeny Smirnov (Institute of Biology and Medical Genetics, Charles University, Prague) for supply of fibrillarin antibody (kindly donated by Dr. U. Scheer, Biocenter of the University of Würzburg, Würzburg, Germany) and HeLa cells.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Funding

This work was supported by The Research Council of Norway, and Ministry of Education, Youth and Sports of the Czech Republic [Norwegian Financial Mechanism 28477/2014, project 7F14156]; by European Regional Development Fund [project BBMRI-CZ III: EF16_013/0001674]; and by Charles University [SVV, project 260367/2017, project PROGRES-Q25 and Q26/LF1].