Corneal endothelial regeneration in human eyes using endothelium-free grafts

Background To report on corneal endothelial regeneration, graft clarity, and vision recovery when using endothelium-free grafts. Methods We evaluated the donor's cell viability using trypan blue staining and dual staining with calcein acetoxy methyl ester and ethidium homodimer-1. To preserve eyeball integrity, we performed therapeutic penetrating keratoplasty using cryopreserved donor tissue without endothelium on 195 consecutive patients who suffered from corneal perforation due to progressive primary corneal disease such as herpes simplex keratitis, fungal keratitis, ocular thermal burns, keratoconus, and phlyctenular keratoconjunctivitis. Of these, 18 eyes recovered corneal graft clarity and underwent periodic slit-lamp microscopy, A-scan pachymetry, and in vivo confocal microscopy to observe the clinical manifestations, variations in corneal thickness, and repopulation of the corneal endothelial cells on the donor grafts. Results No viable cells were detected in the cryopreserved corneas. After the therapeutic penetrating keratoplasty, notable corneal graft edema was observed in all 18 eyes for 1-4 months, and no corneal endothelial cells were detected on the grafts during this period. Thereafter, we observed gradual and progressive regression and final resolution of the stromal edema, with complete recovery of corneal graft clarity. Through periodic confocal microscopy, we observed the corneal endothelium's regenerating process, along with single cells bearing multiple nuclei and cell division-like morphology. The regenerated endothelium on the grafts reached a mean cell density of 991 cells/mm(2). Remarkable vision rehabilitation was achieved in all 18 patients. Conclusions We obtained conclusive evidence that host-derived endothelial cells can regenerate a new endothelium over the endothelium-free graft, which possesses normal functions for corneal clarity and vision recovery.