Abstract
Purpose::
To study the outcome of autologous limbal stem cell (LSC) culture on human denuded amniotic membrane as a treatment for corneal regeneration in a total limbal stem cell deficiency animal model.
Methods::
Complete limbal stem cell deficiency (LSCD) was induced by treatment with n-heptanol in thirty rabbits. After two months animals underwent transplantation of autologous limbal stem cell culture on human denuded amniotic membrane (n=15, Group I) or received symptomatic treatment (n=15, Group II). Culture explants were culture by a combined method including a first part on plastic for 14-21 days and secondly on denuded amniotic membrane (72 hours). Transplanted cells were characterized by immunohistochemistry using antibodies against p63, CK3/12 and CK14. Clinical outcome was based on biomicroscopy examination and impression cytology and was performed monthly in both groups. After 6 months animals were sacrificed and the eyes subjected to histological and IHQ examination.
Results::
6 months post-treatment, histological examination showed complete corneal epithelium regeneration and a significant decrease of goblet cells in the group of animals treated with LSC on AM. Immunohistochemistry revealed a normal distribution of corneal epithelial markers CK3/12 and CK14. Clinical outcome also showed significant corneal epithelization and reduced inflammation and neovascularization on LSCs transplantation group. Corneal ulceration was also resolved on LSCs transplantation group.
Conclusions::
Our results suggest that ex vivo expansion of LSCs can be achieved by using a combined culture method on plastic and denuded human amniotic membrane. Such expanded LSCs can successfully reconstruct corneal surface and improve clinical and histological outcome of total LSCD. Comparing to a control group, treatment with transplantation of LSCs cultured on amniotic membrane improves clinical inflammation and corneal surface regeneration and decreases number of Goblet cells.
Keywords: cornea: epithelium • cornea: basic science • cornea: clinical science