Corneal stromal changes following reconstruction by ex vivo expanded limbal epithelial cells in rabbits with total limbal stem cell deficiency

Aim: To study corneal stromal changes and the presence of myofibroblasts after transplantation of ex vivo expanded limbal epithelium. Methods: A state of limbal deficiency was induced in 16 rabbits. After transplantation with autologous ex vivo expanded limbal epithelium on amniotic membrane ( AM), their clinical outcomes were classified as success, partial success or failure according to surface smoothness, stromal clarity, and vascularisation. Clinical outcomes were correlated with phenotypic outcomes of corneal, conjunctival, or mixed epithelium, defined by expression of K3 keratin or MUC5AC. Immunostaining was performed with antibodies against collagen IV, fibronectin, and alpha- smooth muscle actin (alpha-SMA) to assess stromal wound remodelling. Results: Rabbits were sacrificed after a mean follow up of 10 ( SD 3.3) months. Collagen IV, expressed in the basement membrane of all three groups, was found in the stroma of the partial success, but not in that of the success or the failure. Fibronectin was absent in the success and the failure, but expressed in the stroma of the partial success. alpha-SMA was expressed in superficial stroma of the partial success, but suppressed in areas with AM remnants. Conclusion: Restoration of a clear and transparent cornea is associated with a normal corneal epithelium and complete wound remodelling. In contrast, wound healing remains active and incomplete in conjunctivalised corneas, which remain opaque with myofibroblasts.