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

Abstract

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 a-smooth muscle actin (a-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. a-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.

Update on amniotic membrane transplantation

“…The resultant cultivated cells and AM were transplanted in normal rabbits [61,66], as well as in limbal stem cell deficiency (LSCD) rabbits [61,67] and rat models for short-term study [68]. It has also been used in limbal deficient rabbits for long-term study [69]. Currently, this composite tissue is being transplanted to restore vision as well as the structure and the function of damaged ocular surfaces in humans [45,62,70–72].…”

Signaling-Transduction Pathways Required for Ex Vivo Expansion of Human Limbal Explants on Intact Amniotic Membrane

“…6 Furthermore, restoration of a clear and transparent cornea is associated with a normal corneal epithelium and complete wound remodeling. 7 The success of this new surgical procedure can further be attributed to the fact that ex vivo expanded limbal epithelial cells on intact amniotic membrane (without 3T3 fibroblast feeder layers or air lifting) preserve characteristics of limbal epithelial stem cells such as negative expression of K3 keratin and connexin 43 (Cx 43), label-retaining properties, and p63 expression. 4,8 -10 As a first step to exploring the molecular mechanism governed by AM, we have noted that nerve growth factor (NGF) is abundantly present in AM stromal matrix and that TrkA (the NGF high-affinity receptor) but not p75 NTR (the NGF low-affinity receptor), is expressed at the limbal basal epithelial cell layer, suggesting that the limbal epithelial stem cells may preferentially use TrkA but not p75 NTR to sustain its unique antiapoptotic survival.…”

Long-term follow-up of a supradescemetic keratoprosthesis in rabbits: an immunofluorescence study

“…Previous studies demonstrated deposition of collagen IV in rabbit corneal stroma injured by alkali burns or lacerations, and its association with fibroblast activation and scar tissue [20,21]. Similarly, α-SMA expression is the hallmark for myofibroblast differentiation, the cells responsible for stromal haze and scarring [12,22].…”