Reconstruction of the ocular surface: basic, clinical, and surgical aspects

Abstract

Disorders of the ocular surface and the tear film are considered a heterogenic group of diseases, which result in loss of vision in many patients. In this group, dry-eye syndrome, pterygia, degenerations and dystrophies of the corneal surface, as well as dysplasias are included. The most severe cases of disorders of the ocular surface and the tear film are due to chemical burns, Stevens-Johnson syndrome and ocular cicatricial pemphigoid, frequently inducing seqüelae, which cause difficulties in clinical and surgical management. Due to the high number of cases and the problematic treatment, these diseases constitute a severe social and economic problem, especially in non-developed countries. The thematic project "Reconstruction of the ocular surface: Basic, clinical, and surgical aspects” is a multi-disciplinary project of the Federal University of São Paulo (UNIFESP), with the participation of another two national and one international University. This project includes the development of 16 research projects [5 basic research (PE), 11 clinical research (PCC)] with the following objectives: -The investigation of ultra-structure and biochemical, as well as molecular aspects of the human amniotic membrane in ocular surface reconstruction. -The development of technique for amniotic membrane colonization with human corneal epithelium ex vivo. -The analysis of ocular surface disorders with different diagnostic tools. -The analysis of new treatments for the dry-eye syndrome. -The evaluation of the efficacy of surgical reconstruction of the ocular surface with amniotic membrane alone or in association with limbal stem cells transplantation. In summary, the methods used in each protocol: -PE-1: Transmission + scanning electron microscopy of the human amniotic membrane prior to and after preservation with two different methods (DMSO or glycerol and culture medium 1: 1 ). -PE-2: Quantification of growth factors and cytokines [recombinant epithelial growth factor (rEGF), basic fibroblast growth factor (bFGF), fibroblast growth facto r 4 (FGF-4), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), transforming growth factor bela (TGF-b), interleukins 4 and 10 (IL-4, -10), prostaglandin E2 (PGE2)] in fresh and preserved human amniotic membrane with ELIISA. -PE-3: Identification and quantification of mRNA and proteins of growth factors and cytokines of human amniotic membrane using PCR, Northern 810t, and Western 810t. -PE-4: The use of human amniotic membrane in experimental cicatricial keratoconjunctivitis in rabbits. Randomized study of corneal wound healing after transplantation of preserved human amniotic membrane in corneas with and without limbal deficiency: clinical, biochemical, and immunohistochemical evaluation. -PE-5: Amniotic membrane colonized with human corneal epithelial cells ex vivo: analysis of the ultra-structure, proliferation (immunofluorescence, using Ki-67), apoptosis (morphologic method, using Hoechst 33342), and determination of the expression of epithelial cytokeratins (immunohistochemical, using FACS). -PCC-1: Clinical and cytological investigation of the corneal surface after its reconstruction with transplantation of human limbal epithelial stem cells and amniotic membrane in patients with limbal deficiency secondary to Stevens-Johnson syndrome and ocular cicatricial pemphigoid. -PCC-2: Prospective, randomized, comparative study of ocular surface reconstruction with transplantation of human limbal epithelial stem cells with or without association of human amniotic membrane in cases of totallimbal deficiency due to chemical burn. -PCC-3: Clinical and cytological evaluation of the use of human amniotic membrane colonized ex vivo with human epithelial stem cells for reconstruction of the ocular surface in patients with total limbal stem cell deficiency resistant to previous conservative therapy. -PCC-4: Prospective, randomized study of ocular surface reconstruction in recurrent pterygia using human amniotic membrane graft compared to conjunctival graft. -PCC-5: Clinical investigation of the use of human amniotic membrane in the treatment of corneal and sclera thinning. -PCC-6: Prospective, randomized study of the use of human amniotic membrane compared to micropuncture in the treatment of bullous keratopathy in symptomatic patients. -PCC-7: Prospective, randomized study of clinical treatment in patients with acute chemical or thermal burn (grade III and IV) compared to clinical treatment in association with the use of human amniotic membrane as a biological contact lens. -PCC-8: Investigation of alterations in human corneas at cellular level prior to and after reconstruction of the ocular surface in patients with chemical burn without severe dry-eye using in vivo confocal microscopy. Additionally, patients with total limbal deficiency with severe cicatricial disorders of the cornea and conjunctiva (burn, pemphigoid, Stevens-Johnson syndrome) and patients without ocular disorders (controls) will be included in this study. -PCC-9: Prospective case-control study investigating the efficacy of Schirmer tests I, 11, and fluorescein clearance test of the tear film (CFL) as a differential diagnostic tool in patients with chronic, non-infectious keratoconjunctivitis. -PCC-10: Standardization of impression cytology examination using staining with PAS and Papanicolau for the evaluation of the limbal and conjunctiva removal in donors for limbal stem cell transplantation. -PCC-11: Clinical and cytological investigation of the use of topical Cyclosporin A 0.2% in the treatment of keratoconjunctivitis sicca. (AU)