Chromatin supraorganization, DNA content, nuclear glycoprotein amount, immunophenotypic characterization of corneal limbal epithelial cells cultivated on intact or denuded amniotic membrane versus scaffold composed of fucoidan-alginate-chitosan

Abstract

At the end 1990, the transplantation of substrates colonized by corneal limbal epithelial stem cells emerged as a promising therapeutic option for reconstructing the ocular surface and restoring the vision in patients with limbal deficiency. After 15 years of clinical practice, several aspects related to the security and the standardization of the procedures used for manipulating these substrates sill remain unclear. For example, it is not totally clear if the ideal substrate to cultivate limbal stem cells would be obtained from intact or denuded amniotic membrane. Also, it is not known whether scaffolds constructed with polysaccharides, such as fucoidan, alginate and chitosan, used for the manufacture of bioactive dressings that stimulate the epidermal epithelial cell migration and accelerate the skin wound healing process, could be used for cultivating limbal stem cells and repairing corneal lesions. Many authors have tried to evaluate the effects of intact and denuded amniotic membrane on the expansion of limbal stem cells. Additionally, in view of the increasing use of limbal stem cells, many groups attempted to create new substrates for cell culture. Inadvertently, however, all the research used repetitive or identical analysis methods for evaluating these multidisciplinary biomedical questions. Previous studies have evaluated only the differential expression of molecules correlated with the differentiation course of limbal stem cells. No study was designed for evaluating the supraorganization and the expression of nuclear macromolecules that control the biological activities of corneal limbal epithelial cells expanded ex vivo. In this way, the purpose of this research is to determine the functional status of the chromatin, the DNA and the nuclear glycoproteins of corneal limbal epithelial cell cultivated on intact amniotic membrane, denuded amniotic membrane, and scaffolds composed of fucoidan-alginate-chitosan.