In vitro study of the ideal conditions of enzymatic digestion of the corneas subjected to crosslinking

• Grant number: 16/18648-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): December 01, 2016
• Effective date (End): November 30, 2017
• Field of knowledge: Health Sciences - Medicine - Surgery
• Principal Investigator: Patrícia Alessandra Bersanetti
• Grantee: Luiz Guilherme Ito da Cruz
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

Keratoconus is a non-inflammatory eye disease that affects the shape and thickness of the cornea, resulting in loss of visual acuity and hence quality of life. In this disease, the cornea acquires a cone shape due to the loosening of collagen fibrils. Some treatments are useful in the correction of refractive errors, but without effect on disease progression. The crosslinking procedure using riboflavin, stimulated by ultraviolet radiation at 365 nm, is effective in blocking the progression of keratoconus, causing an increase of the biomechanical strength and corneal resistance and decreasing the progression rate of the disease. Recently, some studies show the possibility of application of natural products such as genipin and proanthocyanidins (class of polyphenols) in the formation of cross-links in the collagen molecule. In the development of this project, after the formation of cross-links with polyphenols of plant extracts, the corneas will be analyzed against its resistance to enzymatic digestion with collagenase. The conditions of enzymatic digestion tests, such as reaction temperature and enzyme concentration, will be optimized by experimental design (DOE). After this, enzymatic digestion assays will be performed in different groups of corneas treated with crosslinking agents, which will be analyzed to determine the mass and area variations, denaturation enthalpy by DSC and hydroxyproline content in the supernatant. The study of in vitro methodologies for determining the success of corneal crosslinking has great importance, since it enables an efficiency analysis of chemical or physical agents for the treatment of keratoconus and other ectasias. (AU)