Generation of a Blister Model by Disruption of the Dermal-Epidermal Junction for Diagnosis of Recessive Dystrophic Epidermolysis Bullosa

Abstract

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a severe skin disorder produced by a mutation in the COL7A1 gene (c.6527inC) and leads to dysfunctional or absent collagen type VII in the dermo-epidermal junction. Consequently, blisters appear throughout the body, including mucous membranes. Currently, diagnosis is routinely performed by rubbing the skin to induce a blister and taking a biopsy for histological analysis. Mutational analysis is possible but it is expensive and difficult. In this work, a suction device has been developed to substitute the current RDEB diagnostic method by a more precise, reproducible and reliable one. An easy to use device represents a less invasive method of diagnosis that betters the life of patients that may have severe conditions such as RDEB. The principal characteristics of the device are its small size and manageability. It is equipped with a vacuum pump for suctioning, connected to a pressure sensor, an OLED screen to provide real-time reading of the sensor, and an incandescent light bulb to provide heat. The device can be turned on and off by a simple pushbutton, and the suction modulated. It is operated by an Arduino-compatible microcontroller. To obtain the combination of pressure and time needed for blister generation, RDEB human skin mice models were developed using human plasma scaffolds as dermal component. Applying a negative pressure of 80-85 kPa for 5 minutes produced a well-defined blister at the DEJ, without damaging the dermis. Results were confirmed by Hematoxylin and Eosin staining and immunohistochemical studies of collagen VII, vimentin and cytokeratins 10 and 14. Temperature influenced blister generation. Higher negative pressures proved to cause dermal disruption, with DEJ and intradermal blistering, as well as tissue deformation. Epidermis remained intact in all cases. To complement this work and explore one of the possible applications of the device, the result was tested on a skin model corrected for the COL7A1 gene mutation that was provided by another research group. Collagen VII was observed, and tissue architecture was conserved after suction without dermal disruption or blisters. In conclusion, determining blistering times for EB represents a great advance in the diagnosis of the disorder. Helping such patients determine their condition may greatly influence their cures. In addition, the data obtained may help in develop specific treatments for the different types and subtypes of RB, including RDEB.