395 Collagen VII is required in both fibroblasts and keratinocytes for anchoring fibril formation in bilayer skin substitutes

Abstract

The blistering disease Recessive Dystrophic Epidermolysis Bullosa (RDEB) is caused by mutations in collagen VII (COL7), which forms anchoring fibrils that attach the epidermis to the dermis. Cutaneous gene therapy to restore COL7 expression in RDEB patient cells has been proposed, and cultured epithelial autograft containing COL7-modified keratinocytes was tested in clinical trials. Because COL7 in normal skin is expressed in both fibroblasts and keratinocytes, cutaneous gene therapy using a bilayer skin substitute may enable faster anchoring fibril restoration. Hypothetically, COL7 expression in both dermis and epidermis of a bilayer skin substitute will be required to enable functional anchoring fibril formation. To test this, engineered skin substitutes (ESS) were prepared using four combinations of normal + RDEB cells: (1) RDEB fibroblasts + keratinocytes; (2) RDEB fibroblasts + normal keratinocytes; (3) normal fibroblasts + RDEB keratinocytes; and (4) normal fibroblasts + keratinocytes. ESS were incubated in vitro at the air-liquid interface for 10 days prior to grafting to full-thickness wounds in immunodeficient mice. Biopsies were analyzed in vitro and at 1, 2, and 3 wks after grafting. COL7 expression was not detected by immunohistochemistry in ESS prepared with all RDEB cells (group 1), and gross blistering was observed by 2 wks after grafting. COL7 was expressed, in vitro and in vivo, in ESS prepared using combinations of normal + RDEB cells (groups 2 & 3) or all normal cells (group 4). However, TEM revealed structurally normal anchoring fibrils, in vitro and in vivo, only in ESS prepared using all normal cells (group 4). The results suggest that although bilayer skin substitutes enable formation of anchoring fibrils in vitro, prior to grafting, this requires expression of COL7 in both fibroblasts and keratinocytes.