Long-term remodeling of a bilayered living human skin equivalent (Apligraf^®) grafted onto nude mice: immunolocalization of human cells and characterization of extracellular matrix

Type I collagen is a clinically approved biomaterial largely used in tissue engineering. It acts as a regenerative template in which the implanted collagen is progressively degraded and replaced by new cell-synthesized tissue. Apligraf, a bioengineered living skin, is composed of a bovine collagen lattice containing living human fibroblasts overlaid with a fully differentiated epithelium made of human keratinocytes. To investigate its progressive remodeling, athymic mice were grafted and the cellular and the extracellular matrix components were studied from 0 to 365 days after grafting. Biopsies were analyzed using immunohistochemistry with species-specific antibodies and electron microscopy techniques. We observed that this bioengineered tissue provided living and bioactive cells to the wound site up to 1 year after grafting. The graft was rapidly incorporated within the host tissue and the bovine collagen present in the graft was progressively replaced by human and mouse collagens. A normal healing process was observed, i.e., type III collagen appeared transiently with type I collagen, the major collagen isoform present at later stages. New molecules, such as elastin, were produced by the living human cells contained within the graft. This animal model combined with species-specific immunohistochemistry tools is thus very useful for studying long-term tissue remodeling of bioengineered living tissues.