685 Study of functional fibrillary collagen network from the cell level to the matrix maturation

Abstract

Dermal collagen fibers are the major structural proteins responsible for a strengthened and functional extracellular matrix. However, the organization and the functionality of the matrix is not restricted to the collagens but involve different key players such as the bone morphogenetic protein-1 (BMP-1). BMP-1 is a specific endo-protease, which is responsible for the C-terminal cleavage of the pro-peptide parts of collagen I and V, a limiting step of fibril formation. With ageing, collagen synthesis decreases, due first to a loss of cell ability to perform synthesis. The consequence is a deterioration of the matrix functionality and 3D organization. As cell stiffening directly results in altered viscoelastic properties of the collagen matrix, we developed methods to study the quality of fibroblast actin cytoskeleton. Therefore different steps of collagen maturation were also studied in order to further find a recovery solution. First, with a specific method of actin immunofluorescence on single micro-patterned fibroblasts, we showed that actin intensity and organization can be modified and normally recovered in order to get active fibroblasts able to synthesize a right amount of matrix proteins. Second, with a new “in House” Delfia method (matrix of normal human fibroblasts based on a time-resolved fluorometric method), we quantified the levels of deposited collagen I and V and of the endo-protease BMP-1 driving functional collagen fibers. Third, once we have shown that collagen I and V were synthesized in a suitable and inducible amount, the second harmonic generation microscopic technique allowed us to show that the collagen fibers were well organized within our reconstructed dermis model (MimedermTM) compared to normal human biopsies. In conclusion, with different in vitro methods, for the first time, we studied collagen maturation from the cell to the matrix level with the different partners of collagen fibers assembly.