RPE Cell Adhesion, Polarization and Directional Migration on Nanostructured Collagen Matrices

Abstract

Purpose: : To determine whether nanostructured collagen matrices can be employed to direct the adhesive and migratory behavior of RPE (retinal pigment epithelial) cells.

Methods: : Ultraflat, self–organizing collagen type I matrices with a defined nanostructure were prepared by addition of collagen I (final concentration 3%) to a KCl–glycine buffer onto a mica surface. In some experiments the collagen matrices were crosslinked by riboflavin/UVA treatment in order to increase their biomechanical rigidity. Primary human RPE, SV–40 transfected human RPE and ARPE–19 cells were seeded on the crosslinked nanostructured collagen I matrices. Cell morphology, adhesion and orientation along the collagen fibrils were studied by video microscopy for up to 20h. The expression of the collagen I–binding integrin α2ß1 was assessed quantitatively by Western blot analysis in all three cell lines.

Results: : All three cell lines adhered to the patterned collagen matrices, although primary and SV–40 transfected RPE cells showed faster attachment than ARPE–19 cells, which retained a rounded shape during the entire observation period. In contrast, primary and SV–40 transfected RPE cells aligned in the direction of the collagen fibrils and developed a stretched cell morphology. While attaching to and aligning along the fibrils, these cells exerted strong tractional forces on the collagen matrix which resulted in the detachment of the cell–matrix–complex from the supporting surface. Crosslinking of the collagen matrix delayed cell detachment by approx. 3h (primary and SV–40 RPE cells) and 12–20h (ARPE–19 cells). Expression of integrin α2ß1 was higher in SV–40 transfected RPE cells than primary RPE cells, whereas ARPE–19 cells showed almost no expression of integrin α2ß1.

Conclusions: : RPE cells align in the direction of the underlying parallel fibrils of the patterned collagen matrix. The collagen–binding α2ß1 integrin promotes efficient cell attachment and polarization on the matrix. The tractional forces exerted by the cells are required for the polarization process but can lead to matrix disruption and cell–matrix–detachment. Photochemical crosslinking of collagen improves the stability of the matrix but does not prevent eventual matrix disruption and detachment from the substratum due to the high tractional forces exerted by RPE cells.

Keywords: retinal pigment epithelium • cell membrane/membrane specializations • retina