DIFFERENTIAL ASSOCIATION OF B-CATENIN, ACTIN AND VINCULIN DURING OSTEOBLAST ADHESION ON GROOVED TA6V SUBSTRATES K. Anselme 1 , M. Bigerelle 2 , B. Noël 1 , A. Iost 2 , and P. Hardouin 1 1 I.R.2.B., Université du Littoral Côte d’Opale, Berck/ mer, France , 2 ENSAM, ,Lille, France INTRODUCTION: Many proteins are involved in osteoblast adhesion on biomaterials; notably the cell adhesion molecules which can be implicated in cell-substrate adhesion (focal contacts) or in cell-cell adhesion (adherens junctions) 1 . Cytoskeletal proteins such as actin are linked at the same time to the focal contacts and the adherens junctions. These interactions play a predominant role in signal transduction, which will regulate cell morphology, migration, gene expression, proliferation and differentiation 2 . The originality of our work is in approaching the respective expressions of cell-cell adhesion and cell-material adhesion proteins of human osteoblastic cells cultured on grooved titanium- based surfaces by using a double immunolabelling technique. METHODS: Titanium alloy Ti6Al4V bars were tool machined in our laboratory to obtain organized and regular grooved surfaces with various roughness amplitudes (Ra) respectively 0.8, 1.21 and 3.35 µm. Primary human osteoblasts from trabecular bone were inoculated onto the substrates at 4 x 10 4 cells/well. Three incubation periods were chosen: 4, 24 and 48 hours. Cells were fixed in 2% paraformaldehyde, permeabilised with 0.2% (v/v) Triton-X100 and rabbit polyclonal anti-β-catenin antibody was used to visualize the cell-cell interactions and mouse monoclonal anti- human vinculin antibody was used to visualize the cell-material interactions. To study the interactions of these proteins with the cytoskeleton, F-Actin microfilaments were directly revealed by FITC- conjugated phalloidin. For double staining, the primary step was with mouse monoclonal anti- human vinculin in combination with rabbit polyclonal anti-β-catenin or with monoclonal anti- human vinculin alone. This was followed respectively by FITC-anti-mouse IgG antibody with TRITC-anti-rabbit IgG antibody or by TRITC-anti-mouse IgG antibody in combination with FITC-conjugated phalloidin. RESULTS: At 4 hours, F-actin labelling immediately showed stress fibres and globular aspects in lamellipodia. These aspects were associated with β-catenin–labelled globular aspects. On the other hand, the vinculin expression did not show any variation from 4h to 48h. No specific differences in protein expression were found between cells cultured on the 3 surfaces. By double staining, we demonstrated that vinculin- labelled focal contacts were situated mainly at the periphery of cells at the termination of F-actin bundles. Cell-cell contacts shown through use of the anti-β-catenin antibody appeared as thin filaments disposed at the cell periphery in a parallel arrangement linking one cell to another. By double staining, in some cases, we demonstrated the co-localization of vinculin- labelled cell-material focal contacts with β- catenin-labelled cell-cell contacts, although in other cases the opposite was observed i.e. the cell- cell contacts alternated with cell material contacts. DISCUSSION & CONCLUSIONS: β-catenin- positive adherens junctions were expressed by human osteoblasts as thin filaments joining cell membranes. Sometimes, they appeared co- localized with vinculin-positive focal contacts and sometimes not. The linkage of catenin/cadherin complex and vinculin-positive focal contacts with actin filaments may explain this apparent co- localization. In this study, we demonstrated for the first time the expression of cell-cell adhesion molecules by osteoblastic cells cultured on a titanium-based substrate. REFERENCES: 1 Anselme K. Osteoblast adhesion on biomaterials. Biomaterials.2000;21:667-681. 2. Yamada KM, Geiger B. Molecular interactions in cell adhesion complexes. Curr Opin Cell Biol.1997;9:76-85. View publication stats View publication stats European Cells and Materials Vol. 1. Suppl. 2, 2001 (page 65 ISSN 1473-2262