Event Abstract Back to Event How cells internalize a growth factor presented by a biomaterial: BMP-2 delivery to cells in a matrix-bound manner using layer-by-layer films Catherine Picart1, 2, Flora Gilde1, 2, Laure Fourel3, Raphael Guillot1, 2, Isabelle Paintrand1, 2, Takaharu Okada1, 2, 4, Thomas Boudou1, 2, Corinne Albigez-Rizo3 and Catherine Picart1, 2 1 Grenoble Institute of Technology, Department of Bioengineering, France 2 UMR5628, CNRS, France 3 Institute Albert Bonniot, France 4 National Institute for Materials Science, Japan Introduction: Bone Morphogenetic Proteins (BMPs) play an essential role in bone formation and regeneration. In the field of orthopedics, delivery of BMP-2 from the surface of implantable materials would enable faster and better bone formation around the implant. A major effort has been recently undertaken to develop biomaterials for local and precise delivery of the osteoinductive bone morphogenetic proteins (BMPs)[5] . As endocytosis and signaling are tightly coupled, a better understanding of how BMP molecules interact with the biomaterial and are effectively processed by the cells is thus crucial. We previously showed that polyelectrolyte films with matrix-bound BMP-2 are bioactive in vivo when deposited on ceramic granules[3] and on titnaium implants[4]. In this study, we investigated the effect of the delivery mode (matrix-bound versus soluble) and the endocytic processes involved in BMP2-mediated Smad and non-Smad signaling in C2C12 cells, which is the early step of bone differentiation. Materials and Methods: We used a biomimetic polyelectrolyte film made by self-assembly of poly(L-Lysine) and hyaluronan, which acts as nanoreservoir to trap BMP-2 and to present it to cells in a "matrix-bound" manner[1],[2]. This polyelectrolyte film was crosslinked to a controlled extent. We used fluorescently labeled BMP-2 to track the possible diffusion of BMP-2 in the biomaterials and its internalization by the cells. We quantified the amount of BMP-2 internalized by the cells. We then used chemical inhibitors to block the major internalization routes (including clathrin,dynamin and caveolin) and knock-down the these pathways to unravel their specific effect on BMP-2 signaling. Results and Discussion: We showed that BMP-2 partially diffused within the film and that it is internalized by C2C12 cells. We then quantified the effect of film cross-linking on the amount of BMP-2 that is internalized by the cells. The use of inhibitors of endocytosis, siRNA-mediated knock-down and immuno-staining of endocytic proteins allowed to investigate the internalization pathways adopted by the cells in comparison to the delivery of BMP-2 in solution. Finally, we investigated the role of endocytosis in the activation of the BMP signaling pathways. Conclusions: We demonstrated that soluble as well as matrix-bound BMP-2 were internalized through both clathrin- and caveolin-dependent pathways. Furthermore, we established that both the initial and final steps of endocytosis process were determinant for BMP-2-induced signaling. These results highlight that a bioactive growth factor delivered via a biomaterial enables the signaling pathways to be transduced in a very efficient manner. European Research council (European Comission, FP7, GA259370, BIOMIM)