Date : 18/02/2009

Laboratory
LMGP-MINATEC
CNRS UMR5628
3 parvis Louis Néel
38016 GRENOBLE
Director : Bernard CHENEVIER

PhD Supervisor
Catherine PICART (Professor at Grenoble INP and Junior Member of the "Institut Universitaire de France for 2007-2012)
email : Cet e-mail est protégé contre les robots collecteurs de mails, votre navigateur doit accepter le Javascript pour le voir
phone : +33 4 56 52 93 11

Subjects / Tools-Methodologies:

1 : thin film mechanical properties / AFM nano-indentations
2 : cell differentiation / specific protein markers
3 : growth factor / quantification of protein expression

Summary of lab's interests

- the first project deals with the preparation of biomimetic thin films as new nanomaterials with controlled properties. In particular, we focus on the nanoscale control of film internal structure and mechanical properties. Our work concerns the physico-chemical characterization of these films as well as the cell behaviour in contact with the films. These films are interesting for both fundamental biological studies, as they are a new kind of thin ECM matrices and for applications in the field of tissue engineering.

- the second project concerns the interactions of ERM proteins (Ezrin, Radixin, Moesin) with biomimetic lipid membranes containing phosphatidylinositol(4,5)bisphosphate (PIP2) on one hand and with actin on the other hand. These proteins have a key role in vivo as they constitute a direct link between the plasma membrane and the cell cytoskeleton. In this project, our aim is to decorticate and to better understand the interactions between these components in vitro in simplified systems.

Summary of project

CONTEXT OF THE PROJECT

In the field of biomaterials and biophysics, the role of the chemical properties of a matrix (presence of adhesive motifs) and of the topography (presence of nano or micro-roughness) is already widely known and studied. More recently, it has been evidenced that the mechanical properties of a matrix (that we will call here a "substrate") play an important role and influence cellular processes such as adhesion, spreading, but also a highly ordered process such as differentiation (1).

In our group, we have developed an expertise on thin multilayered films made of polysaccharides and polypeptides (including hyaluronan and chitosan). We have shown that their rigidity can be tuned depending on their cross-linking extent (2, 3), with only minor modifications in their surface chemical properties. In order to compare the respective effect of rigidity and chemistry, an adhesive ligand can be grafted on one of the polyelectrolytes (4). Very recently, we evidenced that myoblast differentiation in myotubes is highly dependent on the film stiffness (5). In parallel and in an independent manner, we also worked on the modulation of film bioactivity by insertion of a growth factor having osteogenic capacities. We evidenced that the myoblasts can "trans-differentiation" into osteoblasts upon contact with the bioactive films, depending on the amount of bone morphogenetic 2 (BMP-2) loaded in the film (6). The method for preparing these films was also patented (7). This result is particularly interesting on both fundamental and applied point of views.

The objective is now to investigate the interplay between film stiffness/ bioactivity and cell adhesion and differentiation. Toward this end, the films in the presence of adhesive ligands or loaded with the growth factor, will be further characterized by atomic force microscopy both in imaging and force spectroscopy mode to characterize their topography and to measure their mechanical properties in the presence of the growth factor/adhesive molecules. Pluripotent myoblast cells with differentiation capacity toward the myotube and osteoblast phenotypes will be cultured onto these films and their behaviour will be studied (immuno-staining of specificproteins, protein quantification). The growth factor chosen to render the film bioactive is BMP-2, as it is known to have effects on both adhesion (8, 9) and differentiation of myoblasts (10).

Our team has already several ongoing collaborations on different aspects of these thin multilayered films (in the framework of two ANR PNANO grants obtained in 2006 and 2007). A collaboration is starting on this project with Dr Albiges-Rizo from the IAB (Institut Albert Bonniot, Grenoble), who is expert in the dynamics of cell adhesive structures.

SCIENTIFIC ENVIRONMENT

Our team belongs to the LMGP laboratory, which is located in the Minatec building. There are many facilities around, both within the building (AFM, infrared spectrometer, fluorescence microplate reader, quartz crytal microbalance, SEM and TEM electron microscopes) and on the Polygone campus (including CEA biology department and IBS "Institut de Biologie Structurale").

BIBLIOGRAPHIC REFERENCES

1. Engler, A. J., Sen, S., Sweeney, H. L., and Discher, D. E. (2006) Matrix elasticity directs stem cell lineage specification. Cell 126:677-689.
2. Schneider, A., Francius, G., Obeid, R., Schwinté, P., Frisch, B., Schaaf, P., Voegel, J.-C., Senger, B., and Picart, C. (2006) Polyelectrolyte multilayer with tunable Young's modulus : influence on cell adhesion. Langmuir 7:2882-2889.
3. Richert, L., Boulmedais, F., Lavalle, P., Mutterer, J., Ferreux, E., Decher, G., Schaaf, P., Voegel, J.-C., and Picart, C. (2004) Improvement of stability and cell adhesion properties of polyelectrolyte multilayer films by chemical cross-linking. Biomacromolecules 5:284-294.
4. Picart, C., Elkaim, R., Richert, L., Audoin, F., Da Silva Cardoso, M., Schaaf, P., Voegel, J.-C., and Frisch, B. (2005) Primary cell adhesion on RGD functionalized and covalently cross-linked polyelectrolyte multilayer thin films. Adv. Funct. Mater. 15:83-94.
5. Ren, K., Crouzier, T., Roy, C., and Picart, C. (2008) Polyelectrolyte multilayer films of controlled stiffness modulate myoblast differentiation. Adv. Funct. Mater. 18:1378-1389.
6. Crouzier, T., Ren, K., Nicolas, C., Roy, C., and Picart, C. (2008) Layer-by-layer films as biomimetic reservoir for BMP-2 delivery : controlled differentiation of myoblasts to osteoblasts. Small early view, february 13th 2009.
7. Crouzier, T., and Picart, C. (2009) Process for preparing a surface coated by crosslinked polyelectrolyte multilayer films as a biomimetic reservoir for proteins. US provisional application:n° 61/145,158.
8. Nissinen, L., Pirila, L., and Heino, J. (1997) Bone morphogenetic protein-2 is a regulator of cell adhesion. Exp Cell Res 230:377-385.
9. Ozeki, N., Jethanandani, P., Nakamura, H., Ziober, B. L., and Kramer, R. H. (2007) Modulation of satellite cell adhesion and motility following BMP2-induced differentiation to osteoblast lineage. Biochemical Biophysical Research Communications 353:54-59.
10. Katagiri, T., Akiyama, S., Namiki, M., Komaki, M., Yamaguchi, A., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A., and Suda, T. (1997) Bone morphogenetic protein-2 inhibits terminal differentiation of myogenic cells by suppressing the transcriptional activity of MyoD and myogenin. Exp. Cell. Res 230:342-351.