Influence of the substrate-ligand interaction strength on cell adhesion. – FORCELL

In this project, we propose the design of new model substrates of variable stiffness decorated with adhesion peptide ligands. These ligands, peptidic sequences (Arginine-Glycine-Aspartic acid) will be fixed to the substrate by tunable bond strength(covalent or non-covalent bond). The goal of this project is to study the synergetic effect between the mechanical properties (variable Young modulus) and the non-covalent interaction strength of substrate-ligand bounds on the mechanism of cell adhesion and cell fate. The non-covalent interaction is based on host-guest interaction between beta-cyclodextrine and hydrophobic guest molecules such as ferrocene and adamantane. The stability of inclusion complex depends on the nature of the guest molecule and the valency of the interaction. A series of multivalent molecules that are supposed to interact with beta-cyclodextrine grafted on substrates of variable stiffness will be synthesized. Substrates of variable stiffness will be implemented, it will focused on the design of polymer matrix for which mechanical properties will be well-characterized.
The interaction strength between multivalent guest molecules and substrates bearing beta-cyclodextrine host molecule will be measured using force spectroscopy (AFM). We will study the cell adhesion process on the substrates of variable stiffness. We will try in particular, to understand if the cells seeded on stiff substrate decorated with peptide ligands fixed by non-covalent bond exhibit the same behavior than those deposited on soft substrate with adhesive ligands grafted by covalent bonds. This study will bring quantitative informations on the strength exerted by the cells to sense the stiffness of the adhesive substrate. Our project should lead to the development of a novel family of substrate well characterized for applications in tissue engineering.

During this first period, the development of adhesive substrates was conducted along two main axis:
- Designing of a rigid platform bearing beta-cylocextrine (self-assembled monolayer of alkanethiols)
- Designing of polymer hydrogels of tunable stiffness. Three different strategies have been investigated. The scope of the project included the use of agarose hydrogel but the presence of pores make it inappropriate for cell adhesion. Two different alternative strategies are now investigated: polymer multilayers of poly-L-lysine/ Hyaluronic acid and polyacrylamide.
In parallel of these works, a set of molecules has been synthesized, they have been designed based-on a cyclodecapeptide scaffold exhibiting two distinct addressable domains devoted for different function : guest molecules for the anchorage on the cylclodextrine-functionalmized platform and the other the peptidic ligand for cell targeting. The guest motifs valency can be tuned and influences the strength of the host-guest interactions. The characterization of host-guest interactions with rigid substrate bearing host molecule has been initiated. The first experiments performed by force spectroscopy measurements allowed the characterization of host-guest interactions on rigid beta-cyclodextrine functionalized-surface. These experiments have been successfully achieved by the chemical grafting of the multivalent guest molecules on the surface of the AFM.
The mechanical properties of polymer hydrogels have been performed. Their chemical functionalization with peptidic ligand or with beta-cyclodextrine is currently studying. Preliminary, cell assays are investigating by studying the adhesion of fibroblast 3T3 on such polymer films, they will allow the validation of the chemical functionalization with peptidic ligands.

Understand the ways by which cells collect the information from their substrate concerning its mechanical properties could open the door to the design of new hybrid materials. Getting a clear picture on cell behaviour on these original model substrates should allow making use of them in a controlled way for tissue culture.

A manuscript is currently in preparation for the valorisation of the first outcomes in synthesis and in dynamic force spectroscopy. These works could be published Journal of Material chemistry.

Tissue engineering emerges as a new field of engineering science that relies both on biology and materials science. “Living materials” are designed by seeding cells in synthetic matrices or depositing them on synthetic substrates and growing them to regenerate tissues or organs. The correct design of such materials requires a precise knowledge of the response and fate of the cells consecutive to their interaction with the surrounding material. Not only chemical but also mechanical properties of the adhering or surrounding material appear to play a central role in this respect. Until now, studies have concentrated on materials interacting with cells through non-specific interactions or through ligand-receptor interactions where the ligands were covalently fixed to the matrix. In this project we propose to design new substrates encompassing cell adhesion ligands grafted to the adhesion substrate through multivalent non-covalent host-guest interactions. By varying the number of host-guest interactions (multivalency) and their chemical nature we will modulate the interaction strength of these ligands with the adhesion substrate. The stiffness of the underlying substrate will also be tuned. We will use these model systems to investigate how the bond strength of a non-covalent host-guest interaction depends on the stiffness of the substrate, a central issue in understanding cell fate of adhering cells. This model system will also allow comparing cell adhesion and mesenchymal stem cell fate on systems where the ligands are attached covalently on the substrate to those where they are non-covalently anchored. Do cells feel stiff substrates with weakly bound ligands similarly to soft substrates with covalently coupled ligands? Answering this question should allow us to get new information on how cells sense the mechanical properties of a substrate, another central issue in predicting cell adhesion properties. Furthermore, these systems provide a new family of tunable and well controlled substrates for tissue culture and tissue engineering.