Green fibronectin substitute and functionalization of materials – Green Fib

The human (pFN) and plant-derived fibronectin (gFN) are purified by affinity chromatography. The structure of the gFN is determined and compared to the one of the pFN. In particular, the sugar residues, frequent in the plant-derived proteins, are identified and used to develop original grafting methods. The efficiency of gFN to promote cell adhesion but also bacteria adhesion will be evaluated in an original cp-culture model.

The gFN have been purified and the first tests have confirmed its adhesive potential on cells. In spite of needed future optimizations to increase the purification yield, the characterization of gFN has been started in order to know its composition in amino acids and sugar residues and to further select the most interesting fragments. This information will be important to develop original methods to graft it definitively on surface of materials used in medical field. As gFN contains a lot of sugar residues susceptible to favor the adhesion of bacteria, it will be important to test its capacity to favor the adhesion of human cells but also the adhesion of pathogenic bacteria. A co-culture model associating cells and bacteria has been now established. It will allow to test the risk to favor with gFN the adhesion of bacteria to the detriment of cells. The obtained result will be essential for future use of gFN in medical applications. It will also permit to modify the protocol of grafting of gFN on materials in order to limit the adhesion of bacteria for the benefit of cells.

This project may allow to propose a substitute to plasma fibronectin (pFN) derived from plants (gFN) to functionalize biomaterials or cell culture substrates. This gFN would display several advantages compared to the pFN such as a decrease of the risk of disease transmission but above all a significant decrease of the production cost. If the gFN was demonstrated to be equivalent to the pFN, the purification process and adapted grafting techniques could be the object of a transfer to industry.

No production for the moment.

As cells recognize and adhere via integrin receptors to biomacromolecules of extracellular matrix (ECM), recent bio-inspired biomaterial strategies have focused on presenting integrin ligands such as ECM proteins or short bioadhesive motifs derived from these components on implant surfaces. Fibronectin (FN) is a large glycoprotein present in ECM that interacts with cells to control cell adhesion, cytoskeletal organization and cellular signalling. The fibronectin used in biomaterial field is generally purified from human plasma and has been largely used in vitro for increasing the interactions of cells with the material surfaces but also in vivo for improving integration in tissues. FN is also frequently used for functionalization of cell culture dishes or biochips.

Two immobilization strategies exist for proteins on surfaces: protein adsorption (which can hardly give time-resistant chemical modification after immersion in biological fluids in culture or in vivo) and protein grafting (which is more likely to provide a protein layer stable in time). Until now, this last strategy have been applied mainly on RGD-peptides or FN-fragments but not on the whole Fn molecule because of the cost of the purification process of FN from human plasma (pFN) and more surely because of the risk of contaminations by virus or prions after implantation.

We have previously demonstrated that it is possible to find a FN-like protein (gFN for green FN) in plants. Moreover, gFN adhesion potential is comparable to the one of plasma FN for bacteria and eukaryotic cells adhesion. Consequently gFN has a strong potential to replace human plasma FN (pFN) for implant, cell culture dishes or biochips functionalization functionalisation.
As gFN possesses a high percentage of glycosylation (70%), we propose to take advantage of it to develop a new grafting method based on a reductive amination of sugar residues that should permit to maintain the active conformation of the molecule and to leave active the peptidic domains involved in cell/FN interactions. The gFN and pFN molecules will be compared to human vitronectin (pVN) that can be also purified from serum and that has an intermediate glycosylation ratio (30%).

Beside, this high glycosylation ratio of gFN may have a stimulative effect on bacteria adhesion since it is well known that bacteria have a high affinity for sugar residues by the intermediate of lectins. On the other hand, this high glycosylation rate should also influence cell adhesion. Thus it appears essential to evaluate the potential of gFN on eukaryotic cells on one hand, on bacteria on the other hand but also on eukaryotic cells and bacteria together. Indeed, bacteria are frequently introduced onto an implant surface during surgery and the race for the surface start before tissue integration can occur. On the other hand, a bacterial infection in the patient body releases free-living bacteria that are able to further infect an implant surface already colonized by eukaryotic cells. Therefore both competition situations should be affected in a complex manner by the chemical modifications resulting from the surface functionalization of biomedical devices. So far, no study of a new surface functionalization aiming to improve tissue integration has addressed biofilm formation and tissue integration simultaneously.

Finally, we propose to evaluate the potential of the gFN for functionalisation of materials for bone implants (titanium-based implants), of polystyrene dishes for cell culture and of silica wafer for biochips. The interactions of gFN with human healthy bone cells and human cancerous ovarian cells but also with non pathogenic (E. coli, S. epidermidis) and pathogenic bacteria often involved in bone implant infections (P. aeruginosa and P.fluorescens, S. aureus) will be studied separately and in a co-culture model.