Biomimetic peptide tools to crosslink collagen biomaterials and to target a specific collagen-binding integrin for cartilage tissue engineering – CARTEGRIN

The first step involves optimizing the method of collagen photocrosslinking by UV exposure using photoreactive compounds. The collagen is modified with an NHS-Diazirine group attached to lysines, then exposed to 365 nm UV light. The concentration of diazirine, the solvent, the duration of UV exposure, and the distance from the light source are optimized to achieve Young's moduli comparable to that of healthy cartilage (2-20 MPa). This approach covalently bonds the diazirine groups to the collagen backbone without altering the cell recognition sequence, thus leaving the biological activity of collagen intact. The surface topography and rigidity of the films will be analyzed by AFM and mechanical tests. Cell adhesion will also be evaluated to verify that photocrosslinking does not affect the affinity of cells for collagen. EDC/NHS films will be used as controls. Next, porous 3D collagen scaffolds, obtained by freeze-drying, will be cross-linked by adapting this method. The pore size and interconnection will be studied to ensure cell diffusion. We will ensure that this cross-linking prevents scaffold contraction due to cellular forces.

THPs containing a GXX'GEX'' motif that binds to integrins α1β1, α2β1, α10β1, or α11β1 are coated on plates to study the response of mesenchymal stem cells (MSCs) to integrin activation. The effects on MSC attachment, proliferation, morphology, and viability will be evaluated, as well as the gene expression of chondrocyte markers (SOX9, COL2A1, etc.). Discoidin domain receptor binding and activation will also be analyzed. At the same time, mature chondrocytes will be cultured on these THP surfaces to measure extracellular matrix production. A key objective is to target integrin α10β1 to effectively isolate chondrogenic MSCs from adipose tissue, using a specific inhibitor to block other integrins. This process aims to improve chondrocyte differentiation and prevent frequent dedifferentiation in 2D culture. We will also examine the effect of discoidin domain receptors on cellular response on extracellular matrix production by differentiated cells.

This project has led to the development of a new method for cross-linking collagen sponges for tissue engineering. This method is based on the formation of covalent bonds between collagen chains using a photoreactive diazirine group. The diazirine groups are grafted onto the side chains of collagen lysines and then exposed to low-energy UV light, which does not cause cell death. This cross-linking method significantly increases the rigidity and stability of collagen biomaterials without affecting their biological properties. It is also compatible with the seeding of collagen sponges with mesenchymal stem cells, which can then differentiate into chondrocytes for the repair of articular cartilage.

We have highlighted the importance of discoidin domain receptors in chondrocyte differentiation. We are now exploring the role of these receptors in cartilage pathologies such as osteoarthritis. Our goal is to respond to calls for projects to discover new THPs that can bind these receptors with high affinity and specificity, and thus propose candidates for the treatment of osteoarthritis.

The development of a method for photocrosslinking collagen using diazirine groups offers new possibilities for using collagen as a building block for stable and robust biomaterials for tissue engineering. This methodology is not limited to cartilage repair.

The aim of this project is to develop collagen biomaterials for cartilage tissue engineering. To this day, biomaterials for regenerative medicine do not gather the biological and mechanical properties required for cartilage repair. In particular, cell-biomaterial interactions are often inadequate, leading to poor cellular adhesion and a loss in the survival and function of seeded cells. We propose to improve these interactions by functionalizing biomaterials with triple-helical peptides (THPs). THPs are biomimetic peptides that adopt the characteristic triple-helix conformation of collagen, which is essential for both its structural and biological role. In this project, collagen biomaterials will be designed to host mesenchymal stem cells (MSCs), prompt their differentiation into chondrocytes and contribute to the repair of articular cartilage by implantation on damaged cartilage following traumatic injury. These biomaterials will be produced as porous scaffolds (3D porous structures with a controlled architecture) designed to be grafted on cartilage surface defects, or hydrogels that can be injected in cartilage cracks in a non-invasive manner.
First, we will develop a fast and efficient method to crosslink collagen biomaterials. It will occur upon UV exposure in the presence of photoreactive groups, which will not modify the native collagen sequence, in order to improve the biomaterial’s mechanical properties. Using photoreactive THPs, we will notably modulate the rigidity of porous scaffolds and accelerate the sol-gel transition of hydrogels. This novel crosslinking method will lead to stable biomaterials, which possess physical properties compatible with regenerative medicine applications, without altering the natural biological properties of collagen.
THPs will then be used to model interactions between collagen and the collagen-binding integrins (a1ß1, a2ß1, a10ß1 and a11ß1) expressed on the cellular surface of MSCs. Using THPs that contain sequences identified as ligands for a1ß1, a2ß1, a10ß1 or a11ß1, we will determinate the influence of these integrins in MSC activity and in chondrogenesis. In particular, THPs will enable us to target specifically the a10ß1 integrin, which expression increase during chondrogenesis, to select MSC sub-populations with a strong chondrogenic potential. This will provide the scientific community with new tools to elucidate the role of collagen-binding integrins in MSC behavior et to direct the differentiation lineage of MSCs.
Next, THPs will be grafted on porous scaffolds or incorporated into hydrogels seeded with MSCs. The role of these THPs will be to promote the adhesion of chondrogenic MSCs in biomaterials in vitro, as well as favor their differentiation into chondrocytes and the production of extracellular matrix specific to cartilage. The biomaterials developed for this project will subsequently be loaded with MSCs and introduced into an osteochondral block made from human cartilage, in which a defect will be induced (either as a groove in the surface that can host porous scaffolds, or as cracks in which hydrogels can be injected). The block-engineered cartilage construct will finally be implanted in mice to investigate our biomaterial’s stabilization and ability to integrate cartilage in vivo.
THP-functionalized collagen biomaterials that are loaded MSCs differentiated into chondrocytes will constitute, on the one hand, an in vitro platform to model cartilage ; and on the other hand, medical devices that can repair articular cartilage surface lesions (using porous scaffolds) or cracks (using hydrogels) following traumatic injury, as a prevention of osteoarthritis.