Glycosaminoglycans-based biomaterials to study the bioactivity of growth factors 

LE PENNEC Jean, SEFKOW-WERNER Julius, GOUT Eveline, MACHILLOT Paul, PICART Catherine, VIVES Romain, MIGLIORINI Elisa

 Extracellular matrix (ECM) is the surrounding environment of cells, regulating their development and the tissue homeostasis. Among its components, glycosaminoglycans (GAGs) are known to be involved in the bioactivity regulation of numerous proteins 1,2. The effects of GAGs on cellular response are widely studied with classical methods presenting molecules in solution even though in the ECM, the GAGs are naturally bound on proteoglycans, restricting their diffusion and mobility. Yet, the mechanisms of growth factor regulation by the GAGs remain poorly understood. Notably the role of the GAG sulfation content and patterns – crucial for the interaction and bioactivity of proteins – still constitutes a mystery to explore. In this context, we aim to understand the role of GAGs sulfation in the regulation of growth factors, in particular of the osteoinductive Bone Morphogenetic Protein 2 (BMP2) which is known to bind to heparn sultate (HS). Our approach combines molecular interactions studies with Biolayer Interferometry (BLI) to determine binding affinities of BMP2 with GAGs and cellular studies using biomimetic platforms3 that mimics the ECM. These biomaterials, here called platforms, are surfaces composed of a streptavidin monolayer which presents adhesion peptides, immobilized GAGs and growth factors like BMP2.

To explore more precisely the role of GAG sulfation, we prepared a library of HS-derived tetra- and hexa-saccharides through exhaustive digestion of native HS with heprinase III, and purification using combined size-exclusion and strong anion exchange chromatographies.

With BLI we measured the affinities of BMP2 with different GAGs, revealing strong differences between the different GAGs: HS, chondroitin sulfate-A/B/D/E, and hyaluronic acid. Surprisingly we measured a strong affinity of BMP2 with oligosaccharides as short as hexa- and tetra-saccharides. We proved that particular species of hexa-saccharides exhibit an affinity close to full HS chains. To study the role of GAGs on the cellular response, BMP2 was adsorbed onto the GAGs and the cellular response of C2C12 myoblasts seeded on our biomimetic platforms was assessed through immunofluorescence staining.