Mechanical relaxations of hydrogels governed by their physical or chemical crosslinks.

In the field of tissue engineering, in order to restore tissue functionality hydrogels that closely mimic biological and mechanical properties of the extracellular matrix are intensely developed. Mechanical properties including relaxation of the surrounding microenvironment regulate essential cellular processes. However, the mechanical properties of engineered hydrogels are particularly complex since they involve not only a nonlinear elastic behavior but also time-dependent responses. An accurate determination of these properties at microscale, i.e. as probed by cells, becomes an essential step to further design hydrogel-based biomaterials able to induce specific cellular responses. Atomic Force Microscopy (AFM) with contact sizes of the order of few micrometers constitutes an appropriate technique to determine the origin of relaxation mechanisms occurring in hydrogels. In the present study, AFM force relaxation experiments are conducted on chemically and physically crosslinked hydrogels respectively based on a synthetic polymer, polyacrylamide and a natural polymer, a bacterial exopolysaccharide infernan, produced by the deep-sea hydrothermal vent bacterium, Alteromonas infernus. Two distinct relaxation mechanisms are clearly evidenced depending on the nature of hydrogel network crosslinks. Chemically crosslinked hydrogel exhibits poroelastic relaxations, whereas physically crosslinked hydrogel shows time-dependent responses arising from viscoelastic effects. In addition, two relaxation processes are revealed in ionic physical hydrogel originating from chain rearrangement and breaking/reforming of the ionic crosslinks. The effect of the ionic strength on both the long-term elastic modulus and relaxation times of physical hydrogels was also shown. These findings highlight that physical hydrogels with well-defined time-dependent mechanical properties could be tuned for an optimized response of cells.

Interactions between infernan and calcium: From the molecular level to the mechanical properties of microgels.

With the increasing need for hydrogels with tunable properties for specific biomedical applications, a complete understanding of the structure-function relationship of polymers used for hydrogel development remains crucial for their optimal use. In the present study, by combining experimental and theoretical approaches, the structure-function relationship of a bacterial exopolysaccharide, infernan, displaying both glycosaminoglycan-mimetic and gelling properties, was investigated at molecular and microscopic levels. Atomic force microscopy (AFM) experiments and molecular dynamics simulations were applied to determine the persistence length of individual infernan chains before studying their association induced by calcium. Infernan-based microgels were then produced using microfluidics and their mechanical properties were characterized by AFM methods. The mechanical properties of EPS/calcium microgels were finely tuned by varying the crosslinking density of their network, either by calcium or EPS concentrations. The obtained set of viscoelastic microgels with different elastic modulus values opens several possibilities for their applications in tissue engineering.

Microgels based on Infernan, a glycosaminoglycan-mimetic bacterial exopolysaccharide, as BMP-2 delivery systems.

Bone Morphogenetic Protein (BMP-2) is an osteoinductive growth factor clinically used for bone regeneration. Tuneable sustained strategies for BMP-2 delivery are intensely developed to avoid severe complications related to supraphysiological doses applied. To address this issue, we investigated the ability of the bacterial exopolysaccharide (EPS) called Infernan produced by the deep-sea hydrothermal vent bacterium Alteromonas infernus, exhibiting both glycosaminoglycan-mimetic and physical gelling properties, to efficiently bind and release the bioactive BMP-2. Two delivery systems were designed based on BMP-2 retention in either single or complex EPS-based microgels, both manufactured using a microfluidic approach. BMP-2 release kinetics were highly influenced by the ionic strength, affecting both microgel stability and growth factor/EPS binding, appearing essential for BMP-2 bioactivity. The osteogenic activity of human bone-marrow derived mesenchymal stem cells studied in vitro emphasized that Infernan microgels constitute a promising platform for BMP-2 delivery for further in vivo bone repair.