Agarose is a natural polysaccharide known for its ability to form thermoreversible hydrogels. While the effects of curing temperature and polysaccharide concentration on mechanical properties have been discussed in the literature, the role of ionic strength has been less studied. In the present manuscript, we investigate the effects of supporting salt concentration and the role of cation (i.e. Na⁺ or Li⁺, neighbors in the Hofmeister series), on the setting and performance of agarose hydrogels. Compressive and rheological measurements show that the supporting salts reduce the immediate elastic response of agarose hydrogels, with Li⁺ showing a stronger effect than Na⁺ at high ionic strength, while they significantly increase the extent of linear stress-strain response (i.e., linear elasticity). The presence of increasing amounts of added supporting salt also leads to a reduction in hysteresis during mechanical deformation due to loading and unloading cycles, which is more pronounced with Li⁺ than with Na⁺. The combination of rheological measurements and NMR relaxometry shows a mesh size in agarose hydrogels in the order of 6–17 nm, with a thickness of the water layer bound to the biopolymer of about 3 nm. Of note, the different structuring of the water within the hydrogel network due to the different alkali seems to play a role for the final performance of the hydrogels.

2D agarose substrates have recently been surprisingly shown to be permissive for cell adhesion, depending on their mechanics and the use of the adhesive proteins of fetal bovine serum (FBS) in the cell culture medium. Here, we elucidate how the cells exhibit two anchoring mechanisms depending on the amount of FBS. Under low FBS conditions, the cells recognize the surface-coupled adhesive sequences of fibronectin via the binding of the heterodimer α₅β₁ integrin. Functionality of the actomyosin axis and mechanoactivation of focal adhesion kinase (FAK) are essential for the stretching of the protein, thereby accessing the "synergy" PPSRN site and enhancing cell adhesion in combination with the downstream RGD motif. Under high FBS conditions, the specific peptide sequences are much less relevant as the adsorbed serum proteins conceal the coupled fibronectin and the cells recognize the adhesive protein vitronectin, which is constitutively present in FBS, via the binding of the heterodimer α_vβ₃ integrin. Similarly, the intracellular tension and FAK activity are decisive, which collectively indicate that the cells stretch the partially cryptic RGD site of vitronectin and thus make it more accessible for integrin binding. Both anchoring mechanisms only work properly if the agarose substrate is mechanically compliant in terms of linear stress-strain response, unraveling a critical balance between the mechanics of the agarose substrate and the presentation of the adhesive peptides. STATEMENT OF SIGNIFICANCE: In the context of biomaterial design, agarose hydrogels are known to lack intrinsic cell-adhesive peptide motifs and are therefore commonly used for the development of non-permissive 2D substrates. However, we unexpectedly found that agarose hydrogels can become permissive substrates for cell adhesion, depending on a compliant mechanical response of the substrate and the use of fetal bovine serum (FBS) as protein reservoir in the cell culture medium. We describe here two anchoring mechanisms that cells harness to adhere to agarose substrates, depending on the amount of FBS. Our results will have a major impact on the field of mechanobiology and shed light on the central role of FBS as a natural source of adhesive proteins that could promote cell anchoring.

Thanks to its nutritional and mechanical properties, chia seed mucilage is becoming increasingly popular in the food industry as a small biomolecule. The mechanical properties of an ingredient are a key element for food appreciation during chewing. Therefore, with this study, we explore for the first time the structural changes that chia seed mucilage undergoes when treated with α-amylase, the most abundant enzyme in human saliva. First, rheological time-sweep tests were performed on samples with different enzyme and constant chia mucilage concentrations. Then, the effect of increasing the chia mucilage concentration at a constant enzyme content was investigated. The results show that structural changes occur after enzyme treatment. Rheological measurements show a thickening of the material with an increase in the elastic modulus depending on the concentrations of α-amylase and chia used. This effect is attributed to the release and aggregation of insoluble fibrous aggregates that naturally form the mucilage after the cleavage of the α-1,4-glucoside bond between the α-D-glucopyranose residue and the second β-D-xylopyranose residue by α-amylase. Thus, our data suggest an α-amylase-mediated restructuring of the chia mucilage network that could have implications for the commercial processing of this material.