Precision design of dextran-permeated agarose hydrogels matching adipose stem cell adhesion timescales

Viscoelasticity is now recognised as a key parameter in modulating cell behaviour. Tailoring time-dependent materials to elicit specific cellular responses is, however, a challenge because of the intricate relationship between the substrate relaxation time (τrel) and the cell sensing time-window which depends on the time required for the formation of focal adhesions (τb) and the duration of their lifetime (τL). Here, we introduce a novel design approach to guide cell behaviour based on the cell-perceived Deborah number, De = τrel/τL, arguing that for De > 1 and De < 1, substrates promote cell differentiation because stable adhesions and sustained tension drive mechanotransduction and lineage-specific differentiation on the basis of substrate stiffness. Instead, cell stemness is maintained in the De ∼1, whereby excessive mechanical signalling is prevented as cells balance adhesion stability and plasticity. The design workflow consists in modelling substrate τrel, enabling the selection of the optimal gel formulation according to the cell-perceived De. The workflow was applied to agarose gels with different dextran concentrations in the liquid phase, which act as modulators of mechanical time-dependent properties. To predict the relaxation times for these gels, we developed an in-silico model which integrates their structural and transport properties. Our results show that the gels have an almost constant equilibrium elastic modulus, while their τrel decreases with increasing dextran concentration in the liquid phase. Considering adipose-derived mesenchymal stem cells (ADSCs) and their characteristics sensing times, we defined dextran concentrations to mimic the different De conditions in the agarose gels. Experimental cell investigations confirmed the validity of the design approach: ADSC differentiation, highlighted by YAP nuclear translocation, was promoted in the case of De < 1 and De > 1, respectively eliciting adipogenic and osteogenic lineages. On the other hand, cells maintained their stemness when De ∼1. This study provides novel insights on the interplay between hydrogel viscoelasticity and cellular behaviour and paves the way for precision design of viscoelastic biomaterials for in-vitro studies and regenerative medicine.