Transient Polymer Interactions for Dynamic and Reversible Hydrogel Viscoelastic Tuning and Cell Adhesion Control

Cells in multicellular organisms, such as humans, have evolved a mechanically-sensitive signalling pathway called mechanotransduction. Interestingly, mechanotransduction has been shown to direct stem cell differentiation towards different cell types, such as muscle or bone cells, simply by changing the environmental elasticity. It also plays an important role in tumour progression. Despite its importance to cellular and physiological systems, the effect of environmental viscoelasticity on mechanotransduction is still not well understood. While complex, light-based or other polymer-based techniques exist to tune environmental stiffness, they require complex setups or are only useable on a narrow range of hydrogels.<br/> <br/>Here, we present a simple, cost-effective means of dynamically and reversibly tuning polymer hydrogel viscoelasticity using transient interactions using poly(ethylene glycol), or PEG. PEG polymers in solution with a hydrodynamic radius below the size of the hydrogel’s pores can infiltrate it, dynamically interacting with hydrogel polymers. Alginate, a polymeric hydrogel derived from algae, is an ideal candidate for this transient polymer control: at 2% (w/v), alginate has a relatively large (~5 nm) pore size, allowing PEG of molecular weight up to at least 8 kDa to infiltrate and stiffen it. Using rheometry, we demonstrate that PEG interactions can increase hydrogel stiffness by as much as hundreds of kPa. Local elastic modulus measurements confirm rheometry measurements of alginate hydrogel stiffness increase in the presence of PEG. Despite the addition of interacting PEG polymers to the hydrogels, hydrogel viscosity does not increase, further reinforcing the idea that the interactions between PEG and the hydrogel polymers cause the material to stiffen. To determine that this stiffening effect is not due to an increase in alginate hydrogel concentration due to solvent being forced out of the hydrogel from osmotic pressure, we show that stiffness for alginate hydrogels exposed to 300 Da vs. 8 kDa PEG are vastly different, despite similar increases in alginate concentration. PEG uptake into alginate hydrogels is also confirmed using fluorescently tagged PEG, with both confocal microscopy and HPLC demonstrating a change in fluorescence intensity. Interestingly, this dynamic stiffening effect is a function of PEG molecular weight, indicating a size-dependent interaction between PEG molecules and polymer hydrogel molecules. We postulate that this interaction is due to hydrogen bonding between the PEG and hydrogel polymers. Furthermore, we demonstrate the reversibility of the process, an important application as it allows for both stiffening and weakening effects. This reversible and dynamic PEG interaction with hydrogel polymers is also possible with other kinds of hydrogels, such as agarose.<br/> <br/>We also demonstrate the clear effect on the morphology of rat embryonic fibroblast (REF) cells grown on PEG-exposed alginate hydrogels, with cells displaying a stretched-out morphology on PEG-interacting hydrogels that is not present in alginate hydrogels not exposed to PEG. This cell morphology difference is shown to be the result of alginate hydrogel stiffening, as REF cells grown on a very stiff 7% (w/v) alginate hydrogels that are not exposed to PEG show similar cell morphologies as those grown on 2% (w/v) alginate hydrogels exposed to 8 kDa PEG. As a further control to determine that cell morphology differences are due to changes in substrate stiffness, cells grown in the same well as those grown on hydrogels and exposed to the same PEG-containing solution demonstrate similar morphologies to cells grown on alginate but not exposed to PEG. This study is of interest to the biomaterials and polymer materials community, demonstrating a simple means of tuning the cellular environment.