Hydrogels are water-swollen, hydrophilic polymer networks that are popular materials for cell culture matrices and bone scaffolds. Hydrogels placed in water experience mechanical stress due to competing effects of osmotic swelling and network modulus. Such forces can lead to chemical changes at the bonds within the matrix. Here, we show that osmotic stress can initiate covalent mechanochemical reactions that produce reactive oxidative species (ROS), which are known to influence differentiation and proliferation of certain stem cell phenotypes. In particular, hydrogen peroxide (H2O2) is known to affect cell signaling and cellular protein structure, and it has been found to be generated in stressed PDMS. Thus, the relationship between hydrogel stress and ROS production should be studied in more depth. In this work, mechanochemical production of ROS in polyacrylamide hydrogels by osmotic swelling was investigated. A simple enzymatic assay was developed using horseradish peroxidase (HRP) and amplex ultrared (AUR) to quantify the amount of H2O2 formed via a chromogenic readout. Colorless AUR reacts with H2O2 in the presence of HRP to form resorufin, a bright pink compound. Crosslinkers with varying linkage strengths were used to fabricate hydrogels and it was observed that when a crosslinker containing a weak disulfide linkage, such as bis(acryloyl)cystamine (BAC) is used, the H2O2 produced is observed to be in the 12-25 µM range, whereas incorporation of a stronger polyethylene glycol diacrylate (PEGDA) crosslinker resulted in significant lowering of overall H2O2 production. Next, mechanical strain was increased via constrained swelling by chemically conjugating the gel to a glass substrate and swelling in water. Consequentially, biaxial compressive forces developed in the gel, leading to a significant increase in ROS concentration regardless of the crosslinker structure, achieving up to 30 µM H2O2 in the swelling media. The impact of this work is important for the general biomaterials community, especially groups that focus on using hydrogels for stem cell culture.

Mechanochemical phenomena in free and constrained polyacrylamide hydrogels induced by osmotic swelling

2019 Fall National Meeting

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