Preserving the functionality of hepatocytes in vitro poses a significant challenge in liver tissue engineering and bioartificial liver, as these cells rapidly lose their metabolic and functional characteristics after isolation. Inspired by the macroporous structures found in native liver tissues, here we develop synthetic hydrogel scaffolds that closely mimic the liver’s structural organization through the phase separation between polyethylene glycol (PEG) and polysaccharides. Our hydrogels exhibit interconnected macroporous structures and appropriate mechanical properties, providing an optimal microenvironment conducive to hepatocyte adhesion and the formation of sizable aggregates. Compared to two-dimensional hepatocyte cultures, enhanced functionalities of hepatocytes cultured in our macroporous hydrogels were observed for 14 days, as evidenced by quantitative reverse-transcription–polymerase chain reactions (qRT-PCR), immunofluorescence, and enzyme linked immunosorbent assay (ELISA) analyses. Protein sequencing data further confirmed the establishment of cell–cell interactions among hepatocytes when cultured in our hydrogels. Notably, these hepatocytes maintained a protein expression lineage that closely resembled freshly isolated hepatocytes, particularly in the Notch and tumor necrosis factor (TNF) signaling pathways. These results suggest that the macroporous hydrogels are attractive scaffolds for liver tissue engineering.

In soft connective tissues, the extracellular matrix (ECM) provides spatiotemporally well-defined mechanical and chemical cues that regulate the functions of residing cells. However, it remains challenging to replicate these essential features in synthetic biomaterials. Here, we develop a self-sorting double network hydrogel (SDNH) with spatially well-defined bioactive ligands as synthetic ECM. Specifically, the SDNH is made of two peptides that can independently self-assemble into fibers of different microscopic features, mimicking the hierarchical protein assemblies in ECM. Each peptide contains a photo-reactive moiety for orthogonally patterning bioactive molecules (i.e., cyclic arginine-glycine-aspartate (cRGD) and osteogenic growth peptide (OGP)) using UV and visible light. As a proof-of-principle, we demonstrate the engineering of SDNH with spatially separated or colocalized cRGD and OGP molecules to control the response of encapsulated stem cells. Our study represents an important step towards defining the mechanical and biochemical cues of synthetic ECM using advanced chemical biology tools.

Synthetic hydrogels are widely used as biomimetic in vitro model systems to understand how cells respond to complex microenvironments. The mechanical properties of hydrogels are deterministic for many cellular behaviors, including cell migration, spreading, and differentiation. However, it remains a major challenge to engineer hydrogels that recapture the dynamic mechanical properties of native extracellular matrices. Here, we provide a new hydrogel platform with spatiotemporally tunable mechanical properties to assay and define cellular behaviors under light. The change in the mechanical properties of the hydrogel is effected by a photo-induced switch of the cross-linker fluorescent protein, Dronpa145N, between the tetrameric and monomeric states, which causes minimal changes to the chemical properties of the hydrogel. The mechanical properties can be rapidly and reversibly tuned for multiple cycles using visible light, as confirmed by rheological measurements and atomic force microscopybased nano-indentation. We further demonstrated real-time and reversible modulation of cell migration behaviors on the hydrogels through photo-induced stiffness switching, with minimal invasion to the cultured cells. Hydrogels with a programmable mechanical history and a spatially defined mechanical hierarchy might serve as an ideal model system to better understand complex cellular functions.

Hydrogels that can respond to dynamic forces either from endogenous biological activities or from external mechanical stimuli show great promise as novel drug delivery systems (DDS). However, it remains challenging to engineer hydrogels that specifically respond to externally applied mechanical forces with minimal basal drug leakage under normal stressful physiological conditions. Here we present an ultrasound responsive hydrogel-based DDS with special dual-crosslinked nanoscale network architecture. The covalent crosslinks endow the hydrogel high mechanical stability and greatly suppress deformation-triggered drug release. Meanwhile, the dynamic covalent boronate ester linkages between hydrogel backbone and the anti-inflammation compound, tannic acid (TA), allow effective ultrasound-triggered pulsatile release of TA. As such, the hydrogel shows distinct drug release profiles under compression and ultrasound. A proof-of-principle demonstration of the suppression of inflammation activation of macrophage upon ultrasound-triggered release of TA was also illustrated. We anticipate that this novel hydrogel-based drug delivery system can be used for the treatment of inflammatory diseases on load-bearing tissues, such as muscle and cartilage.