The design of reversible hydrogels to capture extracellular matrix dynamics

Abstract

The extracellular matrix (ECM) is a dynamic environment that constantly provides physical and chemical cues to embedded cells. Much progress has been made in engineering hydrogels that can mimic the ECM, but hydrogel properties are, in general, static. To recapitulate the dynamic nature of the ECM, many reversible chemistries have been incorporated into hydrogels to regulate cell spreading, biochemical ligand presentation and matrix mechanics. For example, emerging trends include the use of molecular photoswitches or biomolecule hybridization to control polymer chain conformation, thereby enabling the modulation of the hydrogel between two states on demand. In addition, many non-covalent, dynamic chemical bonds have found increasing use as hydrogel crosslinkers or tethers for cell signalling molecules. These reversible chemistries will provide greater temporal control of adhered cell behaviour, and they allow for more advanced in vitro models and tissue-engineering scaffolds to direct cell fate.

Figure 1. Biological extracellular matrix and synthetic strategies involving reversible chemistries

a | The native extracellular matrix (ECM) is a heterogeneous fibrillar network that provides biochemical and physical cues to cells. b | Synthetic hydrogels are traditionally static, polymeric networks (middle panel). Dynamic hydrogels can capture aspects of the native ECM by temporally controlling ligand presentation (left panel) or reversibly cycling through changes in mechanics (right panel). Although these dynamic chemistries do not fully capture the complexity of the biological environment, they provide an enhanced precision over matrix cues in vitro, which will have a powerful impact on tissue engineering and regenerative medicine.

Figure 2. Irreversible and reversible chemistries for hydrogels

a | Irreversible chemistries for hydrogels include tethered molecules for cell signalling (left panel), degradable crosslinks to mediate cell spreading (middle panel) and the generation (or degradation, which is not shown) of crosslinks to alter matrix mechanics (right panel). b | Reversible chemistries include dynamic presentation of cell signalling molecules (left panel), self-healing or adaptable crosslinks in hydrogels (middle panel) and strategies to alter the crosslinking density without changing the network connectivity of the hydrogel (right panel). c | The reversible chemistries incorporated into synthetic hydrogels and their uses for biofunctionalization, crosslinking and cyclical mechanics are shown in the table.

Figure 3. Light-based strategies for exchangeable ligand presentation

a | Traditional lithographic masks are used for 2D photopatterning (left panel); however, 3D photopatterning (right panel) can access more complex shapes using a confocal laser scanning microscope. b | One strategy for the sequential release of biomolecules, such as proteins, relies on using photocleavable linkers with orthogonal absorption. For example, nitrobenzyl-based linkers cleave in response to 365 nm light and coumarin-based linkers cleave in response to 405 nm light. c | Another strategy for the reversible presentation of biomolecules first uncages a reactive group in the hydrogel, followed by ligation of the molecule (with a photocleavable linker) using click chemistry. The molecule can subsequently be removed using light, which cleaves the photodegradable linker. d | Living radical reactions, such as those mediated with an allyl sulfide functionality, provide opportunities for the reversible conjugation of biomolecules over many cycles. Panel a adapted with permission from REF. , Wiley-VCH. Panel b adapted with permission from REF , Wiley-VCH. Panel c from REF , Nature Publishing Group. Panel d adapted with permission from REF , Wiley-VCH.

Figure 4. Non-covalent strategies for exchangeable ligand presentation

a | Hybridization of leucine zippers allows for the passivation of immobilized biomolecules with poly(ethylene glycol) (PEG) coils. Subsequent removal of the PEG coils can be achieved using a competing leucine zipper. b | Similarly, aptamer hybridization can be utilized to reversibly expose or hide a bioactive site. Panel a adapted with permission from REF. , American Chemical Society. Panel b adapted with permission from REF. , Wiley-VCH.

Figure 5. Crosslinking effects on bulk hydrogel properties

a | Elastic hydrogels typically use covalent linkages, such as thiol-ene chemistry. These linkages constrain polymer conformation in the hydrogel, leading to its behaviour as an elastically active spring. Hence, the material displays a linear stress–strain curve for small deformations (left panel). The modulus is frequency independent (middle panel) and strain independent (right panel). b | Conversely, dynamic linkages (such as the host–guest chemistry) lead to viscoelastic hydrogels because the bond is in equilibrium with its precursors, which imparts liquid-like behaviour to the gels. The stress–strain curve (left panel) shows hysteresis because these linkages can rearrange to accommodate stress. In addition, the modulus displays frequency dependence (middle panel), and many of these gels show shear-thinning behaviour (the decrease in modulus upon the application of high strain; right panel). G′, the storage modulus; G″, the loss modulus.

Figure 6. Covalent adaptable networks

Examples of covalent adaptable chemistries for crosslinking include hydrazone bonds (panel a) and imine bonds (panel b). Depicted schematically in panel c, these chemistries enable cells to push and pull on the matrix without degrading the polymer chains or crosslinkers.

Figure 7. Reversible control of matrix mechanics

a | Hydrogel stiffening can be achieved by increasing the persistence length of the crosslinker using complexation (for example, with DNA hybridization), and subsequent softening can be achieved by removing the hybridizing molecule with a competing strand. b | Conformational changes in hydrogel crosslinkers, such as protein folding and unfolding in response to binding ligands or environmental changes (for example, redox state), can also be used to alter the crosslinking density (and therefore the modulus) over many cycles. c | The number of elastically active crosslinks can be modulated using photoreversible host–guest chemistry. d | Reversible crosslinking between chains can also be modulated with the introduction or removal of ion coordination (for example, calcium ions in alginate networks).