Spatiotemporal hydrogel biomaterials for regenerative medicine

Abstract

Hydrogels mimic many of the physical properties of soft tissue and are widely used biomaterials for tissue engineering and regenerative medicine. Synthetic hydrogels have been developed to recapitulate many of the healthy and diseased states of native tissues and can be used as a cell scaffold to study the effect of matricellular interactions in vitro. However, these matrices often fail to capture the dynamic and heterogenous nature of the in vivo environment, which varies spatially and during events such as development and disease. To address this deficiency, a variety of manufacturing and processing techniques are being adapted to the biomaterials setting. Among these, photochemistry is particularly well suited because these reactions can be performed in precise three-dimensional space and at specific moments in time. This spatiotemporal control over chemical reactions can also be performed over a range of cell- and tissue-relevant length scales with reactions that proceed efficiently and harmlessly at ambient conditions. This review will focus on the use of photochemical reactions to create dynamic hydrogel environments, and how these dynamic environments are being used to investigate and direct cell behavior.

Figure 1

Synthetic hydrogel matrices can provide a blank slate for encapsulated cells. Using photochemical reactions, researchers can modify these scaffolds to impart adhesivity and bioactivity and to change the mechanical properties, which can be used to direct cell behavior through cell-matrix interactions.

Figure 2

Strategies to control spatial illumination of hydrogels. a,b) Photomasks can be used to selectively block collimated light to create a) patterns or b) gradients in illumination that are uniform in the axial direction. c) A maskless photolithography technique is used to project patterns based on graphical inputs to a digital micromirror device (DMD). Reprinted from Ma et al. Copyright (2016) National Academy of Sciences. d) Alternatively, focused laser light can be used to illuminate regions with 3D resolution. The light illumination is illustrated by photobleaching uniformly bound fluorescein within a hydrogel at a single plane with light of 488 nm (single photon absorption) and 800 nm (two photon absorption). Two-photon absorption events are localized to the imaging plane. Photobleaching and confocal imaging performed with a 1.0 NA objective. Scale bar = 25 μm.

Figure 3

Three-dimensional patterning techniques using photoinitiated chain reactions. a) Two-photon patterning is used to immobilize acrylated peptides in 3D with high resolution (scale bar = 25 μm). The acrylated peptides are tagged with AlexaFluorÒ 488, 532 and 633. Reproduced from Hoffmann et al. with permission from the Royal Society of Chemistry. b) A stereolithographic approach using projection based methods to rapidly create three-dimensionally defined hydrogel structures. Cells in alternating layers are labelled with CellTracker® green and orange. Scale bar 50 μm. Reprinted from Gou et al. c) Stereolithography using focused laser light to achieve 3D printing of cell-laden scaffolds. Scale bar 1 mm. Reproduced from Chan et al. with permission from the Royal Society of Chemistry. d) Projection stereolithography used to photopolymerize methacrylated gelatin hydrogel structures. Images show the proliferation of human umbilical vein endothelial cells seeded onto the scaffolds over 4 days. Reprinted from Gauvin et al. copyright (2012) Elsevier.

Figure 4

Thiol-ene photoclick chemistry is a mild and efficient reaction for hydrogel formation and functionalization. a) The thio-ene reaction mechanism, showing the cycle of propagation (thiyl radical addition to alkene) and chain transfer to generate a new thiyl radical. b) Under ambient conditions and identical photoinitiating schemes and functional group concentrations, hydrogels are formed by the thiol-ene reaction within 5 s; comparatively, hydrogels formed by homopolymerization of PEG-diacrylate are delayed. Reproduced from McCall et al. Copyright (2012) American Chemical Society.

Figure 5

Thiol-ene photopolymerizations used to functionalize three-dimensional hydrogels. a) Norbornene-modified PEG can be crosslinked and functionalized with cysteine-containing peptides. hMSCs seeded in MMP-degradable hydrogels spread only in regions with photopatterned RGD. Reproduced from Fairbanks et al. copyright (2009) Wiley. b) Left: nanofibrous norbornene hyaluronic acid hydrogels patterned with three fluorescent peptides. Right: when RGD patterns are created perpendicular to the nanofiber alignment, cells localize to the RGD regions (indicated in red), but align with the fiber orientation. Scale bars 100 μm (left) and 200 μm (right). Reproduced from Wade et al. copyright (2015) Wiley.

Figure 6

Reversible thiol-ene photopatterning via allyl sulfide chemistry. a) Consecutive swapping of thiolated molecules can be performed, forming new thioether bonds and reactive alkenes. b) An RGD peptide containing a cysteine residue and AlexaFluor555 is tethered to the network by two-photon photolithography. c) A second thiolated peptide (with AlexaFluor488 dye) is photopatterned in the buffalo shape, simultaneously releasing the first peptide. d) A third peptide (unlabeled) is patterned in the shape of the CU logo, releasing the second peptide. Scale bar 100 μm. Adapted from Gandavarapu et al. copyright (2014) Wiley.

Figure 7

Secondary photocrosslinking strategies to change hydrogel mechanics in situ. a) Schematic representation of a sequential crosslinking strategy. Hyaluronic acid is functionalized with methacrylate and maleimide groups (MeMaHA). Maleimide reactive groups preferentially react with a dithiol MMP-degradable crosslinker, leaving methacrylates available for subsequent photopolymerization. hMSCs are encapsulated during the initial hydrogel network formation and are allowed to spread in the softer, degradable hydrogel. At a later point in time, methacrylates are photopolymerized (red dashed lines) to stiffen the network. b) Traction force microscopy images of encapsulated hMSCs after 7 days in growth medium and 14 days in mixed medium after stiffening. Although cells retain a spread morphology after secondary photocrosslinking, hMSCs undergo adipogenesis in mixed-media conditions, supporting the proposed hypothesis that hMSC cell fate decisions depend on the ability of the cells to interact with the matrix via remodeling. Scale bars 25 μm. Reprinted from Khetan et al. copyright (2013) Nature Publishing Group. c) Direct printing of a shear-thinning hydrogel into a self-healing network. Guest-host interactions between adamantane and cyclodextrin form a shear-thinning network that can be printed and then covalently crosslinked by photopolymerization of pendant methacrylates. The polymerized construct remains after the support has been washed away. Scale bar 500 μm. Reproduced from Highley et al. copyright (2015) Wiley.

Figure 8

Photocleavage reactions of a) nitrobenzyl and b) coumarin photolabile linkages. When incorporated into the structure of the network, these cleavage reactions result in network degradation. c) Schematic showing the use of a photodegradable hydrogel to vascularize a cell-laden hydrogel construct in situ. Reprinted from Arakawa et al. copyright (2017) Wiley.

Figure 9

Formation and degradation of photodegradable hydrogel networks. a) Idealized structures of chain and step polymerized networks and their photodegradation products. Networks of linear PEG-di(photodegradable acrylate) are formed by radical polymerization, resulting in acrylate kinetic chains (black) connected by PEG spacers (red). PEG-tetra(difluorocyclooctyne) undergoes a spontaneous crosslinking reaction when mixed with a photodegradable di-azide peptide. b) Irradiation of the bulk hydrogel with 365 nm light results in loss of crosslinks that can be tracked with photorheometry. Shuttering the light results in stabilization of the modulus readings. c) Two-photon photodegradation (λ=740 nm) was used to create defined three-dimensional topographies within the hydrogel volume. Scale bar 100 μm. Reprinted from Kloxin et al. copyright (2009) AAAS. d) Orthogonal photochemical strategies are used to pattern RGD (dotted line) and erode the hydrogel network. Fibroblasts migrate through the 3D void space only in the region containing the adhesive ligand. Scale bar 100 μm. Reproduced from DeForest et al. copyright (2011) Nature Publishing Group.

Figure 10

Cellular responses to mechanical surface patterning via photodegradation. a) Photomasks were used to create subcellular stiffness patterns with either regular or disordered spacing. Adherent hMSCs on regularly patterned substrates were more sensitive to changes in the overall fraction of stiff area, indicated by a greater change in the nuclear localization YAP. Scale bars 20 μm. Reproduced from Yang et al. copyright (2016) National Academy of Sciences. b) A maskless light projection technique was used to create repeating gradients in light exposure, leading to patterned photodegradation. hMSCs follow the patterns and localize to the more degraded regions. Scale bar 1 mm. Reprinted with permission from from Norris et al. copyright (2016) American Chemical Society.

Figure 11

Photodegradable and photoadaptable hydrogels based on radical processes. a) Reaction scheme for photodegradation of hydrogels containing disulfide or allyl sulfide linkages. b) A photoadaptable disulfide-crosslinked hydrogel is exposed to light while pressed against a textured surface, resulting in photoinitiated deformation. Scale bar 1.5 mm. Reproduced from Fairbanks et al. copyright (2011) American Chemical Society. c) Radical-mediated photodegradation requires lower concentration of photoactive species, allowing for the photodegradation of thicker monolithic samples. In this example, a 1 cm-thick hydrogel is degraded in approximately 1 min of irradiation (365 nm, 10 mW cm⁻²). Scale bar 5 mm. Reproduced from Brown et al. copyright (2017) Wiley.

Figure 12

Photocaging strategies to reveal reactive groups upon irradiation.

Figure 13

Reversible photopatterning of full length proteins using sequential photoconjugation and photocleavage reactions. a) Network alkoxyamines are uncaged and react with proteins conjugated to photocleavable aldehydes. A secondary round of light exposure releases the protein. b) A second protein can be incorporated during the release of the first, forming intricate patterns. Scale bar 50 μm. c) Sustained vitronectin immobilization leads to osteogenic differentiation of hMSCs, indicated by staining for osteocalcin (green). Reprinted from DeForest et al. copyright (2015) Nature Publishing Group.

Figure 14

Enzymatic photopatterning by the uncaging of the lysine residue in the transglutaminase recognition sequence. This strategy is used to induce migration out of a cluster of MSCs into the surrounding gel in regions patterned with a) RGD and b) fibronectin fragment. Scale bars 200 μm. Reproduced from Mosiewicz et al. copyright (2013) Nature Publishing Group