Chemical Approaches to Dynamically Modulate the Properties of

Dec 22, 2018 - For example, hyaluronic acid hydrogels incorporating hydrazone bonds were 3D-printed into multilayered lattices in the presence of cell...
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Viewpoint Cite This: ACS Macro Lett. 2019, 8, 7−16

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Chemical Approaches to Dynamically Modulate the Properties of Synthetic Matrices Paige J. LeValley† and April M. Kloxin*,†,‡ †

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware 19716, United States Material Science and Engineering, University of Delaware, Newark, Delaware 19716, United States

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‡

ABSTRACT: As knowledge about the dynamic nature of tissues within the human body has increased, the need for cell culture models that mimic the properties of these dynamic microenvironments has grown. Hydrogels are useful platforms for investigating cellular responses to microenvironment cues in disease and regeneration processes and recently have been designed to contain dynamic bonds to regulate the mechanical and biochemical properties of the matrix in three-dimensional cell culture applications. In this Viewpoint, we highlight recent advances in developing hydrogels with dynamic properties for modeling aspects of human tissues, providing control over the properties of the synthetic matrix on multiple length and time scales, and their application for understanding or directing cell response. We conclude by discussing how orthogonal chemistries can be utilized to design dynamic hydrogel platforms for controlling both the mechanical and biochemical environment, affording opportunities to investigate more complex questions associated with disease progression and tissue regeneration.

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known as dynamic reciprocity.9,10 This knowledge has led to the need for hydrogel platforms that can mimic the dynamic nature of human tissue for improving in vitro disease models with the ultimate goal of designing systems whose properties can be tuned when and where needed for probing cell response and elucidating (and ultimately mitigating) key factors that influence the disease state. The biomaterials community has approached this goal with tenacity, developing numerous methods to control the mechanical or biochemical properties of hydrogels where and when desired upon the application of a stimulus. To create matrices with such in situ property control, materials chemistries that respond to either cues produced by cells or applied by the user have been designed and utilized.11,12 Crosslinks that respond to enzymes13 or forces14 generated by cells during their life cycle can be advantageous for mimicking aspects of the dynamic reciprocity that occurs natively between cells and the ECM. For example, hydrazone bonds have been incorporated within poly(ethylene glycol) (PEG)-based hydrogels to permit cell migration through the synthetic matrix by “pulling” on this reversible bond that is integrated within the crosslinks of the hydrogel.15 On the other hand, user-defined cues offer control over when, and potentially where, changes occur in the synthetic matrix by the application of an external stimulus, such as light,16 temperature,17 or a variety of molecules.18 For example,

ver the past decade, knowledge and understanding of a variety of diseases has drastically improved. These advances can be, in part, attributed to great strides made in the development of relevant in vivo1 and in vitro2 model systems that have led to enhanced diagnostics, prognostic platforms, and ultimately treatment strategies. Despite these advances, in vitro models that are more predictive of human diseases remain a need for both fundamental and translational studies, from hypothesis testing to the evaluation of therapeutics. Soft biomaterials, specifically hydrogels, have emerged as a prominent platform for the multidimensional culture of human cells for in vitro studies of disease,3−5 as well as regeneration,6,7 owing to their hydrophilic nature and capacity to mimic aspects of different soft tissues (Figure 1). These water-swollen polymer networks can be formed using natural materials, synthetic materials, or a combination of both. Natural materials, often harvested from animal tissues, inherently provide specific biochemical (e.g., binding) and biophysical (e.g., structure) cues found in human tissues with some batch-to-batch variation. Hybrid or synthetic materials allow the facile installation of reactive handles on harvested proteins or synthesized peptides and polymers for fine-tuned control of both the mechanical (e.g., modulus or “stiffness”) and biochemical cues presented by the matrix.8 These approaches enable the investigation of a diverse array of diseases (e.g., fibrosis, liver disease, cancer, and diabetes) and different diseased states (e.g., healthy wound healing versus fibrotic disease) with base modular building blocks. Beyond the initial properties, the microenvironment of cells within the body is dynamic, with ever-changing properties as cells respond to and remodel the extracellular matrix (ECM), © XXXX American Chemical Society

Received: October 21, 2018 Accepted: December 7, 2018

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particular, materials that are cyclically reconfigurable (i.e., materials whose properties can be cycled between states) are advantageous to mimic the cyclic property changes that occur in the native ECM (e.g., mechanical and biochemical property changes associated with matrix degradation and redeposition during wound healing processes). In this Viewpoint, we discuss progress toward achieving dynamically and cyclically reconfigurable hydrogel systems and their translation to use within in vitro three-dimensional (3D) cell culture models. First, we look at how the crosslinks that comprise the network can be tuned to impart cyclic changes in hydrogel mechanical properties, highlighting in particular how recently developed chemistries present opportunities for incorporation within cell culture platforms. Next, we discuss how the biochemical microenvironment within hydrogel-based matrices can be altered in a cyclic manner, also noting interesting chemistries that have yet to be used with cells but could be applied for controlling chemical cues in synthetic matrices. We conclude with our perspective on the current outlook for designing cyclically reconfigurable hydrogels and opportunities for future work in this field. Cyclic Mechanical Properties. As human tissue undergoes aging, incurs an injury, or experiences other maladies, the mechanical properties of the tissue are altered.8 In the case of aging, the alterations to mechanical properties are often unidirectional; however, for injured or diseased tissue, undergoing reconstruction either naturally or therapeutically, the ultimate goal is to return the tissue to a version of its original state (i.e., homeostasis), making the changes in mechanical properties cyclic. Hydrogel-based synthetic matrices that can cycle between “soft” and “stiff” states will provide opportunities to advance our understanding about the underlying processes regulating these transitions and the design of interventions for directing them. Currently, many hydrogel systems incorporate degradable crosslinks that allow either the cells to modify their environment through cellsecreted factors (e.g., matrix metalloproteinases) or the user to apply stimuli, such as light or biomolecules, to define changes in the stiffness of the matrix, typically producing an irreversible change in the mechanical properties of the system. Dynamic chemistries that can switch between “on” and “off” states offer opportunities for reversibly modulating the mechanical properties of the matrix through incorporation of dynamic bonds (Figure 2) or chain lengths (Figure 3) within the backbone of the hydrogel. Dynamic bonds can be either covalent, such as imine or thioester bonds, or noncovalent, such as host−guest interactions (Figure 2). These bonds naturally exist in an equilibrium between “on” and “off” states, through either changes to the microenvironment, such as switching the oxidative state of the matrix, or forces being applied to the bond, such as a cell “pulling” on the matrix, and the thermodynamics of the system can be altered to shift the equilibrium in one direction or another. This shift in equilibrium can be exploited to provide different levels of transient control over the mechanical properties of the matrix. For example, dynamic crosslinks can afford local (e.g., nanoscale) changes in the hydrogel mechanical properties by cell-exerted forces while maintaining the overall bulk modulus or lead to observable changes in the bulk hydrogel modulus upon the application of a stimulus (Figure 2). An alternative mechanism employed to control the mechanical properties of a system is to use crosslinkers (e.g., functionalized polymers)

Figure 1. Engineering material systems to mimic dynamic cellular environments. The microenvironment of cells within the body, particularly the ECM, is dynamic, with ever-changing properties as cells respond to and remodel the matrix surrounding them (left). Commonly, these changes occur due to a shift in the health of the tissue (e.g., acute injury, aging), where changes in the matrix include the deposition and crosslinking of structural proteins such as collagen (blue), fibronectin (green), and laminin (purple) and release of soluble bioactive proteins (yellow and orange hexagons). The ability to recreate aspects of this environment in vitro using well-defined hydrogel-based materials provides opportunities to more precisely study the factors that influence cell responses in disease states, such as changing the density and crosslinking of the synthetic matrix to mimic various tissue states (right). The use of synthetic platforms to study such complex processes in vitro may aid in the development of better diagnostic, prognostic, and treatment options.

aptamer-patterned hydrogels have been designed for the capture and release of specific proteins to control the chemical composition of the synthetic matrix,19 and light-responsive chemistries, such as azobenzene, have been integrated within hydrogels for triggered softening or stiffening of the matrix with different wavelengths of light.20 By designing hydrogels whose properties can be altered during cell culture, we can begin to investigate the underlying factors that contribute to alterations in cell behavior during changes to their microenvironment (e.g., upon injury, aging, disease initiation, or progression).3,4,21,22 For example, hydrogels that stiffen during cell culture have been exploited for mechanistic studies of fibrosis23 or cancer,24 and systems that allow the addition or removal of biochemical cues have been employed for examining key biochemical cues in cell migration25 and differentiation.26 Broadly, these approaches have addressed a need for reconfigurable cell culture platforms. Yet, many of the materials chemistries established to date offer only a single change in the system properties unlike the local in vivo microenvironment where cells experience a continuum of changes in the mechanical and biochemical cues around them. For studying more complex biological phenomena, hydrogel platforms that can be dynamically reconfigured for in situ control of their physical and chemical properties provide an opportunity for probing bidirectional and reversible interactions between cells and their microenvironments.4 In 8

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Figure 2. Reversible changes in mechanical properties: dynamic bonds. (A) In hydrogel networks formed with dynamic covalent bonds, bonds are continually exchanging and exist in a state of equilibrium; application of a stimulus, such as force applied by a cell (yellow box), can promote network rearrangement and enable cell movement. Here, the green bonds highlight bonds that exist in the “off” state by either natural changes in the equilibrium of the system or a cell-exerted force. When the bond is moved into the “off” state by a cell-exerted force, the bond can reform once the applied force is removed. (B) Dynamic covalent chemistries that have been utilized within hydrogels include imine (top) and thioester (bottom) bonds. (C) Dynamic noncovalent chemistries can also be used for the formation (or removal) of additional crosslinks or a secondary interpenetrating network within an existing hydrogel network to stiffen (or soften) the synthetic matrix, providing an opportunity to investigate the influence of mechanical cues on cell responses. (D) Guest−host interactions, such as that between adamantane and cyclodextrin, are a dynamic noncovalent chemistry that has been shown to allow for hydrogel stiffening and softening in vitro. (E) The difference in bulk hydrogel properties between bonds that respond locally versus bonds that respond broadly can be observed by monitoring the bulk modulus of a hydrogel upon application of a stimulus. Local forces on a bond promote local changes in the network structure without large observable changes in the bulk modulus of the matrix (blue line), whereas broad changes in the hydrogel network afford changes in the bulk modulus that can be observed (red line) through multiple switching cycles.

distance between crosslinks and thereby the crosslink density and mesh size of the hydrogel network, which are directly correlated with the swelling (e.g., Flory−Rehner)27 and mechanical properties of the matrix (e.g., rubber elasticity theory)28,29 (Figure 3). When using crosslinkers with dynamic chain confirmations, large differences in the distance between crosslinks in the extended vs collapsed states lead to larger changes in the modulus than observed with smaller differences. Material systems with both dynamic chemistries and dynamic crosslinker confirmations have been designed to use similar stimuli to induce changes in the mechanical properties of the hydrogel and, accordingly, offer advantages over how the alterations in properties are controlled for a specific application of interest. For example, materials that respond to solution conditions yield bulk control at time points of interest, whereas materials that respond to light often offer control over when and where properties are modified. In either case, the compatibility of the stimulus with the cellular system of interest and the time scale of property control relative to that of the biological response must be considered. In the sections below, recent advances in dynamic bonds and chain confirmations utilized to control the mechanical properties of hydrogels in the presence of encapsulated cells will be discussed. Additionally, we will briefly discuss chemistries that hold the potential for designing new dynamic hydrogelbased cell culture models. Dynamic Crosslinks. The use of dynamic covalent chemistries can permit the formation of hydrogels that enable the long-term study of cells in vitro often while maintaining the bulk mechanical properties of these matrices over time. This can be a difficult task as most synthetic hydrogel formulations incorporate crosslinkers that irreversibly degrade in water or in response to cell-secreted factors (e.g., enzymes or alkyl thiols

Figure 3. Reversible changes in mechanical properties: dynamic crosslinker confirmation. (A) In hydrogels formed with crosslinkers whose lengths can be controlled dynamically, the addition of a stimulus causes a change in the hydrogel crosslink density and mesh size, which in turn alters the hydrogel mechanical properties. (B) Chemistries used to create dynamic crosslinker confirmations within hydrogels include azobenzene moieties and disulfide bonds. (C) The change in hydrogel modulus, as observed by the bulk modulus, is related to the difference in the crosslinker confirmation and distance between crosslinks in the two different states, where small differences in distance between crosslinks (blue) in extended vs contracted state create small changes and large differences (red) create larger changes.

that exhibit a change in chain confirmation (e.g., extended or collapsed) upon the application of a stimulus (e.g., pH or ion concentration). The change in chain confirmation alters the 9

DOI: 10.1021/acsmacrolett.8b00808 ACS Macro Lett. 2019, 8, 7−16

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ACS Macro Letters (GSH)), permitting the encapsulated cells to grow, migrate, and rearrange their environment; while this approach and irreversible mechanism of matrix degradation is useful, robust, and often predictable, irreversible crosslink cleavage is directly linked to the mechanical properties of the matrix (e.g., upon crosslink cleavage, the modulus of the synthetic matrix decreases).8,13,21,30 A system that responds dynamically to forces applied by a cell to rearrange the network of a viscoelastic hydrogel is complementary to such approaches (Figure 2A). Further, recent reports have suggested the importance of considering not only the elastic nature of a hydrogel (e.g., Young’s modulus) but also its viscoelastic properties (e.g., stress relaxation) since the native ECM is viscoelastic in nature.31,32 One system that has achieved this task uses imine/hydrazone bonds: these bonds rapidly break and reform, and the equilibrium between “on” and “off” states can be shifted in response to cell-induced stresses within 3D culture (e.g., cells “pushing” or “pulling” on crosslinks containing these bonds) (Figure 2B). Typically, the nature of these bonds provides a hydrogel with consistent bulk mechanical properties but transient changes in the local mechanical properties in response to cells, permitting cell growth and motility. Additionally, the equilibrium or binding behavior needed to temporarily disrupt the crosslink can be altered by changing the chemical functional groups utilized to form the imine/hydrazone bond, as well as environmental conditions, offering control over how cells respond to the material system.14,15 While useful, this responsiveness of dynamic covalent chemistries to environmental conditions (e.g., pH and reductive potential) can also present challenges in cell culture applications, where a balance must be struck between hydrogel stability and bond dynamics and remains an active area of research in material design.33 Another exciting opportunity presented by hydrogels containing hydrazone bonds is the ability to 3D-print the hydrogels to create more intricate structures. For example, hyaluronic acid hydrogels incorporating hydrazone bonds were 3D-printed into multilayered lattices in the presence of cells using an extrusion methodology. Cells remained viable in these networks after extrusion and were active up to 14 days in culture, providing a mechanism to control not only cell function but also hydrogel structure using self-healing chemistries.34 These works highlight the implementation of imine/hydrazone bonds to allow cells to rearrange and migrate through hydrogels by exerting forces on the matrix; however, these systems generally do not afford user control over the system properties. To incorporate a higher level of user control into dynamic viscoelastic hydrogels, thioester and boronic acid hydrogels have been introduced. For example, a hydrogel system was reported whose viscoelastic properties were controlled by pH through the utilization of a thioester exchange (Figure 2B). At physiological pH, the system is at equilibrium, but by changing the pH or the stoichiometry of the system, this equilibrium can be shifted for modulating the viscoelastic properties of the synthetic matrix. Furthermore, since the bonds are dynamic, cells can exert traction forces on the matrix to rearrange and move through the matrix. To demonstrate this point, human mesenchymal stem cells (hMSCs) were cultured within static and thioester exchange hydrogels, and after 3 days in culture, the cells in the dynamic matrix exhibited elongated morphologies and increased proliferation compared to those in the static network.35

There have been several boronic-acid-based hydrogel systems recently reported for 3D cell culture, where the selection of the boronic acid and diol groups and the valency of the polymer can be used to control the rate of dynamic exchange and hydrogel stability in culture conditions. These systems highlight the biocompatibility of boronic-acid-based chemistries with various cell types and the utility of these chemistries for designing self-healing and fast-relaxing synthetic matrices for 3D cell culture. In one example, the self-healing capabilities of the boronic acid hydrogels, formed using polymers with high valency, were exploited to create dynamic cocultures of complementary cell types (e.g., human breast cancer cells and fibroblasts): hydrogels initially containing only one cell type (either fibroblasts or breast cancer cells) were cut in half, and the opposite halves were healed together, creating a layered hydrogel with two different cell types. Within this dynamic coculture hydrogel, both cell types were observed to migrate across the healed interface into regions initially populated by only the opposite cell type.36 In another example, lower valency boronic acid hydrogels were stabilized with nondynamic bonds to afford control over the hydrogel modulus and stress relaxation while maintaining the dynamic nature of these matrices to permit cell spreading in 3D culture. When hMSCs were encapsulated within the stressrelaxing hydrogels, they exhibited increased spreading and nuclear volume compared to those cultured in a purely elastic hydrogel (i.e., no dynamic covalent crosslinks), demonstrating the utility of dynamic covalent chemistries in controlling cellular response.37 Boronic-acid-based hydrogels have also been designed to respond to glucose, where the dynamic nature of the material is maintained until the concentration of glucose in the surrounding media is sufficiently increased to cause irreversible dissolution of the network.38 Using a responsive chemistry allows for triggered and complete erosion of the hydrogel, providing a useful handle for harvesting cells or proteins from the synthetic matrix for genomic or proteomic studies. Overall, the work to date investigating cell response within dynamic viscoelastic hydrogels has been foundational in establishing the relevance of these systems for 3D cell culture and now presents opportunities for studying cell responses with these materials as well as integration of these chemistries with other existing approaches (e.g., irreversible covalent reactions). Materials systems also are needed that can cycle between “soft” and “stiff” states to mimic the mechanical property changes that occur in the matrix during tissue remodeling processes (e.g., injury and regeneration, disease progression, and regression) (Figure 2C). For this, materials that respond to solution conditions are often utilized as they provide a facile handle for the user to alter the mechanical properties of the synthetic matrix by changing the pH,39 oxidative state,40 temperature,41 or glucose concentration42 of the medium in which the hydrogel is incubated. A bioorthogonal approach that was recently introduced for cyclically controlling mechanical properties is the host−guest interaction between β-cyclodextrin (βCD) and adamantane (AD) (Figure 2D).18,43 In one example, a covalent hydrogel was formed with βCD attached along the polymer backbone, which yielded a handle to stiffen or soften the matrix through the addition of four-arm PEG-AD or free βCD, respectively. Using this system, insulin gene expression of model pancreatic beta cells (MIN6) could be controlled by the stiffness of the hydrogel.43 This study demonstrates the promise of such solution-based approaches 10

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containing disulfide bonds could be designed for control over the formation of dynamic high-ordered structures. Spatially Defined Dynamic Crosslinkers. To gain more exquisite control over the hydrogel properties, light-responsive systems have been developed that allow not only temporal control over the systems properties but also spatial control. The most common photoresponsive molecule used for lightinduced cyclic property changes within 3D cell culture matrices is azobenzene, which undergoes conformational changes upon irradiation with complementary wavelengths of light. Specifically, the azobenzene moiety undergoes a cis to trans isomerization based on irradiation wavelength: irradiation with long wavelength UV induces the cis isomer and irradiation with visible light induces the trans isomer (Figure 3B). Azobenzene has been utilized to control hydrogel mechanical properties through both dynamic noncovalent interactions and crosslinker confirmation. For example, using guest−host interactions between βCD and azobenzene, hyaluronic acid hydrogels were designed that could switch between “stiff” and “soft” states based on the isomerization of azobenzene, which alters the guest−host strength, upon light irradiation.48 For controlling mechanical properties through the crosslinker confirmation, synthetic hydrogels containing azobenzene within the crosslinks were developed that reversibly softened or stiffened upon irradiation with 365 or 420 nm light, respectively. In this work, the amplitude of change between the different mechanical states was shown to be correlated with the concentration of azobenzene within the hydrogel.20 Both systems demonstrated good viability of encapsulated cells, supporting their potential implementation in developing in vitro tissue models. The ability to control not only when but also where stiffening or softening of a hydrogel occurs could allow for studies of cells being exposed to different physical microenvironments within the same culture system. For example, the mechanical properties across a hydrogel could be altered to investigate how varying stiffness alters cell morphology, migration, and protein expression, as has been previously studied unidirectionally with systems that yield irreversible stiffening or softening.49,50 Overall, these bodies of work highlight some of the exciting motifs for dynamically altering the mechanical properties of hydrogel-based matrices and their use for investigating how the mechanical properties of a system influence cell behavior, where potential opportunities have been noted for integrating new chemical handles within these frameworks. Cyclic Biochemical Properties. Similar to physical cues, biochemical cues found within the microenvironment of human tissues are constantly changing to maintain tissue homeostasis or for tissue remodeling during development, regeneration, and disease. Approaches have been introduced that permit the addition or removal of biochemical cues from hydrogels during cell culture through the application of stimuli toward studying such processes in vitro; however, these motifs are typically a “one and done” setup and often do not offer the opportunity to modify the biochemical makeup of the system cyclically. The chemistries introduced to impart a dynamic biochemical environment in hydrogels are similar in both stimulus and time scale to those utilized to facilitate mechanical changes. For example, cell response to the addition or removal of biochemical cues has been investigated in hydrogels whose biochemical composition has been altered temporally in bulk51 or in spatially defined regions.52 Bulk hydrogel modification offers the user the opportunity to

for modulating the mechanical properties of synthetic matrices to better understand not only the effect on cell behavior but also the time scale at which such responses are observed. Several chemical approaches also exist for temporal tuning of hydrogel properties but have yet to be evaluated in the presence of cells, providing opportunities for future investigations. For example, hydrogels have been formed using selfcomplementary and cytosine-rich nucleic acid sequences: the cytosine-rich sequences formed an i-motif that was sensitive to changes in pH, whereas the self-complementary sequences formed hydrogel crosslinks that were insensitive to pH. Combinations of these domains were used to create shape memory hydrogels that could cycle between “soft” and “stiff” states based on the pH-controlled i-motif structure of the cytosine-rich regions.39 To date, this system has not been demonstrated with cells, perhaps owing to the low pH that is needed to regenerate the i-motif confirmation that induces a “stiff” hydrogel matrix. However, such an approach offers the ability to cycle between “soft” and “stiff” hydrogels, where physiologically relevant pH regimes could be achieved by altering the nucleic acid sequence of the i-motif region or designing i-motif regions that respond to metal ions or aptamer complexes. Similarly, histamine-functionalized triblock copolymers have been synthesized that form either bulk hydrogels or micelles based on the polymer concentration and the pH of the system. Slight variations in the pH caused the hydrogel formulation to soften due to the protonation of the histamine and hydrophilic/hydrophobic interactions of the triblock copolymer backbone.44 While this system has not been tested for tissue culture applications, it has been shown to be biocompatible, and the pH values needed to alter the network are physiologically relevant, suggesting its potential utility in both cell and drug delivery applications. Additionally, this system can be completely degraded under slightly acidic conditions, yielding a facile method to release cells from the hydrogel for downstream assays or reseeding. Overall, solutionbased motifs are attractive for cycling hydrogels containing dynamic bonds between “soft” and “stiff” states to investigate a variety of cellular processes. Dynamic Crosslinker Confirmation. As an alternative to hydrogels containing dynamic bonds, crosslinkers that change chain confirmation based on a stimulus can also be used to modify the crosslink density and mesh size of the hydrogel network, thus producing a reversible change in the hydrogel mechanical properties (Figure 3A). In one example, conformational changes produced by the folding and unfolding of engineered proteins, based on the oxidative state of disulfide bonds, were utilized to control the effective length of proteinbased crosslinkers and thereby the mechanical properties of the hydrogel (Figure 3B).40,45 Use of these protein-based systems that respond to alterations in the solution conditions could be expanded for controlling protein stress relaxation or binding by altering the protein sequence, where many proteins are sensitive to single amino acid substitutions.45 There are few reports to date that implement protein-based hydrogels with dynamic crosslinks for 3D cell culture; however, there have been several examples of cell encapsulation within protein and peptide hydrogels, demonstrating biocompatibility and indicating the potential ease of employing dynamic proteins systems to study disease or regenerative processes.46,47 Further, similar approaches could be implemented within synthetic polymerbased systems, where macromers or block copolymers 11

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Figure 4. Tuning biochemical presentation within hydrogels. Hydrogels can be modified in either a bulk (left) or patterned (right) manner based upon the chemistry selected to control biochemical cue presentation within the matrix. Both techniques offer temporal control over the system, though patterned methods permit the user the additional benefit of being spatially controlled.

investigate responses of the entire cell population encapsulated within the hydrogel, whereas patterned modification affords opportunities to investigate single cell or subpopulation responses (Figure 4). For modification of the biochemical content within hydrogel-based matrices, cyclic (Figure 5A) and sequential (Figure 6A) modification techniques have been established.

Cyclic modification allows the removal and addition of biomolecules at multiple times, analogous to the dynamic bond motifs discussed for changing the mechanical properties of synthetic matrices. Generally, this mechanism harnesses changes in the local microenvironment, such as the oxidative state, protein composition, or pH, to induce compositional changes in the hydrogel. An interesting opportunity afforded by cyclic biochemical modification of hydrogels is the ability to either cycle the presentation of one cue or cycle between multiple cues for investigating how the timing and order of biochemical moiety presentation alters cell functions. Sequential modification occurs when a biomolecule is added and subsequently removed (or vice versa). Typically, sequential modification is achieved using external stimuli, such as light or temperature, to both modify and pattern the chemical presentation within the system. Both cyclic and sequential methods have been successfully demonstrated within hydrogelbased cell culture models and will be discussed below. Cyclic Modification with Biochemical Cues. To change the overall biochemical composition of a hydrogel cyclically, techniques that utilize enzyme-responsive motifs have been introduced that enable bulk modification of the hydrogel. For example, systems have been designed that enable the simultaneous addition and removal of biomolecules upon the application of enzymes to catalyze specific reactions. One exciting system employs the transpeptidase Sortase A, an enzyme found in Gram-positive bacteria, to covalently attach any active biomolecule with a GGG motif to molecules containing a LPXTG motif: specifically, the Sortase A enzyme recognizes the amino acid sequence LPXTG, cleaving the amide bond between threonine and glycine residues and subsequently attaching GGG-presenting molecules. The LPXTG peptide has successfully been incorporated into PEG hydrogels and applied to remove human epidermal growth

Figure 5. Reversible changes in biochemical presentation: dynamic modification. (A) Dynamic chemistries can be employed to alter the presentation of biochemical cues within hydrogels for examining the impact of the chemical composition of the microenvironment on cell response. (B) For example, dynamic covalent chemistries, such as allyl-sulfide containing crosslinks, have been used to modulate the presentation of biochemical cues within a hydrogel, using light and thiol-functionalized peptides, providing spatiotemporal control over the molecular composition of the hydrogel. (C) Alternatively, noncovalent chemistries, such as guest−host interactions between β-cyclodextrin and azobenzene, have been utilized to modulate biochemical cue presentation, using light to cause dissociation between the two.

Figure 6. Sequential changes in biochemical presentation. (A) The ability to both add and remove biochemical cues from hydrogels is advantageous for investigating how cells respond to modifications in the microenvironment surrounding them. (B) An example of a light-based chemistry used for spatially defined patterning of hydrogels is shown. Here, the photouncaging of an alkoxyamine functional handle allows for the addition of an aldehyde-functionalized photocleavable molecule that can be subsequently removed through further light irradiation. 12

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over the biochemical environment can be powerful tools for studying how the presentation of different cues alters the phenotype, morphology, and migration of cells. As discussed previously, light-triggered chemistries are of particular interest as they allow spatially defined patterning of hydrogels with precision and often under biocompatible conditions. Nitrobenzyl (NB) groups have been utilized in the biomaterials community to pattern biochemical cues within hydrogels, as many of these groups degrade in response to low, cytocompatible doses of light.56 Additionally, NB groups can be easily modified for use as photocages or as photolabile linkers for incorporation into hydrogels formed by several different crosslinking strategies. For example, one investigation implemented a three-step process for adding and subsequently removing proteins from hydrogels by incorporating combinations of nitrobenzyl groups that degrade in response to light and utilizing oxime ligations between alkoxyamines and aldehydes (Figure 6B). Using photolithographic techniques and varying the light exposure conditions, multiple proteins were patterned into a single hydrogel.26 In another example of sequential modification, hydrogels were photopatterned with aptamers that afforded specific binding to proteins of interest; the proteins then were released from the system at desired time points through the addition of complementary DNA strands that hybridized to the aptamer and displaced the protein.19 While not demonstrated in the presence of cells to date, one could imagine this system being translated to a 3D culture model for introducing and subsequently removing biochemical cues tagged with DNA sequences complementary to aptamers that were patterned into the synthetic matrix. The main limitation to using a sequential patterning mechanism is the ability to change the hydrogel chemical composition a limited number of times (i.e., until all functional handles have been consumed); however, depending on the model system being developed and the biological questions being asked, sequential patterning may provide a variety of options for reconfiguring the hydrogel environment and enable investigations into different cellular responses. Concluding Thoughts. The past decade has seen an increase in the number of reversible chemistries used to form both natural and synthetic hydrogel cell culture platforms. These systems have helped to improve our understanding of biological processes and led to a growing need for in vitro cell culture systems that are multidimensional and dynamic in nature. To address this, the biomaterials community has established several approaches for creating hydrogel-based synthetic matrices that permit cycling between different moduli or biomolecules based on the application of an external stimulus. In this Viewpoint, we have specifically highlighted several chemistries that offer the user cyclic, if not dynamic, control over the biophysical or biochemical microenvironment within synthetic matrices. A few of the chemistries discussed have yet to be demonstrated in the presence of cells but offer exciting possibilities for integration within hydrogels for 3D cell culture. Ultimately, this growing chemical toolbox nicely allows for the selection of one or more chemical handles toward designing a hydrogel cell culture platform with property control across different levels of complexity (e.g., over long or short times, single cells to cell populations, micro- to macroscale) to investigate biological responses. The ability to cycle between material properties holds the potential for dissecting complex systems to aid in elucidating underlying biological mechanisms, such as the role of matrix

factor efficiently and without any deleterious modifications that would impact bioactivity.51 Since the substrate used here is specific to bacteria, this motif can be utilized without fear of altering the surface of mammalian cells, which provides interesting opportunities to dynamically change the biochemical environment of mammalian cells during both long- and short-term 3D cell cultures. While not yet shown within a synthetic matrix, this chemistry is reversible and repeatable over several cycles, providing the user with handles for adding and removing biomolecules from the system. Due to the ease of use and implementation, light is a common stimulus exploited to cyclically modify the biochemical cues being presented within a hydrogel network in both a bulk and patterned format. For example, hydrogels containing allyl-sulfide crosslinks can undergo a radical addition−fragmentation chain transfer (RAFT) reaction with thiol-functionalized molecules upon exposure to free radicals (e.g., light and photoinitiator) (Figure 5B). Upon exposure, the allyl-sulfide crosslinks undergo a thiol exchange reaction that can be utilized to change the biochemical moieties presented within the network (e.g., integrin-binding peptides). Furthermore, under the conditions tested, this system is biocompatible, suggesting its potential as a dynamic matrix for cell culture applications.53,54 An alternative approach is to use azobenzene-containing molecules that switch between an active and inactive state based on the azobenzene conformation (Figure 5C). Specifically, cyclodextrin-modified surfaces have been functionalized with azobenzene-bound antibodies, and cell interactions with the surface were controlled by the azobenzene conformation. Cells could interact with the antibodies until the surface was irradiated with 365 nm light, causing isomerization of the azobenzene that altered the strength of the guest-host interaction, releasing the antibody-bound cells from the surface.55 While this system has only been shown in 2D cell culture, this chemistry is also well suited for modulation of biochemical cues within 3D cell culture systems, as it has already been employed within 3D cell cultures for controlling hydrogel mechanical properties.43 In addition to light, systems can be designed to respond to other stimuli, such as temperature, for achieving cyclic and spatially patterned presentation of biochemical cues. In an elegant example, PNIPAM hydrogel films were formed that could swell and deswell based on the temperature of the system. When in the swollen state, parts of the hydrogel surface were “hidden”, allowing for different surface modifications to be applied and presented in the hydrogel within the swollen and unswollen states. To demonstrate the potential of this system for cell culture, the entire hydrogel surface was functionalized with an adhesive peptide, and by changing the temperature of the system, adhered epithelial cells were cycled between 2D culture in the nonswollen hydrogel state and 3D culture in the swollen hydrogel state.17 While this temperatureresponsive hydrogel system has not been used to study the effects of dynamic biochemical cue patterning on cells, the surface structure could provide an opportunity to cyclically change the presentation of biomolecules without the need to directly add or remove the biomolecules of interest from the hydrogel surface. As a whole, there have been a variety of exciting chemistries introduced to modify the presentation of peptides or proteins within hydrogels utilizing cyclic bulk or patterned motifs. Sequential Addition of Biochemical Cues. Hydrogel systems that provide the user with spatial and temporal control 13

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chemotherapeutic-resistant tumors found in vivo.61 The tunability of this platform introduces the potential for future investigations of cancer progression and therapeutic screening. While not shown here, the Diels−Alder reaction is also reversible based on the temperature of the system, which could be used to modulate the presentation of biochemical cues in the system.62 Additionally, recent reports have introduced sortase-sensitive hydrogels63 and calcium or light-sensitive engineered protein−polymer hydrogels64 to dynamically control the hydrogel modulus, offering additional handles for orthogonal property control. Combining these protein-based property modulation strategies with the suite of available orthogonal chemical handles provides opportunities to develop new and exciting methodologies for studying complex biological environments. Broadly, when selecting orthogonal chemistries for use in the design of hydrogels that are dynamically reconfigurable for studying complex biological phenomena, the main constraints are whether the associated reactions or reconfigurations can be performed in the presence of cells (e.g., cytocompatible processes for hydrogel formation and property modulation). The “ideal” design for such dynamic 3D culture systems will depend on the specific application(s) where observations of how these biological processes occur naturally (e.g., size and time scales of native microenvironment changes) can be used to guide the design criteria for the synthetic matrix (e.g., range of moduli and the concentration and type of biochemical cues). Further, key parameter ranges that have been identified in material systems with unidirectional property control, such as “soft” and “stiff” moduli for controlling the “stem-ness” or differentiation of MSCs, may provide targets for cyclic property changes, as well as benchmarks for potential cell responses.49,50 Overall, by combining different dynamic chemistries, increased control over hydrogel properties can be achieved with the potential for more in-depth investigations into how combinations of critical extracellular cues influence a variety of cellular responses. This Viewpoint has highlighted recent work in the design of dynamic hydrogels utilizing chemistries that can undergo cyclic or dynamic changes upon exposure to physiologically relevant stimuli. There are a suite of dynamic chemistries that can be incorporated within a hydrogel network for cyclic, temporal, and spatial control over matrix properties for investigating cell responses to microenvironmental changes. The hydrogel systems developed to date, as well as the array of new dynamic chemistries being developed across fields, have opened the door to new and exciting opportunities to answer fundamental questions about progressive diseases and regenerative processes, with the potential for creating more predictive model systems for the evaluation and design of new therapeutic strategies.

remodeling in a variety of processes from healing after acute injuries to progressive disease states. For example, by cycling a hydrogel between “soft” and “stiff” states, we may be able to probe regulators of fibroblast activation and persistence in fibrosis, cancer cell phenotypic switching in metastasis and recurrence, or stem cell migration and differentiation in healthy vs chronic wound sites. By combining the results obtained from these platforms with transcriptomic, proteomic, and advanced and real-time imaging technologies, we can begin to further understand how changes in microenvironment properties influence cell phenotype and motility. Additionally, alterations to a system’s mechanical properties not only when but where desired could provide opportunities to study cells at an interface between “soft” and “stiff” microenvironments and to learn how competing factors (e.g., matrix degradation vs. mechanotransduction and durotaxis) influence cell behavior. Alternatively, cyclic control over the biochemical cues presented in cell culture platforms affords the opportunity to better mimic the transient and heterogeneous nature of the biomolecules present in the ECM. The native microenvironment of cells in the body is further complicated by the everchanging biochemical cues (e.g., adherens junctions, growth factors, cytokines) delivered by the myriad of different cell types that reside within specific tissues. The addition and removal of these biochemical cues, or mimics of them, can be used to simulate some of this complexity without the need for direct coculture of different cell types and to provide the ability to investigate the effect of one biochemical cue independent of others.51,57 Similar to controlling the mechanical properties of a system, controlling the biochemical cues can lend us the ability to examine cell responses to changes in either the presentation or concentration of cytokines, enzymes, cadherins, or other physiologically relevant proteins. As the questions we are able to ask become more elaborate, the ability to combine several orthogonal dynamic chemistries into one system could allow for changes in both the mechanical and chemical properties within a single hydrogel system, in concert or independently.58 For example, four dynamic chemistries, boronic ester exchange, thiol addition to a conjugate acceptor, hydrazone exchange, and terpyridine− zinc complexation, have been shown to be orthogonal by controlling the exchange partner added to the system.59 While not all four of these dynamic chemistries have been shown to be biocompatible, the boronic ester exchange and hydrazone exchange reactions have been used for cell culture, signifying the potential utility of these chemistries in orthogonally controlling the mechanical and chemical properties of hydrogel-based 3D culture systems.14,37 Indeed, several investigations have developed hydrogel systems that allow for changes in both the mechanical and biochemical properties to begin asking complex questions about how these properties interact to influence cell response.52,60,61 As an example, recent work has highlighted the usefulness of bioorthogonal chemistries for controlling the biochemical and mechanical properties of hydrogels in the 3D culture of breast cancer spheroids. An oxime ligation mechanism was utilized to form dynamic hydrogels that contained free furan groups that subsequently could be reacted with a maleimide through a Diels−Alder reaction to add in biochemical cues at a desired time point. The properties of the synthetic matrix were tailored utilizing this system to achieve spheroids that responded to drugs in the same manner as

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

April M. Kloxin: 0000-0002-4594-2953 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 14

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ACS Macro Letters Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge support from the Delaware COBRE programs (P20GM104316 and 5 P30 GM110758-02), the PEW Charitable Trusts (00026178), and a National Science Foundation (NSF) CAREER Award (DMR-1253906).

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