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Moving from static to dynamic complexity in hydrogel design

Moving from static to dynamic complexity in hydrogel design REVIEW Received 25 Apr 2012 | Accepted 8 Nov 2012 | Published 11 Dec 2012 DOI: 10.1038/ncomms2271 Moving from static to dynamic complexity in hydrogel design 1 2 Jason A. Burdick & William L. Murphy Hydrogels are water-swollen polymer networks that have found a range of applications from biological scaffolds to contact lenses. Historically, their design has consisted primarily of static systems and those that exhibit simple degradation. However, advances in polymer synthesis and processing have led to a new generation of dynamic systems that are capable of responding to artificial triggers and biological signals with spatial precision. These systems will open up new possibilities for the use of hydrogels as model biological structures and in tissue regeneration. ydrogels are water-swollen polymer networks that have been used for many decades, with applications as varied as contact lenses and super-absorbant materials. As the field Hof biomedical engineering has developed, hydrogels have become a prime candidate for application as molecule delivery vehicles and as carriers for cells in tissue engineering, owing to their ability to mimic many aspects of the native cellular environment (for example, high water content, mechanical properties that match soft tissues). Traditional hydrogels, formed through the covalent and non-covalent crosslinking of polymer chains, were regarded as relatively inert materials, providing a simple biomimetic three-dimensional (3D) environment, either for tissue production by local resident cells or for positioning of cells delivered in vivo. However, the simplicity of these materials may have in fact hindered their application, restricting cellular interactions with the environment and preventing uniform extracellular matrix (ECM) production and proper tissue development. In addition, these materials were limited to modelling static environments and lacked the spatiotemporal dynamic properties relevant for complex tissue processes. Fortunately, during the last decade the concepts of hydrogel design and cellular interaction have evolved, shedding light on how they may control cell behaviour, particularly for tissue engineering applications. Hydrogels with a range of mechanical properties, and capable of incorporating a wide range of biologically relevant molecules, from individual functional groups to multidomain proteins, are currently in development . In addition, hydrogels are being designed with spatial heterogeneity, to either replicate properties in native tissue structures or to produce constructs with distinct regionally specific cell behavior . As a result, studies to date have clearly demonstrated the possibility of creating well-defined microenvironments with control over the 3D presentation of signals to cells . However, recently there has been a focus on the concept of hydrogels that exhibit dynamic complexity. These materials should evolve with time and in response to 1 2 Department of Bioengineering, University of Pennsylvania, 240 Skirkanich Hall, 210 S. 33rd street, Philadelphia, Pennsylvania 19104, USA. Departments of Biomedical Engineering and Orthopedics and Rehabilitation, University of Wisconsin, Madison, Wisconsin 53706, USA. Correspondence should be addressed to J.A.B. (email: [email protected]). NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications 1 & 2012 Macmillan Publishers Limited. All rights reserved. REVIEW NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2271 user-defined triggers or cellular behaviour. Indeed, a cadre of However, the complex combination of signals present in the recent examples demonstrates that hydrogels can be used as well- cellular microenvironment may be mimicked by patterned defined static platforms for presenting signals to cells, and hydrogel systems. Such advances allow the introduction of furthermore as dynamic evolving environments. It is too soon to spatially specific cues in hydrogels, making multicellular con- tell how closely these materials mimic the dynamic complexity structs, either through co-cultures or multilineage differentiation, 16–18 of the cell/hydrogel interface, or that of multicellular constructs a possibility . Spatial patterning of hydrogels also provides or cell condensates, or to what extent dynamic hydrogels an additional tool for the development of high-throughput can regulate emergent biological processes such as tissue screening technology for the rapid investigation of cell–material 19–22 development. However, it is clear that increased material interactions . High-throughput techniques may be complexity is beginning to address such questions. particularly important as cell fate is simultaneously influenced The objective of this review is to highlight the evolution of by cell–cell interactions, soluble signals, cell–ECM interactions, hydrogel design towards dynamic behaviour. We first consider cell shape and mechanical forces, and natural ECMs often present stable and patterned hydrogels, then hydrogels that undergo cells with many distinct signals simultaneously . Therefore, it is either hydrolytic or proteolytic degradation, and finally describe logical to expect that spatially patterned synthetic hydrogels will hydrogels with trigger-responsive properties. We particularly be needed to effectively probe the simultaneous influence of many emphasize recent developments in hydrogel design that offer the distinct signals in order to gain meaningful biological insights or ability to precisely control cell–microenvironment interactions, to direct cellular behaviour. such as those found in cellular processes. Novel chemistry, and the application of orthogonal reactions to networks, now allows the unprecedented patterning of peptides and proteins or the alteration of network structure, even in the Static hydrogels that mimic biophysical cues presence of cells. For example, multiphoton processes were used We are constantly improving our understanding of biochemical to decorate biological moieties throughout 3D systems, relying on and physicochemical signals in the local cellular microenviron- the coupling of acrylate-modified molecules in proteolytically ment, and their role in cellular signalling. This has been difficult degradable polyethylene glycol (PEG) gels containing excess for natural materials, where many cues (for example, adhesive reactive groups . A subsequent study illustrated the use of this and mechanical) are coupled, yet synthetic hydrogels have technique to define regions of precise concentration and to provided a well-defined platform for experimentation. A variety pattern with multiple molecules throughout PEG hydrogels , of biochemical signals have been explored upon or within further expanding their complexity. In another example, hydrogels, taking advantage of hydrogels as ‘blank slates’ that can orthogonal binding pairs of barnase–barstar and streptavidin– 4 5–7 be decorated with cell-interactive , or growth factor-binding , biotin were applied sequentially to pattern multiple growth ligands. Hydrogels are also useful because their physical factors and biological molecules (Fig. 2) . Specifically, one properties are biomimetic, that is, they can be designed with component of the pair was first immobilized into the hydrogel stiffness ranges and topographical features that are analogous to using two-photon chemistry and the corresponding binding natural extracellular environments . As an example, 2D partner was subsequently introduced. By applying different polyacrylamide gels with tunable mechanical properties have binding pairs in sequence, multiple factors could be provided a platform for investigating mechanotransduction immobilized. This approach follows a previously developed 9,10 behavior , suggesting that stem cell fate is strongly influenced method where a tethered photolabile moiety was illuminated to by the mechanics of the target tissue . introduce cell adhesion molecules and guide cell migration . Additional related studies have emphasized the critical role of Light has also been used to introduce patterned channels that hydrogel stiffness in skeletal muscle stem cell self-renewal , permit the migration of cells through a hydrogel . 13 14 neural stem cell behavior and megakaryocyte poiesis .3D Other systems have been developed where orthogonal and mechanical properties are similarly important in controlling adult cytocompatible click chemistry can be used to first form, and then stem cell fate in alginate hydrogels incorporating adhesive pattern, hydrogels . For example, copper-free click chemistry ligands . In this case, hydrogel mechanics alter the ability of a was used to form hydrogels around cells using the reaction of a cell to cluster tethered ligands and exert tension, leading to PEG tetra-azide and a bis(DIFO3) di-functionalized polypeptide different fate decisions (for example, adipogenic fate in softer gels (containing a proteolytic degradable group), then an orthogonal and osteogenic fate in stiffer gels). Several further examples also thiol-ene photocoupling reaction was used to modify the gels demonstrate that 3D hydrogels support the growth of a wide with a peptide. The sequential reactions and spatial patterning variety of tissues ; however, the synthesized tissue properties are permitted the gels to be controllably remodelled by cells (Fig. 2). often inferior to native tissue, for a range of reasons possibly A subsequent study combined the click chemistry with a related to the simplicity of these basic designs. photocleavable functionality, to fabricate spatially patterned These studies illustrate that static hydrogels are a useful, well- hydrogels and then to enable 3D cleavage of crosslinks . defined platform to efficiently probe how cell behaviour is Finally, a sequential crosslinking process was used to pattern influenced by the microenvironment, or by defined physico- cell spreading and remodelling by first encapsulating cells in a chemical and biological inputs. However, biological systems are hydrogel permissive to cell remodelling and then by introducing rarely static and homogeneous, and there is a wealth of biology photo-induced crosslinks. The cells were unable to completely and a cadre of intriguing hypotheses to be addressed with degrade the network, leading to changes in local cell spreading increasing complexity in hydrogel design. and subsequent stem cell differentiation. Introducing spatial heterogeneity into hydrogels Many biological processes are heterogeneous and material design Hydrogels that degrade with time is evolving to emulate them. An example of wound healing from Although stable and patterned hydrogels have been very useful as the perspective of traditional static versus emerging hydrogels is a means of studying fundamental cell–material interactions, they illustrated in Fig. 1. Wound healing is a complex process that over-simplify the complexity of the temporal dynamics present involves spatial and temporal heterogeneity of ECM signals and is during both tissue development and repair processes. Specifically, not recapitulated by static and uniform hydrogel systems. the regulatory cascades during tissue formation and repair are 2 NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications & 2012 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2271 REVIEW Wound healing cascade Inflammation Proliferation Remodelling Traditional hydrogels X X Wound Block cells/proteins Degrade? Erode? Emerging hydrogels Dermis Fat Sequester Respond Induce recruit deliver align Figure 1 | Introducing the complexity of biological processes into hydrogels. Hydrogels are evolving from relatively inert and static materials to those incorporating sophisticated feedback mechanisms. In this theoretical example, hydrogel design is targeted to key steps in the wound healing process, where cellular recruitment, infiltration and organization can be mediated by specific cues introduced into the hydrogel design such as the ability to sequester molecules, respond to cellular signals such as degrading enzymes and organize forming tissue. Generally, hydrogels are increasing in complexity and getting better at targeting specific cascades of biochemical signals and biophysical properties. ab Two-photon irradiation maleimide-barnase Wash Two-photon irradiation Maleimide-streptavidin Wash Barstar-SHH Biotin-CNTF Wash Figure 2 | Patterning biological cues into hydrogels. Techniques are being developed to introduce spatial control of biological cues (for example, ligands and crosslink density) into 3D hydrogels. (a) Sequential introduction of orthogonal binding pairs of barnase–barstar and streptavidin–biotin through two- photon binding of one component of the pairs (that is, barnase or streptavidin) and introduction of molecules bound to the other component (that is, barstar of biotin) to permit the coupling of multiple molecules with precise 3D patterns. Inset image illustrates this approach with fluorescent molecules. (b) Coupling of photocleavable functionality and patterning, via the formation of gels with a click reaction, and then either patterning or photocleaving crosslinks with targeted wavelength light. Examples illustrate directed migration of fibroblasts from a fibrin clot (scale bar, 100 mm). extremely complex and while ‘static’ hydrogels feature some The simplest approach to introduce temporal changes in dynamic components (for example, reversible receptor–ligand hydrogels has been via control of hydrogel degradation, primarily binding and dynamic cell–cell interactions), little has been done to release growth factors and to eliminate the permanent to intentionally manipulate temporal properties in hydrogels, implantation of a material . One of the earliest examples of a potentially owing to a lack of specific enabling technologies. degradable hydrogel features chains of poly(a-hydroxy esters), However, the intrinsic properties of hydrogels lend themselves to such as poly(lactic acid), incorporated at the ends of PEG dynamic physical and biochemical applications, including molecules before the introduction of reactive groups for responsiveness to changes in the signalling environment. There- crosslinking . Other examples include materials based on 34 35 36 fore, the interface between dynamic biological systems and poly(vinyl alcohol) , poly(propylene fumarates) , dextrans hydrogel material science provides an exciting area for future and hyaluronic acid . In all examples, the crosslinking material hydrogel design. changes with time, altering the physical properties and diffusivity NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications 3 & 2012 Macmillan Publishers Limited. All rights reserved. REVIEW NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2271 In an alternative mechanism, hydrogels are also designed to be susceptible to degradation by the proteases used by cells to Low- High- Δt remodel their surroundings; specifically, peptides that may be crosslink crosslink density density cleaved by cell-produced proteases are incorporated into the Hydrolysis 46,47 hydrogel crosslinks . A wide range of sequences were investigated that are degraded by matrix metalloproteinases 2,48,49 (MMPs), elastases and plasmin . The general susceptibility to proteases is controlled by the specific peptide sequence and there are a plethora of sequences that could be used to tailor the specific cell-mediated degradation of hydrogels (Fig. 3). Generally, the hydrogels are formed by reacting a multifunctional polymer (for example, PEG macromers with vinyl sulphones or acrylates) with end groups of protease- ↑ Swelling, diffusivity Polymer chain sensitive peptides (for example, thiols from cysteine moieties), Polymer crosslink ↓ Modulus, crosslink density where cells and molecules can be encapsulated during gelation. The remodelling of hydrogels by cells was observed for hydrogels where both sequences for adhesion and degradation were present , with degradation rates controlled by crosslink density and peptide specificity . This approach has been used in numerous cases to permit cell-mediated degradation of hydrogels, leading to engineered constructs for tissues such as 49,52 bone and vascular structures , including examples of the incorporation of growth factors released via cellular cues . Likewise, these matrices are useful as cell carriers that permit 46,54 cellular morphogenesis into a variety of tissue structures . Hybrid systems of synthetic and biological polymers including 28 55 PEG fibrinogen and hyaluronic acid have also been developed with similar functionality. Polymer chain This approach can also be harnessed to control the delivery of MMP-cleavable crosslink molecules in diseases where protease activity is altered. Some Adhesive peptide 56 57 examples of this include rheumatoid arthritis , cancer and Figure 3 | Hydrolytically and enzymatically degradable hydrogels with 58 after myocardial infarction . In these cases, MMP levels deviate properties that change with time. (a) Hydrolysis occurs relatively from equilibrium after injury. Thus, a system that exhibits MMP homogeneously throughout hydrolytically degradable hydrogels and leads sensitivity may degrade differently depending on local MMP to changes in the overall network crosslink density with time, which levels. This provides a feedback approach where local molecule can influence properties such as diffusivity, swelling and mechanics. delivery is regulated by cellular activity, increasing the available (b) Enzymatic degradation involves local cell-mediated proteases and is intricacy of release profiles. Likewise, MMP-sensitive tethers have used to control cellular infiltration into the hydrogel as long as adhesive been used for release that correlates with increased MMP cues are also present. 59,60 activity . In these cases, a pendant drug is released from matrices in response to local protease levels. These examples represent an initial foray into biological responsiveness, in the sense that hydrogels change their properties in response to a and eventually completely degrading away (Fig. 3). Likewise, non- specific set of biological stimuli. covalently crosslinked hydrogels (for example, via ionic Beyond purely synthetic materials, the development of protein- interactions), such as alginate, can be manipulated to alter based materials is also becoming a possibility. In this case, dissociation kinetics through changes in polymer chemistry . molecular engineering allows for the precise introduction of a One area in which degradation has been particularly useful is range of signal response mechanisms to control hydrogel in controlling the distribution of ECM molecules by encapsulated properties (for example, via crosslinking) or cellular interactions 37 61 cells . In tissue engineering, it is important that these molecules (for example, via degradation sites) . This area is likely to are well distributed, to build up a tissue that can replicate both the expand in upcoming years, as the need for the complexity found form and function of native tissues. In cartilage tissue engineering in native tissues is realized. Approaches such as directed evolution 39 37 approaches (with both PEG and hyaluronic acid hydrogels), will allow the rapid assessment of sequences with specific the inclusion of fractions of hydrolytically degradable functionality. As an alternative to covalently crosslinked components has improved this distribution and led to structures, hydrogels may be formed via the self-assembly of improved properties, particularly functional mechanical peptides designed to form fibrillar structures or even to present properties. Likewise, simple hydrolytic degradation mechanisms biological signals . The same cues for adhesion and degradation have also hinted at the concept of dynamically influencing tissue can be incorporated into such structures , and because self- 40,41 formation via local release of ‘morphogens’ . The adaptability assembling hydrogels are amenable to modular design, it is of hydrogel synthesis allows one to introduce signalling molecules possible to optimize these hydrogels for a particular outcome via covalent linkage, non-covalent tethering or physical such as endothelialization . Proteins can also be specifically 42 40,41,43 entrapment , or as localized depots , leading to spatial engineered for added functionality and cooperativity with morphogen gradients that mimic a common paradigm in tissue integrin-mediated adhesion or for binding to specific development and regeneration. Non-covalent tethering in a molecules . Finally, spatial control, as described in the previous hydrogel can be used as a mechanism to control diffusivity of section, may also be introduced in a dynamic fashion, providing general classes of growth factors such as heparin binders ,or spatiotemporal control of many hydrogel features and adding specific growth factors such as nerve growth factor . increased complexity. 4 NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications & 2012 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2271 REVIEW Triggered changes in hydrogel properties the maturation of cardiomyocytes from mesoderm to adult The use of hydrogels with either user-controlled degradation or myocardium . crosslinking, or biologically responsive feedback mechanisms, Although the majority of studies to date have focused on highlights many novel advances in the field of hydrogel hydrogels that respond to physicochemical stimuli (that is, light, design. Moving with complexity, the field of hydrogel design heat and pH), emerging concepts are also focusing on hydrogels is advancing past systems that change with time owing to that respond to specific biological stimuli, such as protease- simple hydrolysis or through cell-mediated proteolysis. cleavable crosslinks within hydrogels. Beyond this, ‘biologically Although these systems have advanced our understanding of inspired’ mechanisms such as enzyme catalysis , competitive dynamic cell–material interactions, they cannot replicate the ligand–receptor binding and even nanometer-scale protein temporal changes that occur throughout development or permit motions may also be used to trigger changes in hydrogel one to investigate the timed effects of specific cues on cell properties (Fig. 5). In some cases, these hydrogels can respond to function. a biological input by releasing an output, as demonstrated by The most commonly used dynamic hydrogels to date have insulin release in response to glucose catalysis or biochemically 83,84 been designed to respond to changes in temperature and pH. For triggered growth factor release . example, hydrogels composed of poly(N-isopropyl acrylamide) The full potential of bioresponsive materials is becoming and poly(acrylic acid) derivatives can undergo substantial changes apparent, and is likely to become clearer in the coming decade. in volume, shape, mesh size, mechanical stiffness and optical Bioresponsiveness may ultimately enable investigators to mimic transparency in response to temperature and/or pH. Hydrogels key functions of secretory organs in the body, such as the that include temperature-responsive or pH-responsive polymers pancreas. Importantly, they may also allow biologists and have also been shown to dynamically vary ligand presentation to bioengineers to address new hypotheses in cell and developmental 65–67 cells , and to trigger the release of cells and cell sheets for biology. Next-generation hydrogels may be designed to respond tissue engineering applications . These technologies have to cell-secreted biological inputs, in a manner that mimics, or enabled proof-of-concept for new drug delivery and tissue even actively manipulates, the dynamics of tissue development engineering strategies; however, changes in environmental pH, and regeneration. Bioresponsiveness could be particularly rele- temperature or ionic strength may be detrimental in some tissue vant in studying and manipulating ‘emergent’ processes, such as engineering applications and in biological systems that typically stem cell differentiation, in which hydrogel properties that are exist under regulated homeostatic conditions. appropriate in the early stages are likely to be dramatically Alternatively, light is able to present precise control over different from those needed later on. For example, recent studies temporal and spatial signals. Photoinitiated polymerizations have in standard cell culture indicate that human embryonic stem cell 85 86 been widely used to form hydrogels for cell encapsulation and for differentiation into spinal motor neurons or cardiomyocytes the patterning of biological signals within hydrogels . Light has can be amplified by mimicking the timed soluble signalling recently been used as a trigger for the breaking of crosslinks in regimen observed during early tissue development, and that the hydrogel networks, namely through the introduction of timing of signal delivery is critical. One can envision a more photosensitive o-nitro benzyl groups into the crosslinks intelligent embodiment of this approach, in which a hydrogel (Fig. 4). This approach led to control over hydrogel structure in responds to a change in stem cell phenotype (for example, initial space and time, as well as the release of tethered signals. This lineage commitment) by changing local physical properties or by synthetic system was used to probe how dynamic mechanical delivering a specific growth factor, thereby optimizing new tissue properties influence the phenotype of valvular interstitial cells, formation. We have only begun to develop the type of where changes in mechanical properties led to alterations in cell bioresponsiveness needed to manipulate tissue development in phenotype . This material platform is relevant in a wide range of this way, and innovative synthetic strategies are critically studies where dynamic properties are desired and is likely to push important to expand this new area. the boundaries of tunable systems. Beyond degradation, there are many physiological processes where an increase in crosslinking may be of interest. For example, Hydrogels that manipulate tissue formation tissues experience increased mechanics during development and Tissue formation processes include complex and interdependent in various disease states, such as scar formation and tumour changes in diverse environmental parameters, notably cell–ECM development . There are only a few examples of systems where adhesion, cell-mediated ECM remodelling, cell–cell interaction, stiffness can be dynamically increased. Collagen-alginate cell migration, soluble signalling and ECM mechanics. The composite hydrogels have been investigated, demonstrating complex dynamics of natural cell–ECM interactions provide both mechanical properties altered by the introduction of divalent a challenge and a template for hydrogel design. cations . It is important to note that the introduction of calcium Some emerging technologies in hydrogel design relate to may also alter cell signalling. External stimuli such as pH can also bioinspired regulation of tissue formation, such as mimicking the be used to alter matrix stiffness ; however, these approaches may ability of natural ECMs to sequester endogenous, cell-secreted also lead to other undesirable changes in properties such as signals. Sequestering can be used as a mechanism to downregulate hydrophobicity or swelling. Another class of materials involves endogenous signals that negatively impact tissue healing, such as DNA crosslinked hydrogels where the presence of free DNA tumour necrosis factor a (ref. 87). In other circumstances, 75,76 may lead to increased stiffening , with networks that change sequestering may result in local amplification of signals that over a period of hours. Sequential crosslinking systems can also promote healing by ‘harnessing’ endogenous, cell-secreted growth 6,88,89 be used to increase hydrogel crosslinking by applying two factors . Harnessing of endogenous factors may lead to a modes of crosslinking in series , varying crosslink density series of practical advantages when compared with delivery of and in some cases susceptibility to cell-mediated degradation purified recombinant factors. These include enhanced biological (Fig. 4). This was recently used to probe the timing of mechanical activity of native, post-translationally modified molecules, changes on mesenchymal stem cell fate . As a final example, the simplified paths to clinical regulatory approval and the kinetics of gelation (crosslinking of thiolated hyaluronic acid with possibility of manipulating cell–cell paracrine signalling. PEG diacrylate, Fig. 4) has been exploited to temporally alter Interestingly, sequestering and harnessing mechanisms are also substrate mechanics, mimicking changes that occur during dependent on the ever-changing characteristics of the local NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications 5 & 2012 Macmillan Publishers Limited. All rights reserved. REVIEW NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2271 PEG Photo- Acrylate Photolabile Gelation cleaving groups UV Redox light Precursor solution Hydrogel Softer hydrogel MeHA Gelation Light Addition Radical Kinetic Dithiol chains MeHA/Dithiol solution Soft hydrogel Stiffer hydrogel –SH HA Time Gelation PEGDA HA-SH/PEGDA solution Soft hydrogel Stiffer hydrogel Figure 4 | The cleavage or introduction of crosslinks triggers changes in hydrogel properties. (a) Photocleavable groups are incorporated into PEG- based crosslinkers, and light of the appropriate wavelength cleaves crosslinks and leads to decrease in elastic modulus (E) upon exposure. (b) Two-step crosslinking process is used to first form a network and then to introduce additional crosslinks from precursors such as methacrylated hyaluronic acid (MeHA) to increase the mechanical strength in a step-wise manner. (c) Hydrogel modulus can also be increased temporally through the introduction of a crosslinker (polyethylene glycol diacrylate, PEGDA) that slowly crosslinks a modified macromolecule (for example, thiolated-hyaluronic acid, SH–HA). In this example, the kinetics and ultimate moduli can be controlled by the MW of the crosslinker. environment, such as the concentration of cell-secreted growth factors. Therefore, hydrogels that sequester signalling molecules may soon be designed to evolve with time to optimally regulate new tissue formation. For example, it may be possible to sequester particular cytokines or growth factors during early wound healing, and then release them when needed during later stages of tissue formation. These time-dependent and environment- + Ligand dependent regulatory mechanisms are common during natural wound healing, but have yet to be exploited in engineered – Ligand hydrogels. Thus, as the last decade has included major advances in our ability to design complex systems, the next decade is likely to push these systems towards the complexity of biological processes and perhaps exceeding their level of control. In principle, many contemporary hydrogel synthesis schemes combine multiple dynamic, bioresponsive mechanisms into a single hydrogel. For example, glucose catalysis has led to pH-dependent hydrogel swelling and associated insulin release , while protein conformational changes have led to bioresponsive changes in hydrogel swelling and associated growth factor release. The combination of these triggers would lead to unprecedented Figure 5 | Dynamic hydrogels that respond to biochemical inputs. control of bioresponsiveness and molecule delivery for a specific Nanometer-scale biomolecule motions can be built into hydrogels to create application. The field has only begun to explore the design space bioresponsive hydrogels. This example (adapted from ref. 84 with of potential combinations, and the diversity of natural ECMs permission from Wiley–VCH) uses the pronounced ‘hinge motion’ of the suggests that a wide range of dynamic functions are possible and protein calmodulin to create hydrogels that undergo programmable volume perhaps required to achieve functional tissue engineering. transitions in the presence of calmodulin-binding ligands (scale bar, 1 mm). Moving forward in hydrogel design In the future, we may see generic synthetic strategies to produce materials may also evolve as hydrogels mimic many aspects of hydrogels that respond predictably to virtually any desired input. biological tissues, but rarely possess the hierarchical structure (for These systems may be used to achieve independent, dynamic example, fibres) that provides organization and points of contact regulation of multiple parameters during tissue formation and for cells. Advances in hydrogel design over the upcoming years avoid the confounding effects of lurking variables, potentially via will be made by collaboration between material scientists, both advanced material design and high-throughput synthesis bioengineers and cell biologists, and time will tell where the true and characterization technologies. The structure of these utility of such advanced hydrogel systems is found. 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A., Wang, M. X., Tchao, J. & Sia, S. K. Dynamic hydrogels: switching of 3D microenvironments using two-component naturally How to cite this article: Burdick J.A. and Murphy W.L. Moving from static to dynamic derived extracellular matrices. Adv. Mater. 22, 686–691 (2010). complexity in hydrogel design. Nat. Commun. 3:1269 doi: 10.1038/ncomms2271 (2012). 8 NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications & 2012 Macmillan Publishers Limited. All rights reserved. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Communications Springer Journals

Moving from static to dynamic complexity in hydrogel design

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Copyright © 2012 by Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.
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Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
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Abstract

REVIEW Received 25 Apr 2012 | Accepted 8 Nov 2012 | Published 11 Dec 2012 DOI: 10.1038/ncomms2271 Moving from static to dynamic complexity in hydrogel design 1 2 Jason A. Burdick & William L. Murphy Hydrogels are water-swollen polymer networks that have found a range of applications from biological scaffolds to contact lenses. Historically, their design has consisted primarily of static systems and those that exhibit simple degradation. However, advances in polymer synthesis and processing have led to a new generation of dynamic systems that are capable of responding to artificial triggers and biological signals with spatial precision. These systems will open up new possibilities for the use of hydrogels as model biological structures and in tissue regeneration. ydrogels are water-swollen polymer networks that have been used for many decades, with applications as varied as contact lenses and super-absorbant materials. As the field Hof biomedical engineering has developed, hydrogels have become a prime candidate for application as molecule delivery vehicles and as carriers for cells in tissue engineering, owing to their ability to mimic many aspects of the native cellular environment (for example, high water content, mechanical properties that match soft tissues). Traditional hydrogels, formed through the covalent and non-covalent crosslinking of polymer chains, were regarded as relatively inert materials, providing a simple biomimetic three-dimensional (3D) environment, either for tissue production by local resident cells or for positioning of cells delivered in vivo. However, the simplicity of these materials may have in fact hindered their application, restricting cellular interactions with the environment and preventing uniform extracellular matrix (ECM) production and proper tissue development. In addition, these materials were limited to modelling static environments and lacked the spatiotemporal dynamic properties relevant for complex tissue processes. Fortunately, during the last decade the concepts of hydrogel design and cellular interaction have evolved, shedding light on how they may control cell behaviour, particularly for tissue engineering applications. Hydrogels with a range of mechanical properties, and capable of incorporating a wide range of biologically relevant molecules, from individual functional groups to multidomain proteins, are currently in development . In addition, hydrogels are being designed with spatial heterogeneity, to either replicate properties in native tissue structures or to produce constructs with distinct regionally specific cell behavior . As a result, studies to date have clearly demonstrated the possibility of creating well-defined microenvironments with control over the 3D presentation of signals to cells . However, recently there has been a focus on the concept of hydrogels that exhibit dynamic complexity. These materials should evolve with time and in response to 1 2 Department of Bioengineering, University of Pennsylvania, 240 Skirkanich Hall, 210 S. 33rd street, Philadelphia, Pennsylvania 19104, USA. Departments of Biomedical Engineering and Orthopedics and Rehabilitation, University of Wisconsin, Madison, Wisconsin 53706, USA. Correspondence should be addressed to J.A.B. (email: [email protected]). NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications 1 & 2012 Macmillan Publishers Limited. All rights reserved. REVIEW NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2271 user-defined triggers or cellular behaviour. Indeed, a cadre of However, the complex combination of signals present in the recent examples demonstrates that hydrogels can be used as well- cellular microenvironment may be mimicked by patterned defined static platforms for presenting signals to cells, and hydrogel systems. Such advances allow the introduction of furthermore as dynamic evolving environments. It is too soon to spatially specific cues in hydrogels, making multicellular con- tell how closely these materials mimic the dynamic complexity structs, either through co-cultures or multilineage differentiation, 16–18 of the cell/hydrogel interface, or that of multicellular constructs a possibility . Spatial patterning of hydrogels also provides or cell condensates, or to what extent dynamic hydrogels an additional tool for the development of high-throughput can regulate emergent biological processes such as tissue screening technology for the rapid investigation of cell–material 19–22 development. However, it is clear that increased material interactions . High-throughput techniques may be complexity is beginning to address such questions. particularly important as cell fate is simultaneously influenced The objective of this review is to highlight the evolution of by cell–cell interactions, soluble signals, cell–ECM interactions, hydrogel design towards dynamic behaviour. We first consider cell shape and mechanical forces, and natural ECMs often present stable and patterned hydrogels, then hydrogels that undergo cells with many distinct signals simultaneously . Therefore, it is either hydrolytic or proteolytic degradation, and finally describe logical to expect that spatially patterned synthetic hydrogels will hydrogels with trigger-responsive properties. We particularly be needed to effectively probe the simultaneous influence of many emphasize recent developments in hydrogel design that offer the distinct signals in order to gain meaningful biological insights or ability to precisely control cell–microenvironment interactions, to direct cellular behaviour. such as those found in cellular processes. Novel chemistry, and the application of orthogonal reactions to networks, now allows the unprecedented patterning of peptides and proteins or the alteration of network structure, even in the Static hydrogels that mimic biophysical cues presence of cells. For example, multiphoton processes were used We are constantly improving our understanding of biochemical to decorate biological moieties throughout 3D systems, relying on and physicochemical signals in the local cellular microenviron- the coupling of acrylate-modified molecules in proteolytically ment, and their role in cellular signalling. This has been difficult degradable polyethylene glycol (PEG) gels containing excess for natural materials, where many cues (for example, adhesive reactive groups . A subsequent study illustrated the use of this and mechanical) are coupled, yet synthetic hydrogels have technique to define regions of precise concentration and to provided a well-defined platform for experimentation. A variety pattern with multiple molecules throughout PEG hydrogels , of biochemical signals have been explored upon or within further expanding their complexity. In another example, hydrogels, taking advantage of hydrogels as ‘blank slates’ that can orthogonal binding pairs of barnase–barstar and streptavidin– 4 5–7 be decorated with cell-interactive , or growth factor-binding , biotin were applied sequentially to pattern multiple growth ligands. Hydrogels are also useful because their physical factors and biological molecules (Fig. 2) . Specifically, one properties are biomimetic, that is, they can be designed with component of the pair was first immobilized into the hydrogel stiffness ranges and topographical features that are analogous to using two-photon chemistry and the corresponding binding natural extracellular environments . As an example, 2D partner was subsequently introduced. By applying different polyacrylamide gels with tunable mechanical properties have binding pairs in sequence, multiple factors could be provided a platform for investigating mechanotransduction immobilized. This approach follows a previously developed 9,10 behavior , suggesting that stem cell fate is strongly influenced method where a tethered photolabile moiety was illuminated to by the mechanics of the target tissue . introduce cell adhesion molecules and guide cell migration . Additional related studies have emphasized the critical role of Light has also been used to introduce patterned channels that hydrogel stiffness in skeletal muscle stem cell self-renewal , permit the migration of cells through a hydrogel . 13 14 neural stem cell behavior and megakaryocyte poiesis .3D Other systems have been developed where orthogonal and mechanical properties are similarly important in controlling adult cytocompatible click chemistry can be used to first form, and then stem cell fate in alginate hydrogels incorporating adhesive pattern, hydrogels . For example, copper-free click chemistry ligands . In this case, hydrogel mechanics alter the ability of a was used to form hydrogels around cells using the reaction of a cell to cluster tethered ligands and exert tension, leading to PEG tetra-azide and a bis(DIFO3) di-functionalized polypeptide different fate decisions (for example, adipogenic fate in softer gels (containing a proteolytic degradable group), then an orthogonal and osteogenic fate in stiffer gels). Several further examples also thiol-ene photocoupling reaction was used to modify the gels demonstrate that 3D hydrogels support the growth of a wide with a peptide. The sequential reactions and spatial patterning variety of tissues ; however, the synthesized tissue properties are permitted the gels to be controllably remodelled by cells (Fig. 2). often inferior to native tissue, for a range of reasons possibly A subsequent study combined the click chemistry with a related to the simplicity of these basic designs. photocleavable functionality, to fabricate spatially patterned These studies illustrate that static hydrogels are a useful, well- hydrogels and then to enable 3D cleavage of crosslinks . defined platform to efficiently probe how cell behaviour is Finally, a sequential crosslinking process was used to pattern influenced by the microenvironment, or by defined physico- cell spreading and remodelling by first encapsulating cells in a chemical and biological inputs. However, biological systems are hydrogel permissive to cell remodelling and then by introducing rarely static and homogeneous, and there is a wealth of biology photo-induced crosslinks. The cells were unable to completely and a cadre of intriguing hypotheses to be addressed with degrade the network, leading to changes in local cell spreading increasing complexity in hydrogel design. and subsequent stem cell differentiation. Introducing spatial heterogeneity into hydrogels Many biological processes are heterogeneous and material design Hydrogels that degrade with time is evolving to emulate them. An example of wound healing from Although stable and patterned hydrogels have been very useful as the perspective of traditional static versus emerging hydrogels is a means of studying fundamental cell–material interactions, they illustrated in Fig. 1. Wound healing is a complex process that over-simplify the complexity of the temporal dynamics present involves spatial and temporal heterogeneity of ECM signals and is during both tissue development and repair processes. Specifically, not recapitulated by static and uniform hydrogel systems. the regulatory cascades during tissue formation and repair are 2 NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications & 2012 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2271 REVIEW Wound healing cascade Inflammation Proliferation Remodelling Traditional hydrogels X X Wound Block cells/proteins Degrade? Erode? Emerging hydrogels Dermis Fat Sequester Respond Induce recruit deliver align Figure 1 | Introducing the complexity of biological processes into hydrogels. Hydrogels are evolving from relatively inert and static materials to those incorporating sophisticated feedback mechanisms. In this theoretical example, hydrogel design is targeted to key steps in the wound healing process, where cellular recruitment, infiltration and organization can be mediated by specific cues introduced into the hydrogel design such as the ability to sequester molecules, respond to cellular signals such as degrading enzymes and organize forming tissue. Generally, hydrogels are increasing in complexity and getting better at targeting specific cascades of biochemical signals and biophysical properties. ab Two-photon irradiation maleimide-barnase Wash Two-photon irradiation Maleimide-streptavidin Wash Barstar-SHH Biotin-CNTF Wash Figure 2 | Patterning biological cues into hydrogels. Techniques are being developed to introduce spatial control of biological cues (for example, ligands and crosslink density) into 3D hydrogels. (a) Sequential introduction of orthogonal binding pairs of barnase–barstar and streptavidin–biotin through two- photon binding of one component of the pairs (that is, barnase or streptavidin) and introduction of molecules bound to the other component (that is, barstar of biotin) to permit the coupling of multiple molecules with precise 3D patterns. Inset image illustrates this approach with fluorescent molecules. (b) Coupling of photocleavable functionality and patterning, via the formation of gels with a click reaction, and then either patterning or photocleaving crosslinks with targeted wavelength light. Examples illustrate directed migration of fibroblasts from a fibrin clot (scale bar, 100 mm). extremely complex and while ‘static’ hydrogels feature some The simplest approach to introduce temporal changes in dynamic components (for example, reversible receptor–ligand hydrogels has been via control of hydrogel degradation, primarily binding and dynamic cell–cell interactions), little has been done to release growth factors and to eliminate the permanent to intentionally manipulate temporal properties in hydrogels, implantation of a material . One of the earliest examples of a potentially owing to a lack of specific enabling technologies. degradable hydrogel features chains of poly(a-hydroxy esters), However, the intrinsic properties of hydrogels lend themselves to such as poly(lactic acid), incorporated at the ends of PEG dynamic physical and biochemical applications, including molecules before the introduction of reactive groups for responsiveness to changes in the signalling environment. There- crosslinking . Other examples include materials based on 34 35 36 fore, the interface between dynamic biological systems and poly(vinyl alcohol) , poly(propylene fumarates) , dextrans hydrogel material science provides an exciting area for future and hyaluronic acid . In all examples, the crosslinking material hydrogel design. changes with time, altering the physical properties and diffusivity NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications 3 & 2012 Macmillan Publishers Limited. All rights reserved. REVIEW NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2271 In an alternative mechanism, hydrogels are also designed to be susceptible to degradation by the proteases used by cells to Low- High- Δt remodel their surroundings; specifically, peptides that may be crosslink crosslink density density cleaved by cell-produced proteases are incorporated into the Hydrolysis 46,47 hydrogel crosslinks . A wide range of sequences were investigated that are degraded by matrix metalloproteinases 2,48,49 (MMPs), elastases and plasmin . The general susceptibility to proteases is controlled by the specific peptide sequence and there are a plethora of sequences that could be used to tailor the specific cell-mediated degradation of hydrogels (Fig. 3). Generally, the hydrogels are formed by reacting a multifunctional polymer (for example, PEG macromers with vinyl sulphones or acrylates) with end groups of protease- ↑ Swelling, diffusivity Polymer chain sensitive peptides (for example, thiols from cysteine moieties), Polymer crosslink ↓ Modulus, crosslink density where cells and molecules can be encapsulated during gelation. The remodelling of hydrogels by cells was observed for hydrogels where both sequences for adhesion and degradation were present , with degradation rates controlled by crosslink density and peptide specificity . This approach has been used in numerous cases to permit cell-mediated degradation of hydrogels, leading to engineered constructs for tissues such as 49,52 bone and vascular structures , including examples of the incorporation of growth factors released via cellular cues . Likewise, these matrices are useful as cell carriers that permit 46,54 cellular morphogenesis into a variety of tissue structures . Hybrid systems of synthetic and biological polymers including 28 55 PEG fibrinogen and hyaluronic acid have also been developed with similar functionality. Polymer chain This approach can also be harnessed to control the delivery of MMP-cleavable crosslink molecules in diseases where protease activity is altered. Some Adhesive peptide 56 57 examples of this include rheumatoid arthritis , cancer and Figure 3 | Hydrolytically and enzymatically degradable hydrogels with 58 after myocardial infarction . In these cases, MMP levels deviate properties that change with time. (a) Hydrolysis occurs relatively from equilibrium after injury. Thus, a system that exhibits MMP homogeneously throughout hydrolytically degradable hydrogels and leads sensitivity may degrade differently depending on local MMP to changes in the overall network crosslink density with time, which levels. This provides a feedback approach where local molecule can influence properties such as diffusivity, swelling and mechanics. delivery is regulated by cellular activity, increasing the available (b) Enzymatic degradation involves local cell-mediated proteases and is intricacy of release profiles. Likewise, MMP-sensitive tethers have used to control cellular infiltration into the hydrogel as long as adhesive been used for release that correlates with increased MMP cues are also present. 59,60 activity . In these cases, a pendant drug is released from matrices in response to local protease levels. These examples represent an initial foray into biological responsiveness, in the sense that hydrogels change their properties in response to a and eventually completely degrading away (Fig. 3). Likewise, non- specific set of biological stimuli. covalently crosslinked hydrogels (for example, via ionic Beyond purely synthetic materials, the development of protein- interactions), such as alginate, can be manipulated to alter based materials is also becoming a possibility. In this case, dissociation kinetics through changes in polymer chemistry . molecular engineering allows for the precise introduction of a One area in which degradation has been particularly useful is range of signal response mechanisms to control hydrogel in controlling the distribution of ECM molecules by encapsulated properties (for example, via crosslinking) or cellular interactions 37 61 cells . In tissue engineering, it is important that these molecules (for example, via degradation sites) . This area is likely to are well distributed, to build up a tissue that can replicate both the expand in upcoming years, as the need for the complexity found form and function of native tissues. In cartilage tissue engineering in native tissues is realized. Approaches such as directed evolution 39 37 approaches (with both PEG and hyaluronic acid hydrogels), will allow the rapid assessment of sequences with specific the inclusion of fractions of hydrolytically degradable functionality. As an alternative to covalently crosslinked components has improved this distribution and led to structures, hydrogels may be formed via the self-assembly of improved properties, particularly functional mechanical peptides designed to form fibrillar structures or even to present properties. Likewise, simple hydrolytic degradation mechanisms biological signals . The same cues for adhesion and degradation have also hinted at the concept of dynamically influencing tissue can be incorporated into such structures , and because self- 40,41 formation via local release of ‘morphogens’ . The adaptability assembling hydrogels are amenable to modular design, it is of hydrogel synthesis allows one to introduce signalling molecules possible to optimize these hydrogels for a particular outcome via covalent linkage, non-covalent tethering or physical such as endothelialization . Proteins can also be specifically 42 40,41,43 entrapment , or as localized depots , leading to spatial engineered for added functionality and cooperativity with morphogen gradients that mimic a common paradigm in tissue integrin-mediated adhesion or for binding to specific development and regeneration. Non-covalent tethering in a molecules . Finally, spatial control, as described in the previous hydrogel can be used as a mechanism to control diffusivity of section, may also be introduced in a dynamic fashion, providing general classes of growth factors such as heparin binders ,or spatiotemporal control of many hydrogel features and adding specific growth factors such as nerve growth factor . increased complexity. 4 NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications & 2012 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2271 REVIEW Triggered changes in hydrogel properties the maturation of cardiomyocytes from mesoderm to adult The use of hydrogels with either user-controlled degradation or myocardium . crosslinking, or biologically responsive feedback mechanisms, Although the majority of studies to date have focused on highlights many novel advances in the field of hydrogel hydrogels that respond to physicochemical stimuli (that is, light, design. Moving with complexity, the field of hydrogel design heat and pH), emerging concepts are also focusing on hydrogels is advancing past systems that change with time owing to that respond to specific biological stimuli, such as protease- simple hydrolysis or through cell-mediated proteolysis. cleavable crosslinks within hydrogels. Beyond this, ‘biologically Although these systems have advanced our understanding of inspired’ mechanisms such as enzyme catalysis , competitive dynamic cell–material interactions, they cannot replicate the ligand–receptor binding and even nanometer-scale protein temporal changes that occur throughout development or permit motions may also be used to trigger changes in hydrogel one to investigate the timed effects of specific cues on cell properties (Fig. 5). In some cases, these hydrogels can respond to function. a biological input by releasing an output, as demonstrated by The most commonly used dynamic hydrogels to date have insulin release in response to glucose catalysis or biochemically 83,84 been designed to respond to changes in temperature and pH. For triggered growth factor release . example, hydrogels composed of poly(N-isopropyl acrylamide) The full potential of bioresponsive materials is becoming and poly(acrylic acid) derivatives can undergo substantial changes apparent, and is likely to become clearer in the coming decade. in volume, shape, mesh size, mechanical stiffness and optical Bioresponsiveness may ultimately enable investigators to mimic transparency in response to temperature and/or pH. Hydrogels key functions of secretory organs in the body, such as the that include temperature-responsive or pH-responsive polymers pancreas. Importantly, they may also allow biologists and have also been shown to dynamically vary ligand presentation to bioengineers to address new hypotheses in cell and developmental 65–67 cells , and to trigger the release of cells and cell sheets for biology. Next-generation hydrogels may be designed to respond tissue engineering applications . These technologies have to cell-secreted biological inputs, in a manner that mimics, or enabled proof-of-concept for new drug delivery and tissue even actively manipulates, the dynamics of tissue development engineering strategies; however, changes in environmental pH, and regeneration. Bioresponsiveness could be particularly rele- temperature or ionic strength may be detrimental in some tissue vant in studying and manipulating ‘emergent’ processes, such as engineering applications and in biological systems that typically stem cell differentiation, in which hydrogel properties that are exist under regulated homeostatic conditions. appropriate in the early stages are likely to be dramatically Alternatively, light is able to present precise control over different from those needed later on. For example, recent studies temporal and spatial signals. Photoinitiated polymerizations have in standard cell culture indicate that human embryonic stem cell 85 86 been widely used to form hydrogels for cell encapsulation and for differentiation into spinal motor neurons or cardiomyocytes the patterning of biological signals within hydrogels . Light has can be amplified by mimicking the timed soluble signalling recently been used as a trigger for the breaking of crosslinks in regimen observed during early tissue development, and that the hydrogel networks, namely through the introduction of timing of signal delivery is critical. One can envision a more photosensitive o-nitro benzyl groups into the crosslinks intelligent embodiment of this approach, in which a hydrogel (Fig. 4). This approach led to control over hydrogel structure in responds to a change in stem cell phenotype (for example, initial space and time, as well as the release of tethered signals. This lineage commitment) by changing local physical properties or by synthetic system was used to probe how dynamic mechanical delivering a specific growth factor, thereby optimizing new tissue properties influence the phenotype of valvular interstitial cells, formation. We have only begun to develop the type of where changes in mechanical properties led to alterations in cell bioresponsiveness needed to manipulate tissue development in phenotype . This material platform is relevant in a wide range of this way, and innovative synthetic strategies are critically studies where dynamic properties are desired and is likely to push important to expand this new area. the boundaries of tunable systems. Beyond degradation, there are many physiological processes where an increase in crosslinking may be of interest. For example, Hydrogels that manipulate tissue formation tissues experience increased mechanics during development and Tissue formation processes include complex and interdependent in various disease states, such as scar formation and tumour changes in diverse environmental parameters, notably cell–ECM development . There are only a few examples of systems where adhesion, cell-mediated ECM remodelling, cell–cell interaction, stiffness can be dynamically increased. Collagen-alginate cell migration, soluble signalling and ECM mechanics. The composite hydrogels have been investigated, demonstrating complex dynamics of natural cell–ECM interactions provide both mechanical properties altered by the introduction of divalent a challenge and a template for hydrogel design. cations . It is important to note that the introduction of calcium Some emerging technologies in hydrogel design relate to may also alter cell signalling. External stimuli such as pH can also bioinspired regulation of tissue formation, such as mimicking the be used to alter matrix stiffness ; however, these approaches may ability of natural ECMs to sequester endogenous, cell-secreted also lead to other undesirable changes in properties such as signals. Sequestering can be used as a mechanism to downregulate hydrophobicity or swelling. Another class of materials involves endogenous signals that negatively impact tissue healing, such as DNA crosslinked hydrogels where the presence of free DNA tumour necrosis factor a (ref. 87). In other circumstances, 75,76 may lead to increased stiffening , with networks that change sequestering may result in local amplification of signals that over a period of hours. Sequential crosslinking systems can also promote healing by ‘harnessing’ endogenous, cell-secreted growth 6,88,89 be used to increase hydrogel crosslinking by applying two factors . Harnessing of endogenous factors may lead to a modes of crosslinking in series , varying crosslink density series of practical advantages when compared with delivery of and in some cases susceptibility to cell-mediated degradation purified recombinant factors. These include enhanced biological (Fig. 4). This was recently used to probe the timing of mechanical activity of native, post-translationally modified molecules, changes on mesenchymal stem cell fate . As a final example, the simplified paths to clinical regulatory approval and the kinetics of gelation (crosslinking of thiolated hyaluronic acid with possibility of manipulating cell–cell paracrine signalling. PEG diacrylate, Fig. 4) has been exploited to temporally alter Interestingly, sequestering and harnessing mechanisms are also substrate mechanics, mimicking changes that occur during dependent on the ever-changing characteristics of the local NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications 5 & 2012 Macmillan Publishers Limited. All rights reserved. REVIEW NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2271 PEG Photo- Acrylate Photolabile Gelation cleaving groups UV Redox light Precursor solution Hydrogel Softer hydrogel MeHA Gelation Light Addition Radical Kinetic Dithiol chains MeHA/Dithiol solution Soft hydrogel Stiffer hydrogel –SH HA Time Gelation PEGDA HA-SH/PEGDA solution Soft hydrogel Stiffer hydrogel Figure 4 | The cleavage or introduction of crosslinks triggers changes in hydrogel properties. (a) Photocleavable groups are incorporated into PEG- based crosslinkers, and light of the appropriate wavelength cleaves crosslinks and leads to decrease in elastic modulus (E) upon exposure. (b) Two-step crosslinking process is used to first form a network and then to introduce additional crosslinks from precursors such as methacrylated hyaluronic acid (MeHA) to increase the mechanical strength in a step-wise manner. (c) Hydrogel modulus can also be increased temporally through the introduction of a crosslinker (polyethylene glycol diacrylate, PEGDA) that slowly crosslinks a modified macromolecule (for example, thiolated-hyaluronic acid, SH–HA). In this example, the kinetics and ultimate moduli can be controlled by the MW of the crosslinker. environment, such as the concentration of cell-secreted growth factors. Therefore, hydrogels that sequester signalling molecules may soon be designed to evolve with time to optimally regulate new tissue formation. For example, it may be possible to sequester particular cytokines or growth factors during early wound healing, and then release them when needed during later stages of tissue formation. These time-dependent and environment- + Ligand dependent regulatory mechanisms are common during natural wound healing, but have yet to be exploited in engineered – Ligand hydrogels. Thus, as the last decade has included major advances in our ability to design complex systems, the next decade is likely to push these systems towards the complexity of biological processes and perhaps exceeding their level of control. In principle, many contemporary hydrogel synthesis schemes combine multiple dynamic, bioresponsive mechanisms into a single hydrogel. For example, glucose catalysis has led to pH-dependent hydrogel swelling and associated insulin release , while protein conformational changes have led to bioresponsive changes in hydrogel swelling and associated growth factor release. The combination of these triggers would lead to unprecedented Figure 5 | Dynamic hydrogels that respond to biochemical inputs. control of bioresponsiveness and molecule delivery for a specific Nanometer-scale biomolecule motions can be built into hydrogels to create application. The field has only begun to explore the design space bioresponsive hydrogels. This example (adapted from ref. 84 with of potential combinations, and the diversity of natural ECMs permission from Wiley–VCH) uses the pronounced ‘hinge motion’ of the suggests that a wide range of dynamic functions are possible and protein calmodulin to create hydrogels that undergo programmable volume perhaps required to achieve functional tissue engineering. transitions in the presence of calmodulin-binding ligands (scale bar, 1 mm). Moving forward in hydrogel design In the future, we may see generic synthetic strategies to produce materials may also evolve as hydrogels mimic many aspects of hydrogels that respond predictably to virtually any desired input. biological tissues, but rarely possess the hierarchical structure (for These systems may be used to achieve independent, dynamic example, fibres) that provides organization and points of contact regulation of multiple parameters during tissue formation and for cells. Advances in hydrogel design over the upcoming years avoid the confounding effects of lurking variables, potentially via will be made by collaboration between material scientists, both advanced material design and high-throughput synthesis bioengineers and cell biologists, and time will tell where the true and characterization technologies. The structure of these utility of such advanced hydrogel systems is found. 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A., Wang, M. X., Tchao, J. & Sia, S. K. Dynamic hydrogels: switching of 3D microenvironments using two-component naturally How to cite this article: Burdick J.A. and Murphy W.L. Moving from static to dynamic derived extracellular matrices. Adv. Mater. 22, 686–691 (2010). complexity in hydrogel design. Nat. Commun. 3:1269 doi: 10.1038/ncomms2271 (2012). 8 NATURE COMMUNICATIONS | 3:1269 | DOI: 10.1038/ncomms2271 | www.nature.com/naturecommunications & 2012 Macmillan Publishers Limited. All rights reserved.

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Nature Communications – Springer Journals

Published: Dec 11, 2012

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