Bioorthogonal Strategies for Engineering Extracellular Matrices

Hydrogels are commonly used as engineered extracellular matrix (ECM) mimics in applications ranging from tissue engineering to in vitro disease models. Ideal mechanisms used to crosslink ECM‐mimicking hydrogels do not interfere with the biology of the system. However, most common hydrogel crosslinking chemistries exhibit some form of crossreactivity. The field of bioorthogonal chemistry has arisen to address the need for highly specific and robust reactions in biological contexts. Accordingly, bioorthogonal crosslinking strategies are incorporated into hydrogel design, allowing for gentle and efficient encapsulation of cells in various hydrogel materials. Furthermore, the selective nature of bioorthogonal chemistries can permit dynamic modification of hydrogel materials in the presence of live cells and other biomolecules to alter matrix mechanical properties and biochemistry on demand. This review provides an overview of bioorthogonal strategies used to prepare cell‐encapsulating hydrogels and highlights the potential applications of bioorthogonal chemistries in the design of dynamic engineered ECMs.     

Self‐Healing Silk Fibroin‐Based Hydrogel for Bone Regeneration: Dynamic Metal‐Ligand Self‐Assembly Approach

Despite advances in the development of silk fibroin (SF)‐based hydrogels, current methods for SF gelation show significant limitations such as lack of reversible crosslinking, use of nonphysiological conditions, and difficulties in controlling gelation time. In the present study, a strategy based on dynamic metal‐ligand coordination chemistry is developed to assemble SF‐based hydrogel under physiological conditions between SF microfibers (mSF) and a polysaccharide binder. The presented SF‐based hydrogel exhibits shear‐thinning and autonomous self‐healing properties, thereby enabling the filling of irregularly shaped tissue defects without gel fragmentation. A biomineralization approach is used to generate calcium phosphate‐coated mSF, which is chelated by bisphosphonate ligands of the binder to form reversible crosslinkages. Robust dually crosslinked (DC) hydrogel is obtained through photopolymerization of acrylamide groups of the binder. DC SF‐based hydrogel supports stem cell proliferation in vitro and accelerates bone regeneration in cranial critical size defects without any additional morphogenes delivered. The developed self‐healing and photopolymerizable SF‐based hydrogel possesses significant potential for bone regeneration application with the advantages of injectability and fit‐to‐shape molding.     

Injectability of Biosynthetic Hydrogels: Consideration for Minimally Invasive Surgical Procedures and 3D Bioprinting

Injectable hydrogels are often preferred when designing carriers for cell therapy or developing new bio-ink formulations. Biosynthetic hydrogels, which are a class of materials made with a hybrid design strategy, can be advantageous for endowing injectability while maintaining biological activity of the material. The chemical modification required to make these gels injectable by specific crosslinking pathways can be challenging and also make the hydrogels inhospitable to cells. Therefore, most efforts to functionalize biosynthetic hydrogel precursors toward injectability in the presence of cells try to balance between chemical and biological functionality, in order to preserve cell compatibility while addressing the injectability design challenges. Accordingly, hydrogel crosslinking strategies have evolved to include the use of photoinitiated “click” chemistry or bio-orthogonal reactions with rapid gelation kinetics and minimal cyto-toxicity required when working with cell-compatible hydrogel systems. With many new injectable biosynthetic materials emerging, their impact in cell-based regenerative medicine and bioprinting is also becoming more apparent. This review covers the main strategies that are used to endow biosynthetic polymers with injectability through rapid, cyto-compatible physical or covalent crosslinking and the main considerations for using the resulting injectable hydrogels in cell therapy, tissue regeneration, and bioprinting.     

Self‐Assembled Injectable Nanocomposite Hydrogels Stabilized by Bisphosphonate‐Magnesium (Mg2+) Coordination Regulates the Differentiation of Encapsulated Stem Cells via Dual Crosslinking

Nanocomposite hydrogels consist of a polymer matrix embedded with nanoparticles (NPs), which provide the hydrogels with unique bioactivities and mechanical properties. Incorporation of NPs via in situ precipitation in the polymer matrix further enhances these desirable hydrogel properties. However, the noncytocompatible pH, osmolality, and lengthy duration typically required for such in situ precipitation strategies preclude cell encapsulation in the resultant hydrogels. Bisphosphonate (BP) exhibits a variety of specific bioactivities and excellent binding affinity to multivalent cations such as magnesium ions (Mg²⁺). Here, the preparation of nanocomposite hydrogels via self‐assembly driven by bisphosphonate‐Mg²⁺ coordination is described. Upon mixing solutions of polymer bearing BPs, BP monomer (Ac‐BP), and Mg²⁺, this effective and dynamic coordination leads to the rapid self‐assembly of Ac‐BP‐Mg NPs which function as multivalent crosslinkers stabilize the resultant hydrogel structure at physiological pH. The obtained nanocomposite hydrogels are self‐healing and exhibit improved mechanical properties compared to hydrogels prepared by blending prefabricated NPs. Importantly, the hydrogels in this study allow the encapsulation of cells and subsequent injection without compromising the viability of seeded cells. Furthermore, the acrylate groups on the surface of Ac‐BP‐Mg NPs enable facile temporal control over the stiffness and crosslinking density of hydrogels via UV‐induced secondary crosslinking, and it is found that the delayed introduction of this secondary crosslinking enhances cell spreading and osteogenesis.     

Covalently Adaptable Elastin‐Like Protein–Hyaluronic Acid (ELP–HA) Hybrid Hydrogels with Secondary Thermoresponsive Crosslinking for Injectable Stem Cell Delivery

Shear‐thinning, self‐healing hydrogels are promising vehicles for therapeutic cargo delivery due to their ability to be injected using minimally invasive surgical procedures. An injectable hydrogel using a novel combination of dynamic covalent crosslinking with thermoresponsive engineered proteins is presented. Ex situ at room temperature, rapid gelation occurs through dynamic covalent hydrazone bonds by simply mixing two components: hydrazine‐modified elastin‐like protein (ELP) and aldehyde‐modified hyaluronic acid. This hydrogel provides significant mechanical protection to encapsulated human mesenchymal stem cells during syringe needle injection and rapidly recovers after injection to retain the cells homogeneously within a 3D environment. In situ, the ELP undergoes a thermal phase transition, as confirmed by coherent anti‐Stokes Raman scattering microscopy observation of dense ELP thermal aggregates. The formation of the secondary network reinforces the hydrogel and results in a tenfold slower erosion rate compared to a control hydrogel without secondary thermal crosslinking. This improved structural integrity enables cell culture for three weeks postinjection, and encapsulated cells maintain their ability to differentiate into multiple lineages, including chondrogenic, adipogenic, and osteogenic cell types. Together, these data demonstrate the promising potential of ELP–HA hydrogels for injectable stem cell transplantation and tissue regeneration.     

Injectable Hydrogels with In Situ Double Network Formation Enhance Retention of Transplanted Stem Cells

Stem cell transplantation via direct injection is a minimally invasive strategy being explored for treatment of a variety of injuries and diseases. Injectable hydrogels with shear moduli <50 Pa can mechanically protect cells during the injection process; however, these weak gels typically biodegrade within 1–2 weeks, which may be too fast for many therapeutic applications. To address this limitation, an injectable hydrogel is designed that undergoes two different physical crosslinking mechanisms. The first crosslinking step occurs ex vivo through peptide‐based molecular recognition to encapsulate cells within a weak gel that provides mechanical protection from injection forces. The second crosslinking step occurs in situ to form a reinforcing network that significantly retards material biodegradation and prolongs cell retention time. Human adipose‐derived stem cells are transplanted into the subcutaneous space of a murine model using hand‐injection through a 28‐gauge syringe needle. Cells delivered within the double‐network hydrogel are significantly protected from mechanical damage and have significantly enhanced in vivo cell retention rates compared to delivery within saline and single network hydrogels. These results demonstrate that in situ formation of a reinforcing network within an already existing hydrogel can greatly improve transplanted cell retention, thereby enhancing potential regenerative medicine therapies.     

Immune‐Informed Mucin Hydrogels Evade Fibrotic Foreign Body Response In Vivo

The immune‐mediated foreign body response to biomaterial implants can trigger the formation of insulating fibrotic capsules that can compromise implant function. To address this challenge, the intrinsic bioactivity of the mucin biopolymer, a heavily glycosylated protein that forms the protective mucus gel covering mucosal epithelia, is leveraged. By using a bioorthogonal inverse electron demand Diels–Alder reaction, mucins are crosslinked into implantable hydrogels. It is shown that mucin hydrogels (Muc‐gels) modulate the immune response driving biomaterial‐induced fibrosis. Muc‐gels do not elicit fibrosis 21 days after implantation in the peritoneal cavity of C57Bl/6 mice, whereas medical‐grade alginate hydrogels are covered by fibrous tissues. Further, Muc‐gels dampen the recruitment of innate and adaptive immune cells to the gel and trigger a pattern of very mild activation marked by a noticeably low expression of the fibrosis‐stimulating transforming growth factor beta 1 cytokine. Macrophages recruited to Muc‐gels upregulate the gene expression of the protein inhibitor of activated STAT 1 (PIAS1) and SH2‐containing phosphatase 1 (SHP‐1) cytokine regulatory proteins, which likely contributes to their low cytokine expression profiles. With this advance in mucin materials, an essential tool is provided to better understand mucin bioactivities and to initiate the development of new mucin‐based and mucin‐inspired “immune‐informed” materials for implantable devices subject to fibrotic encapsulation.     

Regulation of Stem Cell Fate in a Three‐Dimensional Micropatterned Dual‐Crosslinked Hydrogel System

Micropatterning technology is a powerful tool for controlling the cellular microenvironment and investigating the effects of physical parameters on cell behaviors, such as migration, proliferation, apoptosis, and differentiation. Although there have been significant developments in regulating the spatial and temporal distribution of physical properties in various materials, little is known about the role of the size of micropatterned regions of hydrogels with different crosslinking densities on the response of encapsulated cells. In this study, a novel alginate hydrogel system that can be micropatterned three‐dimensionally is engineered to create regions that are crosslinked by a single mechanism or dual mechanisms. By manipulating micropattern size while keeping the overall ratio of single‐ to dual‐crosslinked hydrogel volume constant, the physical properties of the micropatterned alginate hydrogels are spatially tunable. When human adipose‐derived stem cells (hASCs) are photoencapsulated within micropatterned hydrogels, their proliferation rate is a function of micropattern size. Additionally, micropattern size dictates the extent of osteogenic and chondrogenic differentiation of photoencapsulated hASC. The size of 3D micropatterned physical properties in this new hydrogel system introduces a new design parameter for regulating various cellular behaviors, and this dual‐crosslinked hydrogel system provides a new platform for studying proliferation and differentiation of stem cells in a spatially controlled manner for tissue engineering and regenerative medicine applications.     

Nonswelling,Ultralow Content Inverse Electron‐Demand Diels–Alder Hyaluronan Hydrogels with Tunable Gelation Time: Synthesis and In Vitro Evaluation

Hyaluronan (HA) is a major component of the extracellular matrix and is particularly attractive for cell‐based assays; yet, common crosslinking strategies of HA hydrogels are not fully tunable and bioorthogonal, and result in gels subject to swelling, which affects their physicochemical properties. To overcome these limitations, HA hydrogels based on the inverse electron‐demand Diels–Alder (IEDDA) “click” reaction are designed. By crosslinking two modified HA components together, as opposed to using telechelic components, tunable gelation times as fast as 4.4 ± 0.4 min and as slow as 46.2 ± 1.8 min are achieved for facile use. By optimizing HA molar mass, ultralow polymer content hydrogels of 0.5% (w/v), resulting in minimal (<3–5% mass variation) to nonswelling (<1%), transparent and biodegradable hydrogels are synthesized. To demonstrate their versatility, the newly designed hydrogels are tested as matrices for 3D cell culture and retinal explant imaging where transparency is important. IEDDA hydrogels are cytocompatible with primary photoreceptors and enable multiphoton imaging of embedded retinal explants for double the time (>38 h) than agarose thermogels (<20 h). IEDDA HA hydrogels constitute a new hydrogel platform. They have low polymer content, tunable gelation time, and are stable, thereby making them suitable for a diversity of applications.     

Highly Tunable Elastomeric Silk Biomaterials

Elastomeric, fully degradable, and biocompatible biomaterials are rare, with current options presenting significant limitations in terms of ease of functionalization and tunable mechanical and degradation properties. A new method for covalently crosslinking tyrosine residues in silk proteins, via horseradish peroxidase and hydrogen peroxide, to generate highly elastic hydrogels with tunable properties, is reported. These materials offer tunable mechanical properties, gelation kinetics, and swelling properties. In addition, these new polymers withstand shear strains on the order of 100%, compressive strains greater than 70% and display stiffness between 200–10 000 Pa, covering a significant portion of the properties of native soft tissues. Molecular weight and solvent composition allow control of material mechanical properties over several orders of magnitude while maintaining high resilience and resistance to fatigue. Encapsulation of human bone marrow derived mesenchymal stem cells (hMSC) shows long term survival and exhibits cell‐matrix interactions reflective of both silk concentration and gelation conditions. Further biocompatibility of these materials is demonstrated with in vivo evaluation. These new protein‐based elastomeric and degradable hydrogels represent an exciting new biomaterials option, with a unique combination of properties, for tissue engineering and regenerative medicine.     

Design of Multistimuli Responsive Hydrogels Using Integrated Modeling and Genetically Engineered Silk–Elastin‐Like Proteins

Elastomeric, robust, and biocompatible hydrogels are rare, while the need for these types of biomaterials in biomedical‐related uses remains high. Here, a new family of genetically engineered silk–elastin‐like proteins (SELPs) with encoded enzymatic crosslinking sites is developed for a new generation of stimuli‐responsive yet robust hydrogels. Input into the designs is guided by simulation and realized via genetic engineering strategies. The avoidance of gamma irradiation or chemical crosslinking during gel fabrication, in lieu of an enzymatic process, expands the versatility of these new gels for the incorporation of labile proteins and cells. In the present study, the new SELP hydrogels offer sequence‐dependent, reversible stimuli‐responsive features. Their stiffness covers almost the full range of the elasticity of soft tissues. Further, physical modification of the silk domains provides a secondary control point to fine‐tune mechanical stiffness while preserving stimuli‐responsive features, with implications for a variety of biomedical material and device needs.     

Complex Tuning of Physical Properties of Hyperbranched Polyglycerol‐Based Bioink for Microfabrication of Cell‐Laden Hydrogels

Microfabrication technology has emerged as a valuable tool for fabricating structures with high resolution and complex architecture for tissue engineering applications. For this purpose, it is imperative to develop “bioink” that can be readily converted to a solid structure by the modus operandi of a chosen apparatus, while optimally supporting the biological functions by tuning their physicochemical properties. Herein, a photocrosslinkable hyperbranched polyglycerol (acrylic hyperbranched glycerol (AHPG)) is developed as a crosslinker to fabricate cell‐laden hydrogels. Due to its hydrophilicity as well as numerous hydroxyl groups for the conjugation of reactive functional groups (e.g., acrylate), the mechanical properties of resulting hydrogels could be controlled in a wide range by tuning both molecular weight and degree of acrylate substitution of AHPG. The control of mechanical properties by AHPG is highly dependent on the type of monomer, due to the hydrophilic/hydrophobic balance of polyglycerol backbone and acrylate as well as the dynamic conformational flexibility based on the molecular weight of polyglycerol. The cell encapsulation studies demonstrate the biocompatibility of the AHPG‐linked hydrogels. Eventually, the AHPG‐based hydrogel precursor solution is employed as a bioink for a digital light processing based printing system to generate cell‐laden microgels with various shapes and sizes for tissue engineering applications.     

Combining Mechanical Tuneability with Function: Biomimetic Fibrous Hydrogels with Nanoparticle Crosslinkers

Fibrous networks of biopolymers possess unique properties: mechanical stability at low concentrations, an extremely porous architecture, and strong stiffening at small deformations. An outstanding challenge is to find methods that allow to tailor the mechanical properties of these bionetworks or their synthetic equivalents without changing the polymer concentration, which simultaneously changes all other hydrogel properties. Here, networks of dilute (0.1 wt.%) fibrous hydrogels are prepared and crosslink them with functional rod-shaped nanoparticles. The crosslinking is observed to induce an architectural change that strongly affects the mechanical properties of the hydrogels with a 40-fold increase in stiffness. The effect is strongest at the lowest polymer and particle concentrations (99.8% water) and is tailorable through tuning the crosslink density. Moreover, the nanoparticle components in the composite offer the opportunity to introduce additional functions; gels with magnetic and conductive properties are reported. However, through the generic crosslinking approach of a fibrous network with decorated nanoparticle crosslinkers as presented in this work, virtually any functionality may be introduced in highly responsive hydrogels, providing a guide to design next generations of multi-functional soft materials.     

Tuning the Microenvironment: Click‐Crosslinked Hyaluronic Acid‐Based Hydrogels Provide a Platform for Studying Breast Cancer Cell Invasion

A big challenge in cell culture is the non‐natural environment in which cells are routinely screened, making in vivo phenomena, such as cell invasion, difficult to understand and predict. To study cancer cell invasion, extracellular matrix (ECM) analogs with decoupled mechanical and chemical properties are required. Hyaluronic acid (HA)‐based hydrogels crosslinked with matrix‐metalloproteinase (MMP)‐cleavable peptides are developed to study MDA‐MB‐231 breast cancer cell invasion. Hydrogels are synthesized by reacting furan‐modified HA with bismaleimide peptide crosslinkers in a Diels–Alder click reaction. This new hydrogel takes advantage of the biomimetic properties of HA, which is overexpressed in breast cancer, and eliminates the use of nonadhesive crosslinkers, such as poly(ethylene glycol) (PEG). The crosslink (mechanical) and ligand (chemical) densities are varied independently to evaluate the effects of each parameter on cell migration. Increased crosslink density correlates with decreased MDA‐MB‐231 cell invasion whereas incorporation of MMP‐cleavable sequences within the peptide crosslinker enhances invasion. Increasing the ligand density of pendant GRGDS groups induces cell proliferation, but has no significant impact on invasion. By independently tuning the mechanical and chemical environment of ECM mimetic hydrogels, a platform is provided that recapitulates variable tissue properties and elucidates the role of the microenvironment in cancer cell invasion.     

DNA/Tannic Acid Hybrid Gel Exhibiting Biodegradability,Extensibility, Tissue Adhesiveness,and Hemostatic Ability

DNA has emerged as a novel material in many areas of materials science due to its programmability. Especially, DNA hydrogels have been studied to incorporate new functions into gels. To date, only a few methods have been developed for fabricating DNA hydrogels, such as the use of complementary sequences or covalent bond. Herein, it is demonstrated that one of the most well‐known plant‐derived polyphenols, tannic acid (TA), can form a DNA hydrogel which is named TNA hydrogel ( T A + D NA ). TA plays a role as a “molecular glue” by a new mode of action reversibly connecting between phosphodiester bonds, which is different from the crosslinking utilizing complementary sequences. TA intrinsically degrades due to ester bonds connecting between pyrogallol groups, causing a degradable DNA hydrogel. Furthermore, TNA gel is multifunctional in that the gel is extensible upon pulling and adhesive to tissues because of the rich polyphenol groups in TA (ten phenols per TA). Unexpectedly, TNA gel exhibits superior in vivo hemostatic ability that can be useful for biomedical applications. This new DNA hydrogel preparation method represents a new technique for fabricating a large amount of DNA‐based hemostatic hydrogel without chemically modifying DNA or requiring the crosslinking by complementary sequences.     

Ambient‐Dried, 3D‐Printable and Electrically Conducting Cellulose Nanofiber Aerogels by Inclusion of Functional Polymers

This study presents a novel, green, and efficient way of preparing crosslinked aerogels from cellulose nanofibers (CNFs) and alginate using non‐covalent chemistry. This new process can ultimately facilitate the fast, continuous, and large‐scale production of porous, light‐weight materials as it does not require freeze‐drying, supercritical CO₂ drying, or any environmentally harmful crosslinking chemistries. The reported preparation procedure relies solely on the successive freezing, solvent‐exchange, and ambient drying of composite CNF‐alginate gels. The presented findings suggest that a highly‐porous structure can be preserved throughout the process by simply controlling the ionic strength of the gel. Aerogels with tunable densities (23–38 kg m^?3) and compressive moduli (97–275 kPa) can be prepared by using different CNF concentrations. These low‐density networks have a unique combination of formability (using molding or 3D‐printing) and wet‐stability (when ion exchanged to calcium ions). To demonstrate their use in advanced wet applications, the printed aerogels are functionalized with very high loadings of conducting poly(3,4‐ethylenedioxythiophene):tosylate (PEDOT:TOS) polymer by using a novel in situ polymerization approach. In‐depth material characterization reveals that these aerogels have the potential to be used in not only energy storage applications (specific capacitance of 78 F g^?1), but also as mechanical‐strain and humidity sensors.     

Conductive “Smart” Hybrid Hydrogels with PNIPAM and Nanostructured Conductive Polymers

Stimuli‐responsive hydrogels with decent electrical properties are a promising class of polymeric materials for a range of technological applications, such as electrical, electrochemical, and biomedical devices. In this paper, thermally responsive and conductive hybrid hydrogels are synthesized by in situ formation of continuous network of conductive polymer hydrogels crosslinked by phytic acid in poly(N‐isopropylacrylamide) matrix. The interpenetrating binary network structure provides the hybrid hydrogels with continuous transporting path for electrons, highly porous microstructure, strong interactions between two hydrogel networks, thus endowing the hybrid hydrogels with a unique combination of high electrical conductivity (up to 0.8 S m^?1), high thermoresponsive sensitivity (significant volume change within several seconds), and greatly enhanced mechanical properties. This work demonstrates that the architecture of the filling phase in the hydrogel matrix and design of hybrid hydrogel structure play an important role in determining the performance of the resulting hybrid material. The attractive performance of these hybrid hydrogels is further demonstrated by the developed switcher device which suggests potential applications in stimuli‐responsive electronic devices.     

Tissue Engineering: Scaffold‐Mediated 2D Cellular Orientations for Construction of Three Dimensionally Engineered Tissues Composed of Oriented Cells and Extracellular Matrices (Adv. Funct. Mater. 7/2009)

Various hydrogels, such as poly(γ‐glutamic acid) (γ‐PGA), gelatin (GT), alginic acid (Alg), and agarose (Aga), with 3D interconnected and oriented fibrous pores (OP gels) are prepared for 3D polymeric cellular scaffolds by using silica fiber cloth (SC) as template. After the preparation of these hydrogels with the SC templates, the latter are subsequently removed by washing with hydrofluoric acid solution. Scanning electron microscopy (SEM) clearly shows OP structures in the hydrogels. These various types of OP gels are successfully prepared in this way, independently of the crosslinking mechanism, such as chemical (γ‐PGA or GT), coordinate‐bonded (Alg), or hydrogen‐bonded (Aga) crosslinks. SEM, confocal laser scanning microscopy, and histological evaluations clearly demonstrate that mouse L929 fibroblast cells adhere to and extend along these OP structures on/in γ‐PGA hydrogels during 3D cell culture. The L929 cells that adhere on/in the oriented hydrogel are viable and proliferative. Furthermore, 3D engineered tissues, composed of the oriented cells and extracellular matrices (ECM) produced by the cells, are constructed in vitro by subsequent decomposition of the hydrogel with cysteine after 14 days of cell culture. This novel technology to fabricate 3D‐engineered tissues, consisting of oriented cells and ECM, will be useful for tissue engineering.     

Ascidian‐Inspired Fast‐Forming Hydrogel System for Versatile Biomedical Applications: Pyrogallol Chemistry for Dual Modes of Crosslinking Mechanism

Exploitation of unique biochemical and biophysical properties of marine organisms has led to the development of functional biomaterials for various biomedical applications. Recently, ascidians have received great attention, owing to their extraordinary properties such as strong underwater adhesion and rapid self‐regeneration. Specific polypeptides containing 3,4,5‐trihydroxyphenylalanine (TOPA) in the blood cells of ascidians are associated with such intrinsic properties generated through complex oxidative processes. In this study, a bioinspired hydrogel platform is developed, demonstrating versatile applicability for tissue engineering and drug delivery, by conjugating pyrogallol (PG) moiety resembling ascidian TOPA to hyaluronic acid (HA). The HA–PG conjugate can be rapidly crosslinked by dual modes of oxidative mechanisms using an oxidant or pH control, resulting in hydrogels with different mechanical and physical characteristics. The versatile utility of HA–PG hydrogels formed via different crosslinking mechanisms is tested for different biomedical platforms, including microparticles for sustained drug delivery and tissue adhesive for noninvasive cell transplantation. With extraordinarily fast and different routes of PG oxidation, ascidian‐inspired HA–PG hydrogel system may provide a promising biomaterial platform for a wide range of biomedical applications.