Silk Hydrogels Research Articles

Preparation and Properties of Silk Fibroin Electro Hydrogels via a Low Voltage Electrostatic Field

Protein hydrogels is an important biomaterial for soft tissue repair in biomedical applications. However, the most extracellular matrixes are structured and ordered, the morphology of common hydrogels are of random network structures that impeded their applications in tissue engineering. In this study, silk fibroin hydrogels with different morphologies (i.e., microspheres, regularized beads, nano/micro fibers, intertwined networks, and multi-walls) were prepared under low voltage electrostatic fields by regulating the concentration of silk fibroin solution. Additionally, their stability can be regulated with further processing routes to satisfy the tailored requirements for different applications. Fourier transform infrared spectrometer (FTIR) and X-ray diffraction (XRD) provided evidence of the stability of silk fibroin electro materials was tuned by this method effectively. Therefore, these silk fibroin electro hydrogels with various morphologies, high orientation, and stability-regulatable properties provided a promising candidate for tissue engineering.

Top-down extraction of surface carboxylated-silk nanocrystals and application in hydrogel preparation

Bionanomaterial based hydrogels originated from natural biopolymer have drawn much attention for advanced applications. However, nanosilk-based hydrogels derived from top-down approaches remain in their infancy. First, nanosilks based on existing methods fail to prepare hydrogels; second, both nanosilk extraction and surface modification remain a challenge due to high crystallinity and sophisticated hierarchical structures. To produce nanosilk-based hydrogels, pretreatment and oxidation are necessary. In this work, pretreatments were conducted first to loosen the sophisticated structures of natural silk fibers, NaClO oxidation was utilized in succession to introduce carboxyl groups onto silk fibroin. Combined with moderate mechanical disintegration, silk nanocrystals with additional carboxyl groups were prepared facilely. Finally, silk nanocrystal-based hydrogels were prepared successfully through gas phase coagulation. An optimization of pretreatment approaches and oxidation conditions was carried out. The morphologies, chemical and crystalline structures of original, pretreated and oxidized silk fibroin as well as nanofibrillated silk were investigated. In addition, the silk nanocrystal-based hydrogel exhibited outstanding mechanical properties compared to those of dissolved and regenerated silk fibroin-based hydrogels. Moreover, silk nanocrystal-based aerogels present highly porous, interconnected, and crisscrossed network nanostructures, which are ideal candidates for tissue regeneration and provide new prospects as porous scaffolds for bioengineering applications.

Silk Hydrogels with Controllable Formation of Dityrosine, 3,4-Dihydroxyphenylalanine, and 3,4-Dihydroxyphenylalanine-Fe3+ Complexes through Chitosan Particle-Assisted Fenton Reactions.

The oxidation of tyrosine residues of silk fibroin involves the generation of dityrosine and 3,4-dihydroxyphenylalanine (DOPA). However, it remains a challenge to selectively control the reaction pathway to produce dityrosine or DOPA in a selective fashion. Here, silk hydrogels with controllable formation of not only dityrosine and DOPA but also DOPA-Fe3+ complexes within the cross-linked networks were developed. The use of chitosan particles in the Fenton reaction allowed the interaction of Fe3+ ions with silk fibroin to be limited through the adsorption of Fe3+ ions onto chitosan particles by manipulating contact time between the reaction medium and chitosan particles. This led to significant suppression of the premature formation of β-sheet structures that cause steric hindrance to the collisions between tyrosyl radicals and thus enabled higher selectivity toward the formation of dityrosine than DOPA. Remarkably, the addition of ethylenediaminetetraacetic acid (EDTA) to the chitosan particle-assisted Fenton reactions resulted in hydrogels that significantly favored the formation of DOPA over dityrosine due to the increase in the hydroxylation of phenol in the presence of EDTA. Despite the existence of Fe3+-EDTA complexes, Raman spectra indicated the DOPA-Fe3+ complexation in the hydrogels. Mechanistically, the hydrogel networks with small-sized and uniformly distributed β-sheet structures as well as the abundance of DOPA appear to make non-EDTA-chelated Fe3+ ions more accessible to complexation with DOPA. These findings have important implications for understanding the oxidation of tyrosine residues of silk fibroin by metal-catalyzed oxidation systems with potential benefits for future studies on silk protein-based hydrogels capable of generating intrinsic adhesive features as well as for exploring dual-cross-linked silk hydrogels constructed by chemical cross-linking and metal-coordinate complexation.

Viscoelastic Silk Fibroin Hydrogels with Tunable Strength.

Hydrogels are often used as synthetic extracellular matrices (ECMs) for biomedical applications. Natural ECMs are viscoelastic and exhibit partial stress relaxation. However, commonly used hydrogels are typically elastic. Hydrogels developed from ECM-based proteins are viscoelastic, but they often have weak mechanical properties. Here, biocompatible viscoelastic hydrogels with excellent mechanical performance are fabricated by an all aqueous process at body or room temperature. These hydrogels offer obvious stress relaxation and tunable mechanical properties and gelation kinetics. Their compressive modulus can be controlled between 2 kPa and 1.2 MPa, covering a significant portion of the properties of native tissues. Investigation of the gelation mechanism revealed that silk fibroin gelation is caused by the synergistic effects of hydrophobic interaction and hydrogen bonding between silk fibroin molecules. Newly formed crystals serve as the cross-link sites and form a network endowing the hydrogel with stable structure, and the flexible noncrystalline silk nanofibers connect disparate silk fibroin crystals, endowing hydrogels with viscoelastic properties. The all aqueous gelation process avoids complex chemical and physical treatments and is beneficial for encapsulating cells or biomolecules. Encapsulation of chondrocytes results in high initial survival rate (95% ± 1%). These silk fibroin-based viscoelastic hydrogels, combined with superior biocompatible and tunable mechanical properties, represent an exciting option for tissue engineering and regenerative medicine.

Pressure-driven spreadable deferoxamine-laden hydrogels for vascularized skin flaps.

The development of hydrogels that support vascularization to improve the survival of skin flaps, yet establishing homogeneous angiogenic niches without compromising the ease of use in surgical settings remains a challenge. Here, pressure-driven spreadable hydrogels were developed utilizing beta-sheet rich silk nanofiber materials. These silk nanofiber-based hydrogels exhibited excellent spreading under mild pressure to form a thin coating to cover all the regions of the skin flaps. Deferoxamine (DFO) was loaded onto the silk nanofibers to support vascularization and these DFO-laden hydrogels were implanted under skin flaps in rats to fill the interface between the wound bed and the flap using the applied pressure. The thickness of the spread hydrogels was below 200 μm, minimizing the physical barrier effects from the hydrogels. The distribution of the hydrogels provided homogeneous angiogenic stimulation, accelerating rapid blood vessel network formation and significantly improving the survival of the skin flaps. The hydrogels also modulated the immune reactions, further facilitating the regeneration of the skin flaps. Considering the homogeneous distribution at the wound sites, improved vascularization, reduced barrier effects and low inflammation, these hydrogels appear to be promising candidates for use in tissue repair where a high blood supply is in demand. The pressure-driven spreading properties should simplify the use of the hydrogels in surgical settings to facilitate clinical translation.

Injectable silk nanofiber hydrogels as stem cell carriers to accelerate wound healing

Stem cells have potential utility in wound therapy, however the benefits are often limited due to cell injury from shear stress during injection and poor retention at the wound site. Here, shear-thinning silk nanofiber hydrogels were used to load bone marrow derived mesenchymal stem cells (BMSCs) and inject into wound sites to optimize cell retention and accelerate wound healing. The BMSCs in the silk nanofiber hydrogels maintained stemness better than the cells cultured on plates, and the expression of wound healing-related genes was significantly higher in the hydrogels with higher silk concentrations (2 wt%). The silk nanofibers physically prevented migration of BMSCs from the deposition site in the wound bed. In addition to faster wound healing, these BMSC-loaded hydrogels mediated angiogenesis and inflammation and improved collagen deposition and hair follicle regeneration in vivo in rats. Considering that these silk nanofiber hydrogels were successfully used here as carriers for stem cells to accelerate wound healing, further study for skin regeneration may be warranted.

Novel supramolecular self-healing silk fibroin-based hydrogel via host–guest interaction as wound dressing to enhance wound healing

• A novel approach to prepare supramolecular silk fibroin-based hydrogel. • The hydrogel showed good self-healing, syringeability, and biocompatibility. • The hydrogel exhibited a sustained drug release for hydrophobic drug curcumin. • The hydrogel showed enhanced wound healing efficiency in vivo . Silk fibroin (SF) based hydrogels possess great potential in wound healing. However, they have limitations as wound dressings, such as long-gelation time, high temperature or organic solvent-assisted treatment, and lack of self-healing property. In this study, a supramolecular hydrogel based on SF, acryloyl- β -cyclodextrin (Ac-CD) and 2-hydroxyethyl acrylate was developed through a facile method by photo-polymerization of acrylate under UV irradiation, and showed enhanced performance as wound dressing than commercial dressing. This SF based hydrogel was confirmed to be dually crosslinked by host–guest interaction and hydrophobic β -sheet conformation, and demonstrated improved mechanical properties, long-term stability, rapid self-healing behavior, injectability, and good biocompatibility, benefiting its application as wound dressing. Moreover, as a drug carrier owing abundant CDs, the hydrophobic drug curcumin with antioxidant and anti-inflammatory activity was loaded in SF-based hydrogel, accompanying with a controlled and sustained release profile, therewith boosting better wound healing performance with generating higher granulation tissue thickness, collagen disposition, upregulating vascular endothelial growth factor (VEGF), and decreasing inflammatory response in a full-thickness skin defect model. In conclusion, this novel self-healing SF-based hydrogel is of great value in wound healing and enriches the fabrication methodology of SF-based self-healing biomaterials as well.

A bioinspired mineral-organic composite hydrogel as a self-healable and mechanically robust bone graft for promoting bone regeneration

Despite advances in the development of osteo-regenerative biomaterials, current products are vulnerable to stress-induced formation of cracks, resulting in the loss of functionality and a limited lifespan. In the present study, a strategy based on host-guest assembly was developed to fabricate a silk fibroin-based inorganic-organic hybrid hydrogel (termed SF@HG@HA) in which silk fibroin was used as a polymer template to tether host (β-cyclodextrin) and guest (cholesterol) monomers, respectively. Due to dynamic host-guest interactions, the prepared hydrogel could repair itself spontaneously when damaged, without the assistance of any external stimuli, mimicking the self-healing characteristics of native bone tissue. Furthermore, the efficient energy dissipation mechanism provided by the host-guest crosslinking strategy endowed the hydrogel with robust mechanical properties to bear substantial mechanical loading. SF@HG@HA was shown to support cell proliferation and osteogenic differentiation in vitro and accelerate bone regeneration in critical-size rat femoral defects in vivo. Together, the silk fibroin-based self-healing hydrogel with robust mechanical properties shows potential applications in the reconstruction of bone defects, which may provide new directions for the design of functional biomaterials for tissue regeneration.

Silk fibroin reactive inks for 3D printing crypt-like structures

A reactive silk fibroin ink formulation designed for extrusion three-dimensional (3D) printing of protein-based hydrogels at room temperature is reported. This work is motivated by the need to produce protein hydrogels that can be printed into complex shapes with long-term stability using extrusion 3D printing at ambient temperature without the need for the addition of nanocomposites, synthetic polymers, or sacrifical templates. Silk fibroin from the Bombyx mori silkworm was purified and synthesized into reactive inks by enzyme-catalyzed dityrosine bond formation. Rheological and printing studies showed that tailoring the peroxide concentration in the reactive ink enables the silk to be extruded as a filament and printed into hydrogel constructs, supporting successive printed layers without flow of the construct or loss of desired geometry. To enable success of longer-term in vitro studies, 3D printed silk hydrogels were found to display excellent shape retention over time, as evidenced by no change in construct dimensions or topography when maintained for nine weeks in culture medium. Caco-2 (an intestinal epithelial cell line) attachment, proliferation, and tight junction formation on the printed constructs was not found to be affected by the geometry of the constructs tested. Intestinal myofibroblasts encapsulated within reactive silk inks were found to survive shearing during printing and proliferate within the hydrogel constructs. The work here thus provides a suitable route for extrusion 3D printing of protein hydrogel constructs that maintain their shape during printing and culture, and is expected to enable longer-term cellular studies of hydrogel constructs that require complex geometries and/or varying spatial distributions of cells on demand via digital printing.

Self-powered artificial skin made of engineered silk protein hydrogel

Abstract Engineered silk protein hydrogel that resembles skin tissue is a promising material for artificial electronic skin; it can be interfaced with real biological tissues seamlessly and used as an artificial tissue in soft robotics. Herein, we report a soft, biocompatible, and skin-adhesive silk hydrogel incorporating ZnO nanorods (ZnONRs) for a tribo- and piezo-electric energy-generating skin (EG-skin) that can harvest biomechanical energy and sense biomechanical motions. Incorporation of ZnONRs mediates an eight-fold enhancement of piezoelectricity compared to pristine silk hydrogel. An additional two-fold increase in the electrical response is possible when it is encapsulated in silk protein layers because of the hybrid effect of tribo- and piezo-electricity. The high power generated (~1 mW/cm2) is sufficient to activate low-power electrical devices, such as LEDs, oximeters, and stopwatches. Additionally, the EG-skin can be used as a tactile identifier for finger movements with quantized real-time electrical signals. The softness and skin-adhesive properties provide conformal interfaces with human skin and biological tissues, and we can harvest energies of approximately 6.2 and 0.9 μW/cm2, respectively, from their mechanical stimulation. The silk-protein-based artificial EG-skin can be effectively utilized in human–machine interfaces, tactile sensors, soft robotics, and biomedical implants.

4D-bioprinted silk hydrogels for tissue engineering

Recently, four-dimensional (4D) printing is emerging as the next-generation biofabrication technology. However, current 4D bioprinting lacks biocompatibility or multi-component printability. In addition, suitable implantable targets capable of applying 4D bioprinted products have not yet been established, except theoretical and in vitro study. Herein, we describe a cell-friendly and biocompatible 4D bioprinting system including more than two cell types based on digital light processing (DLP) and photocurable silk fibroin (Sil-MA) hydrogel. The shape changes of 3D printed bilayered Sil-MA hydrogels were controlled by modulating their interior or exterior properties in physiological conditions. We used finite element analysis (FEA) simulations to explore the possible changes in the complex structure. Finally, we made trachea mimetic tissue with two cell types using this 4D bioprinting system and implanted it into a damaged trachea of rabbit for 8 weeks. The implants were integrated with the host trachea naturally, and both epithelium and cartilage were formed at the predicted sites. These findings demonstrate that 4D bioprinting system could make tissue mimetic scaffold biologically and suggest the potential value of the 4D bioprinting system for tissue engineering and the clinical application.

Development and novel design of clustery graphene oxide formed Conductive Silk hydrogel cell vesicle to repair and routine care of myocardial infarction: Investigation of its biological activity for cell delivery applications

Cell delivery therapy is a talented and hopeful strategic approach to repair the damaged cardiac tissues after myocardial infarction. The biocompatible injectable hydrogel with bioactive nanomaterials with effective self-healing capability has been highly required for the cell delivery and regeneration therapy. In the present study, we have developed the biopolymeric and biocompatible self-healing silk protein hydrogel matrix with graphene oxide nanoformulations as cell delivery vehicles for the treatment of myocardial infarction regeneration. The materials combinations, structural interactions, thermal stability and morphology of the designed hydrogel were investigated and elaborated. To improve therapeutic potential of the hydrogel matrix, growth factor was loaded and investigated its biological activity such as cell survival and cell proliferations properties by using endothelial progenitor cells. Collected, these developed materials are excellent vesicle for cell therapy in myocardial infarction.

Hydrogel−Solid Hybrid Materials for Biomedical Applications Enabled by Surface‐Embedded Radicals

AbstractCombinations of hydrogels and solids provide high level functionality for devices such as tissue engineering scaffolds and soft machines. However, the weak bonding between hydrogels and solids hampers functionality. Here, a versatile strategy to develop mechanically robust solid−hydrogel hybrid materials using surface embedded radicals generated through plasma immersion ion implantation (PIII) of polymeric surfaces is reported. Evidence is provided that the reactive radicals play a dual role: inducing surface‐initiated, spontaneous polymerization of hydrogels; and binding the hydrogels to the surfaces. Acrylamide and silk hydrogels are formed and covalently attached through spontaneous reactions with the radicals on PIII activated polymer surfaces without cross‐linking agents or initiators. The hydrogel amount increases with incubation time, monomer concentration, and temperature. Stability tests indicate that 95% of the hydrogel is retained even after 4 months in PBS solution. T‐peel tests show that failure occurs at the tape−hydrogel interface and the hydrogel‐PIII‐treated PTFE interfacial adhesion strength is over 300 N m−1. Cell assays show no adhesion to the as‐synthesized hydrogels; however, hydrogels synthesized with fibronectin enable cell adhesion and spreading. These results show that polymers functionalized with surface‐embedded radicals provide excellent solid platforms for the generation of robust solid−hydrogel hybrid structures for biomedical applications.

Mechanical Sensors: Body‐Integrated, Enzyme‐Triggered Degradable, Silk‐Based Mechanical Sensors for Customized Health/Fitness Monitoring and In Situ Treatment (Adv. Sci. 13/2020)

In article number 1903802, Tiger H. Tao and co-workers report a set of body-integrated and enzyme-triggered degradable mechanical sensors using carbon nanotubes-doped conductive silk hydrogels. The sensors can real-timely monitor physiological signals and sporting motions by sensing the forces-induced resistance change of their carbon nanotube networks, and simultaneously control drug release through laser radiation-triggered rapid degradation of the hydrogel-based sensors for in situ chronic disorders treatment.

Photo-Electro Active Nanocomposite Silk Hydrogel for Spatiotemporal Controlled Release of Chemotherapeutics: An In Vivo Approach toward Suppressing Solid Tumor Growth.

Conventional systemic chemotherapeutic regimens suffer from challenges such as nonspecificity, shorter half-life, clearance of drugs, and dose-limiting toxicity. Localized delivery of chemotherapeutic drugs through noninvasive spatiotemporally controllable stimuli-responsive drug delivery systems could overcome these drawbacks while utilizing drugs approved for cancer treatment. In this regard, we developed photoelectro active nanocomposite silk-based drug delivery systems (DDS) exhibiting on-demand drug release in vivo. A functionally modified single-walled carbon nanotube loaded with doxorubicin (DOX) was embedded within a cross-linker free silk hydrogel. The resultant nanocomposite silk hydrogel showed electrical field responsiveness and near-infrared (NIR) laser-induced hyperthermal effect. The remote application of these stimuli in tandem or independent manner led to the increased thermal and electrical conductivity of nanocomposite hydrogel, which effectively triggered the intermittent on-demand drug release. In a proof-of-concept in vivo tumor regression study, the nanocomposite hydrogel was administered in a minimally invasive way at the periphery of the tumor by covering most of it. During the 21-day study, drastic tumor regression was recorded upon regular stimulation of nanocomposite hydrogel with simultaneous or individual external application of an electric field and NIR laser. Tumor cell death marker expression analysis uncovered the induction of apoptosis in tumor cells leading to its shrinkage. Heart ultrasound and histology revealed no cardiotoxicity associated with localized DOX treatment. To our knowledge, this is also the first report to show the simultaneous application of electric field and NIR laser in vivo for localized tumor therapy, and our results suggested that such strategy might have high clinical translational potential.

High biocompatible AuNCs-silk fibroin hydrogel system for visual detection of H2O2

Hydrogen peroxide (H2O2) quantification in biomedicine is an important indicator for different biological tissues. In this study, we developed a visual detection for H2O2 sensing based on gold nanozyme-silk fibroin (AuNCs-SF) hydrogel hybrid system with good compatibility between silk fibroin hydrogel and BSA-AuNCs. The AuNCs-SF hydrogel exhibited a fast response to H2O2 directly by fluorescence change and showed high sensitivity (limit of detection of 0.072 mM) and well stability. Moreover, the hydrogel possessed high selectivity for H2O2 in FBS solution and excellent biocompatibility to L929 cells (mouse fibroblasts). Therefore, the AuNCs-SF hydrogel hybrid system has a great potential in the fields of clinical diagnosis for visual detection of H2O2.

Immobilized laccase-catalyzed coupling for construction of silk fibroin-lignin composite hydrogels

Laccase (Lac) is an environmentally friendly commercially available low-cost biocatalyst that can act on phenolic groups at room temperature using ambient air. In this study, Lac immobilized silk fibroin (SF)-based hydrogels were employed to simultaneously absorb alkaline lignin (AL) and graft AL on SF to fabricate SFAL composite hydrogels. The immobilized Lac retained highly active, and mainly catalyzed inter- and intra-molecular covalent coupling between the phenolic groups of AL and the tyrosine residues of SF. Through imparting the unique structure of lignin, the SFAL composite hydrogels exhibited improved mechanical strength and anti-ultraviolet properties over the SF-based hydrogels. By making good use of Lac and turning diffusional limitations in Lac immobilization into advantages, this study developed a new efficient approach for production of sustainable value-added materials based on silk and lignin. This study offers a promising strategy to exploit the enormous potential of these natural biopolymers and enzymes.

Recombinant Silk Hydrogel as a Novel Dermal Filler Component: Preclinical Safety and Efficacy Studies of a New Class of Tissue Fillers.

Hyaluronic acid-based tissue fillers are commonly utilized in reconstructive surgery as well as for aesthetic augmentation. A new type of recombinant silk-based tissue filler might pose a beneficial alternative for surgeons and patients. The aim of this study was to compare injectability, reshaping, tolerability, and postimplantation behavior of dermal filler preparations containing recombinant silk hydrogel with a commercially available hyaluronic acid filler in 2 different animal models. Recombinant silk hydrogel as standalone preparation or as a mixture with commercial stabilized hyaluronic acid was tested in rodent and porcine animal models. The preparations were analyzed in detail and administered subdermally followed by clinical, volumetric, and histological monitoring of the subdermal depots over several months. Applicability, dosing, and tissue distribution of the filler preparations were facilitated in the presence of silk hydrogel. No clinical complications attributable to tissue filler application were recorded. State-of-the art methods, such as high-performance magnetic resonance imaging, were applied successfully to monitor the volumetric development of the filler depots in live animals. The preclinical data demonstrate the basic suitability of recombinant silk hydrogel as safe and convenient tissue filler ingredient. Due to its shear thinning properties, recombinant silk hydrogel has the potential for less painful application, comfortable aesthetic reshaping immediately after administration, and negligible postoperative discomfort.

A Skin‐Inspired, Interactive, and Flexible Optoelectronic Device with Hydrated Melanin Nanoparticles in a Protein Hydrogel–Elastomer Hybrid

AbstractMelanin, a biologically occurring pigment featuring broadband optical absorption, ion‐binding affinity including antioxidative and radical‐scavenging properties, and hydration‐dependent electrical conductivity is an ideal natural semiconducting material for interfacing electronics with biological systems. Here, a skin‐mimicking optoelectronic device with a melanin nanoparticle (MNP) dispersion in a protein hydrogel–elastomer hybrid (a mimic of epidermis/dermis layers with melanin) and its application as a dopamine sensor and a UV index meter are reported. MNPs dispersed in water are placed in the elastomer reservoir with integrated silver nanowire electrodes and a transparent silk hydrogel window cover; they exhibit n‐type semiconducting behavior with increased conductivities compared with those of dehydrated MNPs. Enhanced generation of free radicals by light illumination results in an increase of the electrical conductivity. In addition, the artificial optoelectronic skin interacts with its environment through the silk hydrogel window. Applications of the device, including its use as a dopamine sensor and a UV index meter to detect dopamine and UV light passing through the window, are demonstrated. The sensitivity of the dopamine sensor is enhanced by the light illumination.

Rapid Photocrosslinking of Silk Hydrogels with High Cell Density and Enhanced Shape Fidelity.

Silk fibroin hydrogels crosslinked through di-tyrosine bonds are clear, elastomeric constructs with immense potential in regenerative medicine applications. In this study, demonstrated is a new visible light-mediated photoredox system for di-tyrosine bond formation in silk fibroin that overcomes major limitations of current conventional enzymatic-based crosslinking. This photomediated system rapidly crosslinks silk fibroin (<1 min), allowing encapsulation of cells at significantly higher cell densities (15 million cells mL-1 ) while retaining high cell viability (>80%). The photocrosslinked silk hydrogels present more stable mechanical properties which do not undergo spontaneous transition to stiff, β-sheet-rich networks typically seen for enzymatically crosslinked systems. These hydrogels also support long-term culture of human articular chondrocytes, with excellent cartilage tissue formation. This system also facilitates the first demonstration of biofabrication of silk fibroin constructs in the absence of chemical modification of the protein structure or rheological additives. Cell-laden constructs with complex, ordered, graduated architectures, and high resolution (40 µm) are fabricated using the photocrosslinking system, which cannot be achieved using the enzymatic crosslinking system. Taken together, this work demonstrates the immense potential of a new crosslinking approach for fabrication of elastomeric silk hydrogels with applications in biofabrication and tissue regeneration.