Silk Fibroin-Based Shape-Memory Organohydrogels with Semicrystalline Microinclusions.

Inspired by nature, we designed organohydrogels (OHGs) consisting of a silk fibroin (SF) hydrogel as the continuous phase and the hydrophobic microinclusions based on semicrystalline poly(n-octadecyl acrylate) (PC18A) as the dispersed phase. SF acts as a self-emulsifier to obtain oil-in-water emulsions, and hence, it is a versatile and green alternative to chemical emulsifiers. We first prepared a stable oil-in-water emulsion without an external emulsifier by dispersing the n-octadecyl acrylate (C18A) monomer in an aqueous SF solution. To stabilize the emulsions for longer times, gelation in the continuous SF phase was induced by the addition of ethanol, which is known to trigger the conformational transition in SF from random coil to ß-sheet structures. In the second step, in situ polymerization of C18A droplets in the emulsion system was conducted under UV light in the presence of a photoinitiator to obtain high-strength OHGs with shape-memory function, and good cytocompatibility. The incorporation of hydrophilic N,N-dimethylacrylamide and noncrystallizable hydrophobic lauryl methacrylate units in the hydrogel and organogel phases of OHGs, respectively, further improved their mechanical and shape-memory properties. The shape-memory OHGs presented here exhibit switchable viscoelasticity and mechanics, a high Young's modulus (up to 4.3 ± 0.1 MPa), compressive strength (up to 2.5 ± 0.1 MPa), and toughness (up to 0.68 MPa).

4D Printing of Body Temperature-Responsive Hydrogels Based on Poly(acrylic acid) with Shape-Memory and Self-Healing Abilities.

Additive manufacturing of smart materials that can be dynamically programmed with external stimuli is known as 4D printing. Among the 4D printable materials, hydrogels are the most extensively studied materials in various biomedical areas because of their hierarchical structure, similarity to native human tissues, and supreme bioactivity. However, conventional smart hydrogels suffer from poor mechanical properties, slow actuation speed, and instability of actuated shape. Herein, we present 4D-printed hydrogels based on poly(acrylic acid) that can concurrently possess shape-memory and self-healing properties. The printing of the hydrogels is achieved by solvent-free copolymerization of the hydrophilic acrylic acid (AAc) and hydrophobic hexadecyl acrylate (C16A) monomers in the presence of TPO photoinitiator using a stereolithography-based commercial resin printer followed by swelling in water. The printed hydrogels undergo a reversible strong-to-weak gel transition below and above human body temperature due to the melting and crystallization of the hydrophobic C16A domains. In this way, the shape-memory and self-healing properties of the hydrogels can be magically actuated near the body temperature by adjusting the molar ratio of the monomers. Furthermore, the printed hydrogels display a high Young's modulus (up to ∼215 MPa) and high toughness (up to ∼7 MJ/m3), and their mechanical properties can be tuned from brittle to ductile by reducing the molar fraction of C16A, or the deformation speed. Overall, the developed 4D printable hydrogels have great potential for various biomedical applications.

Cryogelation reactions and cryogels: principles and challenges.

Cryogelation is a powerful technique for producing macroporous hydrogels called cryogels. Although cryogelation reactions and cryogels were discovered more than 70 years ago, they attracted significant interest only in the last 20 years mainly due to their extraordinary properties compared to the classical hydrogels such as a high toughness, almost complete squeezability, a mechanically stable porous structure with honeycomb arrangement, poroelasticity, and fast responsivity against external stimuli. In this mini review, general properties of cryogelation systems including the cryoconcentration phenomenon responsible for the unique properties of the cryogels are discussed. The squeezability and poroelasticity of cryogels comparable to those seen with articular cartilage are also discussed. Cryogelation reactions conducted within the pores of preformed cryogels and some novel cryogels with attractive properties are then discussed in the last section.

Re-Entrant Conformation Transition in Hydrogels.

Hydrogels are attractive materials not only for their tremendous applications but also for theoretical studies as they provide macroscopic monitoring of the conformation change of polymer chains. The pioneering theoretical work of Dusek predicting the discontinuous volume phase transition in gels followed by the experimental observation of Tanaka opened up a new area, called smart hydrogels, in the gel science. Many ionic hydrogels exhibit a discontinuous volume phase transition due to the change of the polymer-solvent interaction parameter χ depending on the external stimuli such as temperature, pH, composition of the solvent, etc. The observation of a discontinuous volume phase transition in nonionic hydrogels or organogels is still a challenging task as it requires a polymer-solvent system with a strong polymer concentration dependent χ parameter. Such an observation may open up the use of organogels as smart and hydrophobic soft materials. The re-entrant phenomenon first observed by Tanaka is another characteristic of stimuli responsive hydrogels in which they are frustrated between the swollen and collapsed states in a given solvent mixture. Thus, the hydrogel first collapses and then reswells if an environmental parameter is continuously increased. The re-entrant phenomenon of hydrogels in water-cosolvent mixtures is due to the competitive hydrogen-bonding and hydrophobic interactions leading to flow-in and flow-out of the cosolvent molecules through the hydrogel moving boundary as the composition of the solvent mixture is varied. The experimental results reviewed here show that a re-entrant conformation transition in hydrogels requires a hydrophobically modified hydrophilic network, and a moderate hydrogen-bonding cosolvent having competitive attractions with water and polymer. The re-entrant phenomenon may widen the applications of the hydrogels in mechanochemical transducers, switches, memories, and sensors.

Solvent-Free UV Polymerization of n-Octadecyl Acrylate in Butyl Rubber: A Simple Way to Produce Tough and Smart Polymeric Materials at Ambient Temperature.

One of the most fascinating challenges in recent years has been to produce mechanically robust and tough polymers with smart functions such as self-healing and shape-memory behavior. Here, we report a simple and versatile strategy for the preparation of a highly tough and highly stretchable interconnected interpenetrating polymer network (c-IPN) based on butyl rubber (IIR) and poly(n-octadecyl acrylate) (PC18A) with thermally induced healing and shape-memory functions. Solvent-free UV polymerization of n-octadecyl acrylate (C18A) at 30 ± 2 °C in the presence of IIR leads to IIR/PC18A c-IPNs with sea-island or co-continuous morphologies depending on their IIR contents. The lamellar crystals with a melting temperature Tm of 51-52 °C formed by side-by-side packed octadecyl (C18) side chains are responsible for more than 99% of effective cross-links in c-IPNs, the rest being hydrophobic associations and chemical cross-links. The c-IPNs exhibit varying stiffness (9-34 MPa), stretchability (72-740%), and a significantly higher toughness (1.9-12 MJ·m-3) than their components, which can be tuned by changing the IIR/PC18A weight ratio. The properties of c-IPNs could also be tuned by incorporating a second, noncrystallizable hydrophobic monomer, namely, lauryl methacrylate (C12M), in the melt mixture. We show that the lamellar clusters acting as sacrificial bonds break at the yield point by dissipation of energy, while the ductile amorphous continuous phase keeps the structure together, leading to the toughness improvement of c-IPNs. They exhibit a two-step healing process with >90% healing efficiency with respect to the modulus and a complete shape-recovery ratio induced by heating above Tm of alkyl crystals. The temperature-induced healing occurs via a quick step where C18 bridges form between the damaged surfaces followed by a slow step controlled by the interdiffusion of C18A segments in the bulk. We also show that the strategy developed here is suitable for a variety of rubbers and n-alkyl (meth)acrylates of various side-chain lengths.

Performance of butyl rubber-based macroporous sorbents as passive samplers.

In this study, two macroporous butyl rubber (BR)-based sorbents prepared in benzene (H-BR) and in cyclohexane (L-BR) with different porosities were synthesized by cryogelation technique. Their performances as a passive sampler were studied and then compared with commercially available silicon rubber (polydimethylsiloxane, PDMS) passive sampler. For that aim, polycyclic aromatic hydrocarbon (PAH) absorption rates of the sorbents in the short-term and their accumulation capacities in the long-term periods were investigated. Four PAHs (naphthalene, phenanthrene, fluoranthene, and pyrene) with a different number of aromatic rings were utilized. The concentrations of the PAHs in solutions were quantified by fluorescence spectrophotometer. The results showed that the BR sampler prepared in benzene (H-BR) generally has the highest absorption rates for all PAHs. The rate constants k (h-1) of the H-BR, L-BR, and PDMS samplers were found as 1.07, 0.55, and 0.55 for naphthalene; 0.73, 0.16, and 0.09 for phenanthrene; 0.24, 0.26, and 0.08 for fluoranthene; and 0.97, 0.38, and 0.17 for pyrene, respectively. The highest PAH absorption capacity was found for the BR sorbents prepared in benzene for all PAHs. Thus, benzene was selected as the organic solvent rather than cyclohexane for further studies in the preparation of butyl rubber-based samplers. The H-BR possessing the highest absorption rate and capacity underlines their usage as a capable passive sampler for both short- and long-term monitoring activities in the aquatic environments.

Macroporous methacrylated hyaluronic acid cryogels of high mechanical strength and flow-dependent viscoelasticity.

We present here a new approach for the fabrication of macroporous hyaluronic acid (HA) cryogels with a tunable porous structure, flow-dependent viscoelasticity, and a high mechanical strength. They were synthesized from methacrylated HA in aqueous solutions at -18 °C by free-radical mechanism using in situ prepared poly(N, N-dimethylacrylamide) (PDMAA) as a spacer. Both the porosity and the average diameter of the pores decrease from 99 to 90% and from 150 to 90 µm, respectively, with increasing PDMAA content of the cryogels due to the simultaneous decrease in the amount of ice template during cryogelation. The cryogels also exhibit reversible strain-dependent apparent gel-to-sol transition due to the flowing-out and flowing-in water through the pores. This flow-dependent viscoelasticity is of great interest as it protects HA network from damage under large strain and hence acts as a self-defense mechanism.

Single-, Double-, and Triple-Network Macroporous Rubbers as a Passive Sampler.

Over the past decades, large quantities of organic compounds including polycyclic aromatic hydrocarbons (PAHs) entering aquatic systems create acutely toxic effects and chronic abnormalities in aquatic organisms. Passive sampling is an effective technique to detect organic compounds at very low concentrations in water by accumulating them in their structure to a measurable concentration level. Polymeric passive samplers reported so far have a nonporous structure, and hence, the absorption of organic compounds into the passive sampler is governed by their slow diffusion process. We present here novel macroporous rubber sorbents as monophasic passive samplers with tunable pore morphologies, extraordinary mechanical properties, and high sorption rates and capacities for PAHs. Sorbent materials based on single-network (SN), double-network (DN), and triple-network (TN) butyl rubber were prepared via the cryogelation technique from butyl rubber solutions in benzene as the solvent at -18 °C using a sulfur monochloride cross-linker. To obtain macroporous rubbers with DN and TN structures, the reactions were conducted in the macropores of SN and DN rubber networks, respectively. The porous morphology and the mechanical behavior of the rubbers can be tuned by adjusting the weight ratio wR of the network components. The rubbers exhibit two generations of pores, namely, large and small pores with diameters 40-240 and 14-54 µm, respectively. The sizes of both large and small pores decrease and approach each other as wR is increased. Four PAH compounds, namely, naphthalene, phenanthrene, fluoranthene, and pyrene with two to four aromatic rings, dissolved in filtered seawater with a salinity of 22 ppt were used to highlight the correlations between the properties of macroporous rubbers and their absorption rates and capacities. Nonporous silicone rubber reported before as a passive sampler has the lowest absorption rate and capacity as compared to the macroporous rubbers. The SN rubber absorbs most rapidly PAHs because of its largest porosity, whereas the TN rubber with the smallest pores exhibits the highest sorption capacity.

Cryogel composites based on hyaluronic acid and halloysite nanotubes as scaffold for tissue engineering.

We present here preparation of mechanically strong and biocompatible cryogel composites based on hyaluronic acid (HA) and halloysite nanotubes (HNTs) of various compositions, and their applications as scaffold for different cell growing media. Uniaxial compression tests reveal that the incorporation of HNTs into HA cryogels leads to a ~2.5-fold increase in their Young moduli, e.g., from 38 ±â€¯1 to 99 ±â€¯4 kPa at a HA:HNTs weight ratio of 1:2. Although HA:HNTs based cryogels were found to be blood compatible with 1.37 ±â€¯0.11% hemolysis ratio at a HA:HNTs weight ratio of 1:2, they trigger thrombogenic activity with a blood clotting index of 17.3 ±â€¯4.8. Remarkably, HA:HNTs cryogel composites were found to be excellent scaffold materials in the proliferation of rat mesenchymal stem cells (MSC), human cervical carcinoma cells (HeLa), and human colon cancer cells (HCT116). The cell studies revealed that an increased amount of HNT embedding into HA cryogels leads to an increase of MSC proliferation.

Highly Stretchable and Rapid Self-Recoverable Cryogels Based on Butyl Rubber as Reusable Sorbent.

Cryogels based on hydrophobic polymers combining good mechanical properties with fast responsivity are attractive materials for many applications, such as oil spill removal from water and passive sampler for organic pollutants. We present, here, cryogels based on butyl rubber (BR) with a high stretchability, rapid self-recoverability, and excellent reusability for organic solvents. BR cryogels were prepared at subzero temperatures in cyclohexane and benzene at various BR concentrations in the presence of sulfur monochloride (S2Cl2) as a crosslinker. Although the properties of BR cryogels are independent of the amount of the crosslinker above a critical value, the type of the solvent, the cryogelation temperature, as well as the rubber content significantly affect their properties. It was found that benzene produces larger pore volumes as compared to cyclohexane due to the phase separation of BR from benzene at low temperatures, producing additional pores. Increasing cryogelation temperature from -18 to -2 °C leads to the formation of more ordered and aligned pores in the cryogels. Increasing BR content decreases the amount of unfrozen microphase of the frozen reaction solution, leading to a decrease in the total porosity of the cryogels and the average diameter of pores. Cryogels formed at -2 °C and at 5% (w/v) BR in cyclohexane sustain up to around 1400% stretch ratios. Cryogels swollen in toluene can completely be squeezed under strain during which toluene is released from their pores, whereas addition of toluene to the squeezed cryogels leads to recovery of their original shapes.

Mechanically robust and stretchable silk/hyaluronic acid hydrogels.

Combining the material and biological properties of hyaluronic acid (HA) and silk fibroin (SF) in a single hydrogel would expand the range of applications available to HA and SF individually. Here, we present a novel strategy to prepare mechanically robust and stretchable SF/HA hydrogels. The hydrogels were prepared from methacrylated HA (MeHA) and SF in aqueous solutions in the presence of a radical initiator. N, N-dimethylacrylamide (DMAA) monomer was also included into the reaction solution as a spacer to connect MeHA's through their pendant vinyl groups. The presence of SF significantly enhances the mechanical strength of HA hydrogels due to its ß-sheet domains acting as physical cross-links. The damage in SF network under large strain leads to a significant energy dissipation, which is responsible for the improved mechanical properties of SF/HA hydrogels.

Semicrystalline physical hydrogels with shape-memory and self-healing properties.

Synthetic hydrogels are generally amorphous in nature without any order at the molecular level. This is in contrast to biological gels containing ordered aggregates contributing significantly to their mechanical performance. Semicrystalline hydrogels, first developed in 1994, are moderately water-swollen hydrogels containing crystalline domains. Recent work shows that physically cross-linked semicrystalline hydrogels belong to one of the groups of mechanically strong and highly stretchable hydrogels exhibiting melt-processability, self-healing and shape-memory functions. They can undergo an abrupt and reversible change from a solid-like to a liquid-like state at the melting temperature, opening up several applications such as shape-memory hydrogels, injectable gels, chemical motors, and smart inks for 3D or 4D printing. In this review article, recent advances in the field of semicrystalline physical hydrogels prepared from hydrophilic and hydrophobic vinyl monomers via a free-radical mechanism are summarized. Synthesis-molecular structure-property relations of semicrystalline hydrogels, current challenges and future directions are also discussed.

High-strength and self-recoverable silk fibroin cryogels with anisotropic swelling and mechanical properties.

Creating mechanically strong macroporous hydrogels with anisotropic properties as observed in many biological tissues is a major challenge in the gel science. Here we describe a directional freezing/cryogelation method of producing high-strength and rapid self-recoverable silk fibroin scaffolds with a high degree of mechanical anisotropy similar to that of tendon. By adjusting the synthesis parameters, we were able to create fibroin scaffolds exhibiting the highest modulus anisotropy so far reported, 21 ±â€¯5, with moduli E = 2.3 ±â€¯0.5 and 0.11 ±â€¯0.03 MPa measured along parallel and perpendicular to the freezing direction, respectively. The cryogels are squeezable under load whereas, upon unloading, the squeezed-out water is taken back immediately. It was shown that the squeezability of the cryogels results in significant viscous stresses and energy dissipation. Cyclic mechanical tests reveal that the friction between the fibroin pore walls is the primary factor responsible for the energy dissipation. Independent on the fibroin concentration or direction of the measurements, 60% of the mechanical energy given to the cryogels are dissipated due to the friction between the pore walls, which is responsible for their almost complete squeezability and self-recoverability.

Monitoring the Instant Creation of a New Fluorescent Signal for Evaluation of DNA Conformation Based on Intercalation Complex.

Here we report the monitoring the instant creation of a new fluorescent signal (FS) aroused from a positively charged water-soluble fluorogenic probe, ethidium bromide (EtBr) in the presence of a radical initiator, ammonium persulfate (APS) and an accelerator, tetraethylmetilendiamine (TEMED) for evaluation of deoxyribonucleic acid (DNA) conformation. The results revealed that the occurred FS (λex = 430 nm; λmax = 525 nm) is a reduced form of EtBr (λex = 480 nm; λmax = 617 nm) and it is completely distinct from hydroethidine (λex = 350 nm; λmax = 430 nm), which is two-electron reduced form of EtBr. It was noticed that EtBr was reduced to a new FS during the polymerization of N, N dimethyacrylamide (DMAA) too, at 25 °C in the presence of APS and TEMED or at 55 °C with only APS, and the rate of formation of FS was increased upon treatment time. The effect of nanoclays such as Laponite XLG® and Laponite XLS®, which provide a protective environment for DNA in nature, were also investigated through the reduction process of EtBr in the absence and presence of a water soluble monomer DMAA. We demonstrated that DNA conformation might be evaluated by monitoring FS effectuated during the reduction of EtBr in the presence of nanoclays having positively and negatively charged surfaces. Protective property of DNA against the formation of reduced product was elucidated by carrying out the polymerization at 55 °C. The results revealed that the monitoring of formation of FS in the presence of radical initiator could lead to elucidate the conformation of DNA upon formation of intercalator complex.

Highly Stretchable DNA/Clay Hydrogels with Self-Healing Ability.

We present mechanically strong and self-healable clay hydrogels containing 2-8 w/v % ds-DNA together with a synthetic biocompatible polymer, poly( N, N-dimethylacrylamide). Clay nanoparticles in the hydrogels act like a chemical cross-linker and promote their elastic behavior, whereas DNA contributes to their viscoelastic energy dissipation. The extent of mechanical hysteresis during cyclic tensile tests reveals that the strength of intermolecular bonds in DNA/clay hydrogels is in the range of the strength of hydrogen bonds. The hydrogels exhibit a high stretchability (up to 1500%) and a tensile strength between 20 and 150 kPa. They have the ability to self-heal, which is induced by heating the damaged gel samples above the melting temperature of ds-DNA. When comparing the mechanical properties of the hydrogels before and after healing, the healing efficiency is greater than 100%. We also demonstrate that ds-DNA molecules entrapped in the gel network undergoes thermal denaturation/renaturation cycles, leading to a further improvement in the mechanical properties of the hydrogels.

Bisphosphonic Acid-Functionalized Cross-Linkers to Tailor Hydrogel Properties for Biomedical Applications.

Two bisphosphonic acid-functionalized cross-linkers (one novel) with different spacer chain characteristics were synthesized and incorporated into hydrogels by copolymerization with 2-hydroxyethyl methacrylate at different ratios to control the hydrogels' swelling, mechanical properties, and ability to support mineralization for biomedical applications. The cross-linkers were synthesized by reaction of 2-isocyanatoethyl methacrylate and bisphosphonated diamines followed by selective dealkylation of the bisphosphonate ester groups. The hydrogels provide in vitro growth of carbonated apatite, morphology affected by the cross-linker structure. The hydrogels exhibit a high Young's modulus E (up to 400 kPa) and can sustain up to 10.2 ± 0.1 MPa compressive stresses. E and hence the cross-link density significantly increases upon mineralization reflecting the formation of many bisphosphonate BP-Ca2+ bonds acting as additional cross-links. Cyclic mechanical tests reveal self-recoverability of hydrogels because of reversible nature of BP-Ca2+ bonds. The results suggest that these cross-linkers can add calcium-binding abilities to hydrogels synthesized from any monomer and improve their mechanical, swelling, and mineralization properties and hence are potentially useful materials for biomedical applications.

Reversibility of strain stiffening in silk fibroin gels.

We investigate the linear and nonlinear viscoelastic properties as well as the reversibility of strain-stiffening behavior of silk fibroin gels. The gels are prepared from 4.2w/v% fibroin solution in the presence of butanediol diglycidyl ether and N,N,N',N'-tetramethylethylenediamine (TEMED) as a cross-linker and catalyst, respectively. By changing the concentration of TEMED in the gelation system, fibroin gels exhibiting a storage modulus G' between 10-1-105Pa and a loss factor tan δ between 10-2 and 10° could be obtained. We observe a strong stiffening (up to 900%) in fibroin gels with increasing strain above 10% deformation, but reversibly if the strain is removed, the gel recovers its initial viscoelastic properties. The strain induced formation of transient intermolecular domains acting as reversible cross-links are responsible for the stiffening behavior of fibroin gels. These additional cross-links formed in the hardened fibroin gels have a temporary nature with lifetimes of the order of seconds. The nonlinear behavior of fibroin gels can be reproduced by a wormlike chain model taking into account the entropic elasticity of fibroin molecules and the strain induced increase in the cross-link density of fibroin gels.

Mechanically strong triple network hydrogels based on hyaluronan and poly(N,N-dimethylacrylamide).

Hyaluronan (HA) is a natural polyelectrolyte with distinctive biological functions. Cross-linking of HA to generate less degradable hydrogels for use in biomedical applications has attracted interest over many years. One limitation of HA hydrogels is that they are very brittle and/or easily dissolve in physiological environments, which limit their use in load-bearing applications. Herein, we describe the preparation of triple-network (TN) hydrogels based on HA and poly(N,N-dimethylacrylamide) (PDMA) of high mechanical strength by sequential gelation reactions. TN hydrogels containing 81-91% water sustain compressive stresses above 20 MPa and exhibit Young's moduli of up to 1 MPa. HA of various degrees of methacrylation was used as a multifunctional macromer for the synthesis of the brittle first-network component, while loosely cross-linked PDMA was used as the ductile, second and third network components of TN hydrogels. By tuning the methacrylation degree of HA, double-network hydrogels with a fracture stress above 10 MPa and a fracture strain of 96% were obtained. Increasing the ratio of ductile-to-brittle components via the TN approach further increases the fracture stress above 20 MPa. Cyclic mechanical tests show that, although TN hydrogels internally fracture even under small strain, the ductile components hinder macroscopic crack propagation by keeping the macroscopic gel samples together.

Macroporous silk fibroin cryogels.

Silk fibroin cryogels with remarkable properties were obtained from frozen fibroin solutions (4.2-12.6%) at subzero temperatures between -5 and -22 °C. This was achieved by the addition of ethylene glycol diglycidyl ether (EGDE) into the cryogelation system. EGDE triggers the conformational transition of fibroin from random coil to ß-sheet structure and hence fibroin gelation. One of the unique features of fibroin cryogels is their elasticity that allows them to resist complete compression without any crack development, during which water inside the cryogel is removed. The compressed cryogel immediately swells during unloading to recover its original shape. The scaffolds obtained by freeze-drying of the cryogels consist of regular, interconnected pores of diameters ranging from 50 to 10 µm that could be regulated by the synthesis parameters. The mechanical compressive strength and the modulus of the scaffolds increase with decreasing pore diameter, that is, with decreasing gelation temperature or, with increasing fibroin or EGDE concentrations in the feed. The scaffolds produced at 12.6% fibroin exhibit a very high compressive modulus (50 MPa) making them good candidates as bone scaffold materials.

Diepoxide-triggered conformational transition of silk fibroin: formation of hydrogels.

Silk fibroin hydrogels with tunable properties could be obtained from aqueous fibroin solutions (4.2 w/v %) in a short period of time. This was achieved by the addition of ethylene glycol diglycidyl ether (EGDE) into the reaction solution. Introduction of EGDE cross-links between the fibroin molecules decreases the mobility of the chains, which triggers the conformational transition from random-coil to ß-sheet structure and hence fibroin gelation. Dynamic rheological measurements conducted at 50 °C show the formation of strong to weak hydrogels depending on the pH of the reaction solution. Although EGDE attacks the amino groups of fibroin and forms interstrand cross-links, ß-sheets acting as physical cross-links dominate the elasticity of the hydrogels. Mechanical response of low-modulus fibroin hydrogels formed above pH 9.7 is highly nonlinear with strong strain hardening behavior (700%) arising from the alignment of the crystallizable amino acid segments.