Nanochitin: Chemistry, Structure, Assembly, and Applications.

Chitin, a fascinating biopolymer found in living organisms, fulfills current demands of availability, sustainability, biocompatibility, biodegradability, functionality, and renewability. A feature of chitin is its ability to structure into hierarchical assemblies, spanning the nano- and macroscales, imparting toughness and resistance (chemical, biological, among others) to multicomponent materials as well as adding adaptability, tunability, and versatility. Retaining the inherent structural characteristics of chitin and its colloidal features in dispersed media has been central to its use, considering it as a building block for the construction of emerging materials. Top-down chitin designs have been reported and differentiate from the traditional molecular-level, bottom-up synthesis and assembly for material development. Such topics are the focus of this Review, which also covers the origins and biological characteristics of chitin and their influence on the morphological and physical-chemical properties. We discuss recent achievements in the isolation, deconstruction, and fractionation of chitin nanostructures of varying axial aspects (nanofibrils and nanorods) along with methods for their modification and assembly into functional materials. We highlight the role of nanochitin in its native architecture and as a component of materials subjected to multiscale interactions, leading to highly dynamic and functional structures. We introduce the most recent advances in the applications of nanochitin-derived materials and industrialization efforts, following green manufacturing principles. Finally, we offer a critical perspective about the adoption of nanochitin in the context of advanced, sustainable materials.

Rate-Limited Reaction in TEMPO/Laccase/O2 Oxidation of Cellulose.

The environment-friendly oxidation of cellulose by the 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO)/laccase/O2 system is an alternative route with huge potential to prepare cellulose nanofibers. It is found that the concentration of TEMPO significantly affects the oxidation efficiency. An effective method for improving the oxidation effect is to increase the TEMPO concentration and prolong the oxidation time. To clarify the rate-limited step of TEMPO/laccase/O2 oxidation of cellulose, the academically accepted oxidation process is divided into individual pathways. A series of experiments is conducted with laccase and the three forms of organocatalyst (TEMPO, oxoammonium (TEMPO+), and hydroxylamine (TEMPOH)) to simulate individual reactions and calculate the reaction rates. The concentrations of TEMPO and oxoammonium are monitored by EPR spectroscopy. The oxidation rate of TEMPO by laccase varies at different pH conditions, and laccase activity is much higher at pH 4.5. Other reactions without laccase involved express a higher reaction rate when the pH value increased. TEMPO is mainly regenerated through a comproportionation reaction between oxoammonium and hydroxylamine. The acceleration of TEMPO regeneration by laccase is not obvious. The results indicate that the rate-limited reaction in TEMPO/laccase/O2 oxidation is cellulose oxidation by TEMPO+.

Preparation of Silk Nanowhisker-Composited Amphoteric Cellulose/Chitin Nanofiber Membranes.

Native biopolymer nanofibers (cellulose, chitin, and silk nanofiber) are one of the most important contributors to the outstanding functions and mechanical properties of natural materials. To enhance the mechanical performance, A great deal of top-down routes have been reported to prepare biopolymers nanofibers/nanowhiskers that retaining their nanostructures. Compared with advances in cellulose and chitin nanofibers/nanowhiskers, it remains difficult for direct downsizing the natural silk fibers into silk nanofibers/nanowhiskers (SNFs/SNWs) because of their high crystallinity and sophisticated structures. In this work, environmentally friendly and recyclable deep eutectic solvents (DESs) were used to direct pretreat and downsize natural silks into silk nanowhiskers with high yield. SNWs with similar diameter (3.1-22 nm for OA/ChCl DES treated SNWs, 2.7-20 nm for CA/ChCl DES treated SNWs) and contour length (329 ± 140 nm for OA/ChCl DES treated SNWs, 365 ± 200 nm for CA/ChCl DES treated SNWs) to individual nanofibers in natural silk fibers were obtained. In addition, the separated DES with a recovery yield of at least 92% could be reused four times to produce SNWs, indicating the possibility of DESs as green solvents for sustainable biopolymer nanomaterial extraction. Based on the inherent amphoteric properties of SNWs, multicompatibility was explored to facilely composite SNWs with various polymers for preparation of coextruded membranes with enhanced performance and endowed the composites with protein-endowed double adsorption properties. Overall, this work demonstrated that the DES pretreatment process is promising for green and low-cost biopolymer nanomaterial extraction and that the SNWs prepared via DES have good prospects as nanoscale materials in the environmental field and in development of smart biomaterials and drug delivery in biomedicine.

Multiplexing microelectrodes for dielectrophoretic manipulation and electrical impedance measurement of single particles and cells in a microfluidic device.

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single-cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 µm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 µm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP-release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single-cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP-release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single-cell analysis.

Cellulose Nanofibers Prepared Using the TEMPO/Laccase/O2 System.

The 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)/laccase/O2 system was used to prepare cellulose nanofibers from wood cellulose without requiring any chlorine-containing oxidant. Laccase was degraded by oxidized TEMPO (TEMPO+) formed by laccase-mediated oxidation with O2, which competed with the oxidation of wood cellulose. Thus, large amounts of laccase and TEMPO and a long reaction time were needed to introduce ∼0.6 mmol g-1 of C6-carboxylate groups onto wood cellulose. The TEMPO/laccase/O2 system underwent one-way reaction from TEMPO to reduced TEMPO through TEMPO+. When the oxidation was applied again to the oxidized wood cellulose following isolation and purification, the C6-carboxylate groups increased to ∼1.1 mmol g-1, which was sufficient to convert the sample to cellulose nanofibers by sonication in water. However, the higher the carboxylate content of the oxidized celluloses, the lower their degree of polymerization.

Preparation of strong, UV-blocking and sustainable glucose-cross-linked cellulose plastics via hydroxyl-yne click reaction.

The construction of functional cellulose plastics possessing strong UV-blocking, hydrophilicity, and biodegradability is challenging. Therefore, we provide a novel strategy to successfully prepare sustainable and hydrophilic glucose-cross-linked cellulose (GC) plastics showing effective UV-blocking and excellent mechanical properties via hydroxyl-yne click reaction at room temperature. The results demonstrated that hydroxyl-yne click chemistry enabled efficient crosslinking of cellulose with glucose using 4-dimethylamino pyridine (DMAP) as a catalyst. Moreover, the DMAP residue imparted good UV-shielding properties to GC films exhibiting nearly 100 % UVC (200-275 nm) and 100 % UVB (320-275 nm) shielding ratios. The introduction of glucose imparted superior hydrophilicity (water contact angle of 40.3-43.2°) and improved water adsorption. Additionally, the mechanical properties of the GC films increased with the increasing crosslinking density, and the highest tensile stress was 94 MPa. The water-induced breaking and hydrogen bond reforming strategy led to a stress of 127 MPa and a strain of 25.6 % for the final GC2 film, which were excellent compared to those of the most reported cellulose films. Additionally, GC films were biosafe, exhibited improved oxygen barrier, and good biodegradability. Hence, this study provides a promising and efficient approach for preparing high-performance cellulose plastics.

Ferulic acid triggering a co-production of 4-vinyl guaiacol and fumaric acid from lignocellulose-based carbon source by Rhizopus oryzae.

Plant secondary metabolites have attracted considerable attention due to the increasing demand for finite fossil resources and environmental concerns. However, the biosynthesis of aromatic aldehydes or alcohols from renewable resources remains challenging and costly. This study explores a novel approach performed by the aromatic catabolizing organism Rhizopus oryzae, which enables a ferulic acid-activated co-production of 4-vinyl guaiacol (4-VG) and fumaric acid. The strain produced 4.60 g/L 4-VG and 11.25 g/L fumaric acid from a mixed carbon source of glucose and xylose, suggesting that this new pathway allows the potential production of natural 4-VG from low-cost substrates. This green route, which utilizes Rhizopus oryzae's ability to efficiently convert various renewable resources into valuable chemicals, paves the way for improved catalytic efficiency in 4-VG production.

Promoting microbial fermentation in lignocellulosic hydrolysates by removal of inhibitors using MTES and PEI-modified chitosan-chitin nanofiber hybrid aerogel.

To further enhance the removal efficiency for furanic and phenolic compounds in lignocellulosic hydrolysates, a new detoxification strategy was proposed, which retained fermentable sugars and promoted the growth and metabolism of subsequent bacteria. The best adsorbent (P/M-CCA) was prepared by hybrid chitosan-chitin nanofiber, graft modification with polyethylenimine, and silanization with methyl triethoxylsilane in order. Taken corn cob hydrolysate as object, the removal rates of HMF and furfural were 85.1 % and 99.0 %, respectively. The removal rates of six out of nine phenolic inhibitors were 100 %, and the other three were more than 65 %. Even better, the retention rates of glucose and xylose were both 100 %. In contrast to no growth in undetoxified hydrolysates, Bacillus coagulans grew normally in detoxified hydrolysates, and lactic acid reached 19.1 g/L after 12 h fermentation. P/M-CCA achieves both removal of multiple inhibitors and retain sugars, which would promote the valorization of highly toxic lignocellulosic hydrolysates.

Dissolving-co-catalytic strategy for the preparation of flexible and wet-stable cellulose membrane towards biodegradable packaging.

In the realization of the goal of circular economy, cellulose as one of sustainable biomass resources, have attracted much attention because of their abundant sources, biodegradability and renewability. However, the mechanical and waterproof performance of cellulose-based materials are usually not satisfying, which limits their high-value utilization. In this study, cellulose membrane with high-performance from the aspects of mechanical properties, water-resistance ability, oxygen barrier capacity and biodegradability, was prepared from bleached hardwood pulp (HBKP) in a AlCl3/ZnCl2/H2O solution. The AlCl3/ZnCl2/H2O acted as both solvent and catalyst to dissolve cellulose and facilitate the chemical crosslinking of epichlorohydrin (EPI) with cellulose, thus improved the overall performance of the obtained cellulose membrane. The addition sequence, amount and crosslinking time of EPI during chemical crosslinking had important effects on the properties of the membranes. When 7 wt% EPI was crosslinked for 24 h, the tensile stress reached 133 MPa and the strain reached 17 %. Moreover, the membrane had excellent oxygen insulation down to (1.1 ± 0.31) × 10-4 cm3/m2·d·Pa, and good water-resistance ability, no obvious swelling behavior after 450 days of immersion in distilled water. Furthermore, the membrane could be degraded by microorganisms in about 20 days. This cellulose-based membrane offers a sustainable and biodegradable packaging material.

Comparison of cellulose and chitin nanofibers on Pickering emulsion stability-Investigation of size and surface wettability contribution.

There is an increasing concern about developing biobased colloid particles for Pickering stabilization due to the environment-friendliness and health-safety needs. In this study, Pickering emulsions were formed by using TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidized cellulose nanofibers (TOCN) and chitin nanofibers prepared by TEMPO-mediated oxidation (TOChN) or partial deacetylation (DEChN). The physicochemical characterizations of Pickering emulsions demonstrated that the higher cellulose or chitin nanofiber concentrations, surface wettability, and zeta-potential, the higher effectiveness in Pickering stabilization. Specifically, even though DEChN was at a shorter size (with a length of 254 ± 72 nm) as compared to TOCN (with a length of 3050 ± 1832 nm), it showed an excellent stabilization effect on emulsions at the concentration of 0.6 wt% due to its higher affinity to soybean oil (water contact angle of 84.38 ± 0.08°) and large electrostatic repulsion between oil particles. Meanwhile, when the concentration was 0.6 wt%, long TOCN (water contact angle of 43.06 ± 0.08°) formed a three-dimensional network in the aqueous phase, which produced a superstable Pickering emulsion resulting from the limited moving of droplets. These results provided important information on the formulation of Pickering emulsions stabilized by polysaccharide nanofibers with suitable concentration, size and surface wettability.

Utilization of different wood-based microfibril cellulose for the preparation of reinforced hydrophobic polymer composite films via Pickering emulsion: A comparative study.

Pickering emulsion is a promising strategy for the preparation of hydrophobic polymer composite using hydrophilic nanocellulose. Herein, two types of microfibril cellulose, pure mechanical pretreated microfibril cellulose (P-MFC) and Deep eutectic solvents pretreated microfibril cellulose (DES-MFC), were used to fabricate reinforced hydrophobic polystyrene (PS) composites (MFC/PS) with the aid of Pickering emulsion. The results showed that both oil/water ratio and the content as well as surface hydrophilicity of MFC were playing an important role in emulsifying capacity. 8 % MFC/PS emulsion showed the smallest and most uniform emulsion droplets which is similar to nanofibril cellulose (NFC)/PS at the oil/water ratio of 3:1. The mechanical performance of MFC/PS composites verified that the reinforcement effect was closely related to the emulsifying capacity of MFC. Specially, when the content of P-MFC was 8 wt%, the composite exhibited the best mechanical properties with the tensile strength of 44.7 ± 4.4 MPa and toughness of 1162 ± 52.8 kJ/m3 and Young's modulus of 13.5 ± 0.8 GPa, which was comparable to NFC/PS composite. Moreover, the effective enhancement role of P-MFC in hydrophobic polymethyl methacrylate and polycarbonate composites were also realized via Pickering emulsion strategy. Overall, this work constituted a proof of concept of the potential application of P-MFC in nano-reinforced hydrophobic composite.

One-pot and one-step preparation of "living" cellulose nanofiber hydrogel with active double-bond via chemical vapor deposition.

Due to the existence of water, it is still a challenge to conduct chemical modification on cellulose nanofiber (CNF) hydrogels with active double bonds. A simple one-pot and one-step method for constructing "living" CNF hydrogel with double bond was created at room temperature. The chemical vapor deposition (CVD) of methacryloyl chloride (MACl) was used to introduce physical-trapped, chemical-anchored and functional double bonds into TEMPO-oxidized cellulose nanofiber (TOCN) hydrogels. TOCN hydrogel could be fabricated within just 0.5 h, the minimum dosage of MACl could be reduced to 3.22 mg/g (MACl/TOCN hydrogel). Furthermore, the CVD methods showed high efficiency for mass production and recyclability. Moreover, the chemical "living" reactivity of the introduced double bonds were verified by the freezing and UV crosslinking, radical polymerization and thiol-ene click reaction. Compared with pure TOCN hydrogel, the obtained functionalized TOCN hydrogel exhibited remarkable improvements in mechanical properties, with enhancements of 12.34 times and 2.04 times, as well as an increase in hydrophobicity by 2.14 times and a fluorescence performance improvement of 2.93 times.

High internal phase Pickering emulsions stabilized by ε-poly-l-lysine grafted cellulose nanofiber for extrusion 3D printing.

An effective method for preparing food-grade three-dimensional (3D) printing materials was the use of highly concentrated oil-in-water emulsions. This research reported 3D printable materials constructed from food-grade high internal phase Pickering emulsions (HIPPEs) that were stabilized by ε-poly-l-lysine grafted cellulose nanofiber (ε-PL-TOCNs). The ε-PL-TOCNs were prepared via ε-poly-l-lysine grafting of 2, 2, 6, 6-tetramethylpiperidine-N-oxyl (TEMPO)-oxidized cellulose (TOC) and the successive mechanical treatment. Subsequently, the chemical structure, microstructure and surface properties of ε-PL-TOCNs were characterized. The results showed that the prepared ε-PL-TOCNs had excellent dispersion performances, cationic properties brought by amino groups, and hydrophilic/hydrophobic functions of chain structure, which confirmed the feasibility of preparing HIPPEs. The HIPPEs with an internal phase volume fraction of 82 % were obtained at 0.8 wt% ε-PL-TOCNs concentration and pre-emulsification followed by continuous oil feeding. The HIPPEs' storage stability, morphology, and rheological behavior were further discussed. The ultra stable HIPPEs with apparent shear-thinning behavior and high solid viscoelasticity were successful produced, which was suitable for 3D printing. This work expanded the application of nanocellulose in emulsions field and provided a new thinking to prepare food-grade 3D printable materials and porous foam.

Multifunctional coating films by layer-by-layer deposition of cellulose and chitin nanofibrils.

An environmentally benign surface modification process for plastic films was demonstrated by fabricating composite coatings through layer-by-layer assembly with green solid materials: aqueous dispersions of two kinds of crystalline polysaccharide nanofibrils. Anionic cellulose nanofibrils were obtained by the TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation of native cellulose, while cationic ß-chitin nanofibrils were prepared by the protonation of squid pen chitin. Uniform layer depositions, driven by electrostatic attraction and enhanced by hydrogen bonding, were observed on silicon wafers and then reproduced onto poly(ethylene terephthalate) films. Contact angle measurements and dyeing tests on the resulting films revealed their hydrophilic nature and the sorption of both charged and uncharged substances. Antireflection properties were also confirmed via the light transmittance measurements. As might be presumed from all these properties, this composite coating exhibited its unique behavior largely due to its structure, which was distinct from both those of nanofibril cast films and polymer films.

Nanochitin and poly(N-isopropylacrylamide) interpenetrating network hydrogels for temperature sensor applications.

A strengthened interpenetrating network of 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO)-oxidized nanochitin (TOChN) with poly(N-isopropylacrylamide) (PNIPAm) chains was constructed in an acetic acid coagulation bath to fabricate hydrogels. The TOChN increased the water retention of the PNIPAm hydrogels while maintaining the lower critical solution temperature (LCST) at approximately 34 °C. The storage modulus and compression stress of the 0.62% TOChN@PNIPAm hydrogel were increased by about 32 and 13.47 times, respectively, compared among those as the pure PNIPAm hydrogel. These effects were accompanied by the transformation of the dominant network structure from PNIPAm molecular chain interactions to TOChN nanofibrillar cross-linking. In addition, TOChN@PNIPAm showed a shrinking-reswelling ability with a reserved shape. Fe3+ was further introduced to chelate with carboxyl groups on TOChN, which not only substantially increased the mechanical properties but additionally provided good conductivity for the hydrogels. Therefore, a temperature sensor was designed and showed a good thermal-resistance response, providing potential biosensor applications.

Zwitterionic chitin nanocrystals mediated composite and self-assembly with cellulose nanofibrils.

Zwitterionic dispersed chitin nanocrystals and TEMPO oxidized cellulose nanofibrils can be well mixed and self-assembled to be hydrogels/membranes. Active carboxyl groups ensure the well mixing of zwitterionic chitin nanocrystals and cellulose nanofibrils under neutral and alkaline condition. Electrostatic attraction between amino groups in chitin nanocrystals and carboxyl groups in chitin nanocrystals and cellulose nanofibrils further endows self-assemble property of composite suspensions. Simple standing for 12 h at room temperature is found enough for preparing self-assembled composite hydrogels. By 1-(3-dimethy-laminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxy succinimide (EDC/NHS) mediated chemical crosslinking, the storage modulus of composite hydrogel can achieve almost 8 times higher than self-assembled hydrogel. Well dispersed composite suspensions also can be transformed to be membranes via filtration treatment. The strain increases almost 2.3 times higher with similar tensile strength for cellulose nanofibril rich samples, and chitin nanocrystals mainly contributes to the enhancement in strain of composite membranes.

Tiny NaOH Assisted Facile Preparation of Silk Nanofibers and Their Nanotube-Compositing Strong, Flexible, and Conductive Films.

Natural silk nanofibers (SNFs) can not only be used as good building blocks for two- or three-dimensional biomaterials but also provide a clue for understanding the theory of structure-function relationships. Nevertheless, it is still difficult to directly extract SNFs from natural silk fibers due to their high crystallinity and recalcitrant complex structures. In the present study, a dilute alkali-assisted separation of high-yield SNFs is proposed. The degummed silk was first treated with a tiny amount of alkali at a mild temperature, followed by high-pressure homogenization. Under the optimized conditions (2% sodium hydroxide, 0 °C, 48 h), SNFs with diameters of 8-42 nm and lengths of 0.9 ± 0.3 µm were prepared with yields higher than 75%, which retained the natural structures at the nanoscale and some inherent properties of silk fibers. Interestingly, SNFs can be used as a stabilizing matrix to assist carbon nanotubes (CNTs) to disperse, aiming to form a uniform and stable CNT/SNF dispersion. Thereafter, a strong and flexible conductive composite film was fabricated with good mechanical properties. The composite film showed good piezoelectric properties and electric thermal response, which has promising application prospects for SNFs, such as in optical devices, nanoelectronics, and biosensors.

Removal of inhibitory furan aldehydes in lignocellulosic hydrolysates via chitosan-chitin nanofiber hybrid hydrogel beads.

To obtain fermentable sugars from lignocellulose, various inhibitors, especially furan aldehydes, are usually generated during the pretreatment process. These inhibitors are harmful to subsequent microbial growth and fermentation. In this study, a novel detoxification strategy was proposed to remove 5-hydroxymethylfurfural (HMF) and furfural while retaining glucose and xylose using self-prepared chitosan-chitin nanofiber hybrid hydrogel beads (C-CNBs). After C-CNBs treatment, the removal rates of HMF and furfural from sugarcane bagasse hydrolysates reached 63.1% and 68.4%, while the loss rates of glucose and xylose were only 6.3% and 8.2%, respectively. Two typical industrial strains grew well in monosaccharide-rich detoxified hydrolysates, with a specific growth rate at least 4.1 times that of undetoxified hydrolysates. Furthermore, adsorption mechanism analysis revealed that the Schiff base reaction and mesopore filling were involved in furan aldehyde adsorption. In total, C-CNBs provide an efficient and practical approach for the removal of furan aldehydes from lignocellulosic hydrolysates.

An optimized preparation of nanofiber hydrogels derived from natural carbohydrate polymers and their drug release capacity under different pH surroundings.

Cellulose and chitin, as the two important natural carbohydrate polymers, have possibility to disassemble to biomass derived polysaccharide nanofibers. The 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) oxidized nanocellulose and nanochitin based hydrogel was fabricated via acid gas phase coagulation. It was observed that hydrogels began to form when the pH was lower than 3. When 0.1 mL of acetic acid coagulation bath was provided, 10 h were enough to form sufficient physical crosslinking. Moreover, the release time of amygdalin loaded in the hydrogel could be more than 60 h with a release amount of 80 % due to the uniform network and water-bearing structure. Meanwhile, the release capacity of hydrogels showed diversity at different pH surroundings, which was attributed to the existence of carboxyl groups on the oxidized nanofiber. The results suggested the possible application of the produced nanofiber hydrogels in some specific areas, such as drug delivery, wound dressing, and food packaging.

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.