Developing exquisite collagen fibrillar assemblies in the presence of keratin nanoparticles for improved cellular affinity.

Recently, the collagen-keratin (CK) composites have received much attention for the purpose of biomedical applications due to the intrinsic biocompatibility and biodegradability of these two proteins. However, few studies have reported the CK composites developed by the self-assembly approach and the influence of the keratin on the collagen self-assembly in vitro was still unknown. In this study, the keratin nanoparticles (KNPs) were successfully prepared by the reduction method, and we focused on investigating the effect of the varying concentrations of KNPs on the mechanism of the fibrillogenesis process of collagen. The intermolecular interaction between the two proteins revealed by the ultraviolet spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy and circular dichromatic (CD) spectroscopy showed that KNPs would interact with the collagen, and keratin significantly influenced the hydrogen bonding interaction existed in collagen molecules. The SEM images exhibited the formation of exquisite fibrillar networks after incorporating the KNPs into collagen, and it was conspicuous that the KNPs could uniformly distribute on the surface of collagen fibrils via electrostatic interaction, for both of the two proteins possessed many charged moieties. In addition, the AFM images confirmed the presence of the characteristic D-periodicity of collagen fibrils, indicating that the introduction of KNPs did not disrupt the self-assembly nature of the native collagen. The cell adhesion, proliferation and migration experiments on the CK fibrils were also performed in this study. The results demonstrated that the CK composites showed a better cellular affinity compared with the collagen, thus it might be a promising candidate for the biomedical applications.

Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy.

Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery, which provides resistance to mechanical stresses. Mutations in keratin genes are related to a variety of epithelial tissue diseases. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost mouse keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Computational reconstitution of filaments of substantial length uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross-section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex and remarkable heterogenic architecture of keratin filaments, suggesting that they are highly flexible, dynamic cytoskeletal structures.

Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins.

BACKGROUND: Profilaggrin belongs to the S100 fused-type protein family expressed in keratinocytes and is important for skin barrier integrity. Its N-terminus contains an S100 ("A") domain and a unique "B" domain with a nuclear localization sequence. OBJECTIVE: To determine whether profilaggrin B domain cooperates with the S100 domain to bind macromolecules. To characterize the biochemical and structural properties of the profilaggrin N-terminal "AB" domain and compare it to other S100 fused-type proteins. METHODS: We used biochemical (protease protection, light scattering, fluorescence spectroscopy, pull-down assays) and computational techniques (sequence analysis, molecular modeling with crystallographic structures) to examine human profilaggrin and S100 fused-type proteins. RESULTS: Comparing profilaggrin S100 crystal structure with models of the other S100 fused-type proteins demonstrated each has a unique chemical composition of solvent accessible surface around the hydrophobic binding pocket. S100 fused-type proteins exhibit higher pocket hydrophobicity than soluble S100 proteins. The inter-EF-hand linker in S100 fused-type proteins contains conserved hydrophobic residues involved in binding substrates. Profilaggrin B domain cooperates with the S100 domain to bind annexin II and keratin intermediate filaments in a calcium-dependent manner using exposed cationic surface. Using molecular modeling we demonstrate profilaggrin B domain likely interacts with annexin II domains I and II. Steric clash analysis shows annexin II N-terminal peptide is favored to bind profilaggrin among S100 fused-type proteins. CONCLUSION: The N-terminal S100 and B domains of profilaggrin cooperate to bind substrate molecules in granular layer keratinocytes to provide epidermal barrier functions.

Keratin Biomembranes as a Model for Studying Onychomycosis.

Difficulties in obtaining human nails that are large enough for examining the penetration of drug formulations led us to produce keratin films regenerated from human hair. We assume that these films can simulate human nail plates in drug penetration and permeation tests and can serve as a biological model for studying onychomycosis. The films were formed from keratin extracted from human hair using dithiothreitol, urea and thiourea. The obtained keratin extract was dispensed into Teflon rings and dried at 40 °C and then cured at 110 °C. The structure, surface morphology, chemical characterization and thermal stability of the films were characterized and were compared to those of human nail, hair and bovine hoof samples using SDS-electrophoresis, scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The structure of the obtained films was found to be closer to human nails than to hair or bovine hooves. The keratin films were infected with Trichophyton rubrum and were proven to be appropriate for serving as a model for studying onychomycosis.

FT-IR investigation of Terbinafine interaction with stratum corneum constituents.

Terbinafine (Tbf) is a well-established anti-fungal agent used for management of a variety of dermal conditions including ringworm and athlete's foot. Both the biochemical mechanism of Tbf fungicidal action (based on squalene epoxidase inhibition) and the target region for Tbf in vivo (the stratum corneum (SC)) are well determined. However, the biochemical and pharmacokinetic approaches used to evaluate Tbf biochemistry provide no biophysical information about molecular level physical changes in the SC upon Tbf binding. Such information is necessary for improved drug and formulation design. IR spectroscopic methods were used to evaluate the effects of Tbf on keratin structure in environments commonly used in pharmaceutics to mimic those in vivo. The Amide I and II spectral regions (1500-1700 cm-1) provided an effective means to monitor keratin secondary structure changes, while a Tbf spectral feature near 775 cm-1 provides a measure of relative Tbf levels in skin. Interaction of Tbf with the SC induced substantial ß-sheet formation in the keratin, an effect which was partially reversed both by ethanol washing and by exposure to high relative humidity. The irreversibility suggests the presence of a Tbf reservoir (consistent with kinetic studies), permitting the drug to be released in a controlled manner into the surrounding tissue.

An intact keratin network is crucial for mechanical integrity and barrier function in keratinocyte cell sheets.

The isotype-specific composition of the keratin cytoskeleton is important for strong adhesion, force resilience, and barrier function of the epidermis. However, the mechanisms by which keratins regulate these functions are still incompletely understood. In this study, the role and significance of the keratin network for mechanical integrity, force transmission, and barrier formation were analyzed in murine keratinocytes. Following the time-course of single-cell wound closure, wild-type (WT) cells slowly closed the gap in a collective fashion involving tightly connected neighboring cells. In contrast, the mechanical response of neighboring cells was compromised in keratin-deficient cells, causing an increased wound area initially and an inefficient overall wound closure. Furthermore, the loss of the keratin network led to impaired, fragmented cell-cell junctions, and triggered a profound change in the overall cellular actomyosin architecture. Electric cell-substrate impedance sensing of cell junctions revealed a dysfunctional barrier in knockout (Kty-/-) cells compared to WT cells. These findings demonstrate that Kty-/- cells display a novel phenotype characterized by loss of mechanocoupling and failure to form a functional barrier. Re-expression of K5/K14 rescued the barrier defect to a significant extent and reestablished the mechanocoupling with remaining discrepancies likely due to the low abundance of keratins in that setting. Our study reveals the major role of the keratin network for mechanical homeostasis and barrier functionality in keratinocyte layers.

Imaging techniques for observing laminar geometry in the feather shaft cortex.

Bird feather shafts are light, stiff and strong, but the fine details of how their structure, mechanics and function relate to one another remains poorly understood. The missing piece in our understanding may be the various fibrous layers that make up the shaft's cortex. Detailed imaging techniques are needed to enable us to capture, analyse and quantify these layers before we can begin to unravel the relationship between their structure, mechanics and function. We show that Serial-Block-Face scanning electron microscopy, scanning confocal polarised microscopy and synchrotron-based computed tomography are three suitable techniques to investigate layer thickness and fibre orientation in the feather cortex. These techniques and other are discussed in terms of their ability to resolve the fibrous laminar structure of the feather cortex, on sample preparation, and on throughput. Annotated images are presented for each and less suitable techniques are presented in the Supplementary Material. LAY DESCRIPTION: Bird feathers have a light, stiff and strong central shaft. However, the fine details of how their structure, mechanics and function relate to one another remains poorly understood. The missing piece in our understanding may have to do with how fibrous layers within the shaft vary in thickness and alignment. Detailed imaging techniques are needed so that we can quantify some of this variation before we can revisit some long-unanswered questions about the feather shaft's structure, mechanics and function. We investigate a number of microscopy techniques and show that three techniques are suitable for the sort of investigation that is required. These techniques and others are discussed in terms of their ability to resolve the layers' thickness and alignment, on sample preparation, and on the sample sizes they are able to process. Annotated images are presented and discussed for each of the three techniques and unsuitable techniques receive the same examination in the Supplementary Material.

Lepidosaur ß-keratin chains with four 34-residue repeats: Modelling reveals a potential filament-crosslinking role.

ß-keratin chains contain a characteristic and homologous 34-residue sequence, which is believed to adopt a twisted ß-sheet conformation that assembles in an antiparallel manner with a similar sheet in a second chain to form a ß-sandwich. These sandwiches are, in turn, related to one another by a left-handed four-fold screw axis to generate a helical structure that forms the core of the 3.4 nm diameter filaments observed by electron microscopy and deduced from X-ray fibre diffraction. Recently, it has been shown that one ß-keratin chain, with a molecular weight approximately twice that of the majority of ß-keratin chains, is conserved across the lepidosaurs (lizards, snakes and tuatara). Uniquely, it contains four 34-residue repeats. Although this chain is a minor component the observation that the entire chain shows a high degree of sequence conservation between species suggests an important structural/functional role in vivo. Modelling shows that only six families of structures are physically possible. In three of these the repeats exist within a single filament and might therefore act in a filament nucleation role. In the second three families the repeats exist in two, three or four filaments, implying that their function may be to act as an inter-filament crosslinker, thereby providing lateral reinforcement to the epidermal appendage. The favoured model is one in which the first two repeats form a ß-sandwich in one filament and the second two repeats form a ß-sandwich in a neighbouring filament. Links between alternating up- and down-pointing ß-sheets would provide optimum connectivity.

Analysis of the green turtle esophagus Chelonia mydas (Linnaeus, 1758), Testudines, Cheloniidae / Análisis del esófago de la tortuga verde Chelonia mydas (Linnaeus, 1758), Testudines, Cheloniidae

The esophagus is a muscular tube whose function is to transport food from the mouth to the stomach and basically contain the same layers as the rest of the digestive tract. The morphological knowledge of structures involved in basic physiological processes in organisms is extremely important in the acquisition of ecological and evolutionary knowledge about species. This study produces morphological information proving the structures found in the green turtle's esophagus. The animals were dissected according to the methodology used by Wyneken, allowing analysis of external and internal morphological characteristics of the esophagus. The samples were processed for microscopic analysis by light and scanning electron microscopy. It was observed that the esophagus presents thin and conical dermal papillae facing the stomach throughout the mucosa to the gastroesophageal portion, decreasing its size at the end of its extension, covered by a thick keratin layer on keratinized pavement epithelial tissue. It is concluded that the esophagus of Chelonia mydas is covered by a layer of conical keratinized dermal papillae important to exert the mechanical and protective function of the mucosa of this organ, considering that the feeding of these animals is extremely abrasive, besides serving as protection so that the food does not return.

El esófago es un tubo muscular cuya función es transportar los alimentos desde la cavidad oral hasta el estómago y, básicamente, contienen las mismas capas que el resto del tracto digestivo. El conocimiento morfológico de las estructuras involucradas en los procesos fisiológicos básicos en los organismos es extremadamente importante en la adquisición de conocimientos ecológicos y evolutivos sobre las especies. Este estudio produce información morfológica que demuestra las estructuras encontradas en el esófago de la tortuga verde. Los animales se disecaron de acuerdo con la metodología utilizada por Wyneken, lo que permitió el análisis de las características morfológicas internas y externas del esófago. Las muestras se procesaron para análisis microscópico mediante microscopía electrónica de barrido y de luz. Se observó que el esófago presenta papilas dérmicas delgadas y cónicas que se enfrentan al estómago a través de la mucosa hasta la porción gastroesofágica, disminuyendo su tamaño al final de su extensión, cubierta por una capa gruesa de queratina sobre el tejido epitelial queratinizado. Se concluye que el esófago de Chelonia mydas está cubierto por una capa de papilas dérmicas queratinizadas cónicas importantes para ejercer la función mecánica y protectora de la mucosa de este órgano, considerando que la alimentación de estos animales es extremadamente abrasiva, además de servir de protección para que la comida no sea devuelta.

Helical twist direction in the macrofibrils of keratin fibres is left handed.

Macrofibrils, the main structural features within the cortical cells of mammalian hair shafts, are long composite bundles of keratin intermediate filaments (KIFs) embedded in a matrix of keratin-associated proteins. The KIFs can be helically arranged around the macrofibril central axis, making a cylinder within which KIF helical angle relative to macrofibril axis increases approximately linearly from macrofibril centre to edge. Mesophase-based self-assembly has been implicated in the early formation of macrofibrils, which first appear as liquid-crystal tactoids in the bulb of hair follicles. Formation appears to be driven initially by interactions between pre-keratinized KIFs. Differences in the nature of these KIF-KIF interactions could result in all macrofibrils being internally twisted in a single handedness, or a 50:50 mixture of handedness within each cortical cell. We data-mined 41 electron tomograms containing three-dimensional macrofibril data from previously published studies of hair and wool. In all 644 macrofibrils examined we found that within each tomogram all macrofibrils had the same handedness. We concluded that earlier reports of left- and right-handed macrofibrils were due to artefacts of imaging or data processing. A handedness marker was used to confirm (using re-imaged sections from earlier studies) that, in both human and sheep, all macrofibrils are left-handed around the macrofibril axis. We conclude that this state is universal within mammalian hair. This also supports the conclusion that the origin of macrofibril twist is the expression of chiral twisting forces between adjacent KIFs, rather than mesophase splay and bending forces relaxing to twisting forces acting within a confined space.

Structure of intermediate filament assembly in hair deduced from hydration studies using small-angle neutron scattering.

Intermediate filaments (IFs) are ubiquitous in biological structures including hair. Small-angle neutron scattering (SANS) data from hydrated samples were used in this study to investigate the distribution of water in hair, and model the structure of the IF assembly. A main diffraction peak at a d-spacing of ∼90 Å, and two weaker reflections show that IFs are arranged in a ∼105 Šquasi-hexagonal lattice. Changes in the diffraction peaks show that only a small fraction of the water absorbed by hair enters between the IFs, and little water diffuses into the core of the IFs. The amount of water in the IF assembly increases rapidly up to 10% relative humidity (RH), and then slowly with further increase in RH. Most of the water appears to reside outside the IF assembly, in the voids and at the interfaces, and contribute to the central diffuse scattering. The IF assembly in the decuticled hair absorbs more water and is more ordered than that the native hair. This suggests that cuticle acts as a barrier, and might constrain the structure by compressing the cortex radially. Treatments with oils that are hydrophobic, heat treatment, and reduction of the S-S linkages that opens up the matrix by disulfide bond cleavage, all affect structure and water permeability. Coconut oil was found to impede hydration more than the soybean oil because of its ability to penetrate deeper into hair. A new model for the IF assembly that is sterically more favorable than the previous models is proposed.

Highly polydisperse keratin rich nanofibers: Scaffold design and in vitro characterization.

The use of bioactive proteins such as keratin has been successfully explored to improve the biological interface of scaffolds with cells during the tissue regeneration. In this work, it is optimized the fabrication of nanofibers combining wool keratin extracted by sulfitolysis, with polycaprolactone (PCL) in order to design bicomponent fibrous matrices able to exert a self-adapting pattern of signals-morphological, chemical, or physical-confined at the single fiber level, to influence cell and bacteria interactions. It is demonstrated that the blending of highly polydisperse keratin with PCL (50:50) improves the stability of the electrospinning process, promoting the formation of nanofibers-144.1 ± 43.9 nm-without the formation of defects (i.e., beads, ribbons) typically recognized in the fabrication of keratin ones. Moreover, keratin drastically increases the fiber hydrophilicity-compared with PCL fiber alone-thus improving the hMSC adhesion and in vitro proliferation until 14 days. Moreover, the growth of bacterial strains (i.e., Escherichia coli and Staphylococcus aureus) seems to be not specifically inhibited by the contribution of keratin, so that the integration of further selected compounds (i.e., metal ions) is suggested to more efficiently fight against bacteria resistance, to make them suitable for the regeneration of different interfaces and soft tissues (i.e., skin and cornea). © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1803-1813, 2019.

Self-assembly behavior of the keratose proteins extracted from oxidized Ovis aries wool fibers.

Water soluble keratose proteins were obtained from an Ovis Aries wool using peracetic acid oxidation. The wool samples and the extracted keratose proteins were characterized by using FTIR, XRD, SEM and TGA techniques. Fractions of α-keratose (MW = 43-53 kDa) along with protein species with molecular weights between 23 kDa and 33 kDa were identified in the SDS-PAGE analysis result of the extracted protein mixture. DLS and AFM experiments indicated that self-assembled globular nanoparticles with diameters between 15 nm and 100 nm formed at 5 mg/ml keratose concentration. On the other hand, upon incubation of 10 w % keratose solutions at 37 °C and 50 °C, interconnected keratose hydrogels with respective storage modulus (G') values of 0.17 ±â€¯0.03 kPa and 3.7 ±â€¯0.5 kPa were obtained. It was shown that the keratose hydrogel prepared at 37 °C supported L929 mouse fibroblast cell proliferation which suggested that these keratose hydrogels could be promising candidates in soft tissue engineering applications.

Utilization of 3D printing technology to facilitate and standardize soft tissue testing.

Three-dimensional (3D) printing has become broadly available and can be utilized to customize clamping mechanisms in biomechanical experiments. This report will describe our experience using 3D printed clamps to mount soft tissues from different anatomical regions. The feasibility and potential limitations of the technology will be discussed. Tissues were sourced in a fresh condition, including human skin, ligaments and tendons. Standardized clamps and fixtures were 3D printed and used to mount specimens. In quasi-static tensile tests combined with digital image correlation and fatigue trials we characterized the applicability of the clamping technique. Scanning electron microscopy was utilized to evaluate the specimens to assess the integrity of the extracellular matrix following the mechanical tests. 3D printed clamps showed no signs of clamping-related failure during the quasi-static tests, and intact extracellular matrix was found in the clamping area, at the transition clamping area and the central area from where the strain data was obtained. In the fatigue tests, material slippage was low, allowing for cyclic tests beyond 105 cycles. Comparison to other clamping techniques yields that 3D printed clamps ease and expedite specimen handling, are highly adaptable to specimen geometries and ideal for high-standardization and high-throughput experiments in soft tissue biomechanics.

Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

Macrofibril Formation.

Macrofibrils are the main structural component of the hair cortex, and are a composite material in which trichokeratin intermediate filaments (IFs) are arranged as organised arrays embedded in a matrix composed of keratin-associated proteins (KAPs) and keratin head groups. Various architecture of macrofibrils is possible, with many having a central core around which IFs are helically arranged, an organisation most accurately described as a double-twist arrangement. In this chapter we describe the architecture of macrofibrils and then cover their formation, with most of the material focusing on the theory that the initial stages of macrofibril formation are as liquid crystals.

The structure of the "amorphous" matrix of keratins.

Various keratin fibers, particularly human hairs, were investigated by transmission electron microscopy, TEM, solid-state 1H NMR and Transient Electro-Thermal Technique, TET. The results converge to suggest that the matrix of keratin fiber cortex, far from being amorphous, has a well-defined nano-scale grainy structure, the size of these grains being around 2-4nm. The size of the grains appears to strongly depend on the chemical treatment of the fiber, on the temperature and on the relative humidity of the environment, as well as on the physiological factors at the level of fiber production in follicle. By suggesting an organization at the nano-scale of the protein chains in these grains, likely to be Keratin Associated Proteins, the results challenge the view of matrix as a homogeneous glassy material. Moreover, they indicate the potential of further investigating the purpose of this structure that appears to reflect not only chemical treatments of keratins but also biological processes at the level of the follicle.

Morphological analyses in fragility of pili torti with Björnstad syndrome.

Pili torti is an extremely rare hair phenotype characterized by short length of hairs with hair shafts being easily broken. However, the mechanism of fragility in pili torti is unclear. In this study, we examined the underlying morphological features responsible for pili torti formation using transmission electron microscopy (TEM). We used pili torti samples from a patient with Björnstad syndrome and normal hairs from a healthy subject as a comparison. The macroscopic morphological features of the samples agreed with the results of a previous study showing that pili torti is twisted, flattened, thin and with partial trichorrhexis. Young's modulus of the samples was lower than that of normal hairs. Because the cross-sectional area of the pili torti samples was also smaller than that of normal hairs, it was clarified that the tensile strength of pili torti is 2.1-times lower than that of normal hair. Assessment of morphological features by TEM showed that the cuticle layers of the samples had wavy shapes with different thicknesses. Additionally, the cortex in the samples showed loose keratin intermediate filaments (IF). Our results suggested that these abnormalities in pili torti had already occurred below the infundibulum. Thus, the weakness of pili torti in tensile strength is thought to result from loose IF because of dysformation of disulfide bonds.

Comparative Study of Ultrasonication-Induced and Naturally Self-Assembled Silk Fibroin-Wool Keratin Hydrogel Biomaterials.

This study reports the formation of biocompatible hydrogels using protein polymers from natural silk cocoon fibroins and sheep wool keratins. Silk fibroin protein contains ß-sheet secondary structures, allowing for the formation of physical cross-linkers in the hydrogels. Comparative studies were performed on two groups of samples. In the first group, ultrasonication was used to induce a quick gelation of a protein aqueous solution, enhancing the ability of Bombyx mori silk fibroin chains to quickly entrap the wool keratin protein molecules homogenously. In the second group, silk/keratin mixtures were left at room temperature for days, resulting in naturally-assembled gelled solutions. It was found that silk/wool blended solutions can form hydrogels at different mixing ratios, with perfectly interconnected gel structure when the wool content was less than 30 weight percent (wt %) for the first group (ultrasonication), and 10 wt % for the second group (natural gel). Differential scanning calorimetry (DSC) and temperature modulated DSC (TMDSC) were used to confirm that the fibroin/keratin hydrogel system was well-blended without phase separation. Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structures of blended protein gels. It was found that intermolecular ß-sheet contents significantly increase as the system contains more silk for both groups of samples, resulting in stable crystalline cross-linkers in the blended hydrogel structures. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to analyze the samples' characteristic morphology on both micro- and nanoscales, which showed that ultrasonic waves can significantly enhance the cross-linker formation and avoid phase separation between silk and keratin molecules in the blended systems. With the ability to form cross-linkages non-chemically, these silk/wool hydrogels may be economically useful for various biomedical applications, thanks to the good biocompatibility of protein molecules and the various characteristics of hydrogel systems.