Self-assembly Of Silk Fibroin Research Articles

Designing of a Multifunctional 3D-Printed Biomimetic Theragenerative Aerogel Scaffold via Mussel-Inspired Chemistry: Bioactive Glass Nanofiber-Incorporated Self-Assembled Silk Fibroin with Antibacterial, Antiosteosarcoma, and Osteoinductive Properties.

Biomaterial-mediated bone tissue engineering (BTE) offers an alternative, interesting approach for the restoration of damaged bone tissues in postsurgery osteosarcoma treatment. This study focused on synthesizing innovative composite inks, integrating self-assembled silk fibroin (SF), tannic acids (TA), and electrospun bioactive glass nanofibers 70SiO2-25CaO-5P2O5 (BGNF). By synergistically combining the unique characteristics of these three components through self-assembly and microextrusion-based three-dimensional (3D) printing, our goal was to produce durable and versatile aerogel-based 3D composite scaffolds. These scaffolds were designed to exhibit hierarchical porosity along with antibacterial, antiosteosarcoma, and bone regeneration properties. Taking inspiration from mussel foot protein attachment chemistry involving the coordination of dihydroxyphenylalanine (DOPA) amino acids with ferric ions (Fe3+), we synthesized a tris-complex catecholate-iron self-assembled composite gel. This gel formation occurred through the coordination of oxidized SF (SFO) with TA and polydopamine-modified BGNF (BGNF-PDA). The dynamic nature of the coordination ligand-metal bonds within the self-assembled SFO matrix provided excellent shear-thinning properties, allowing the SFO-TA-BGNF complex gel to be extruded through a nozzle, facilitating 3D printing into scaffolds with outstanding shape fidelity. Moreover, the developed composite aerogels exhibited multifaceted features, including NIR-triggered photothermal antibacterial and in vitro photothermal antiosteosarcoma properties. In vitro studies showcased their excellent biocompatibility and osteogenic features as seeded cells successfully differentiated into osteoblasts, promoting bone regeneration in 21 days. Through comprehensive characterizations and biological validations, our antibacterial scaffold demonstrated promise as an exceptional platform for concurrent bone regeneration and bone cancer therapy, setting the stage for their potential clinical application.

Bioactive self-assembling silk fibroin–sericin films for skin tissue engineering

The quest for an ideal wound dressing material has been a strong motivation for researchers to explore novel biomaterials for this purpose. Such explorations have led to the extensive use of silk fibroin (SF) as a suitable polymer for several applications over the years. Unfortunately, another major silk protein—sericin has not received its due attention yet in spite of having favorable biological properties. In this study, we report an approach of blending SF and silk sericin (SS) without the usage of chemical crosslinkers is made possible by the usage of formic acid which evaporates to induce β-sheets formation to form cytocompatible films. Raman spectroscopy confirms the presence of SF/SS components in blend and formation of β-sheet in films. In situ, gelation kinetics studies were conducted to understand the change in gelation properties with addition of sericin into SF. Methyl thiazolyl tetrazolium and live/dead assays were performed to study cellular attachment, viability and proliferation on SF/SS films. The antibacterial properties of SF/SS films were tested using Gram-negative and Gram-positive bacteria. The re-structured SF/SS films were stable, transparent, show good mechanical properties, antibacterial activity and cytocompatibility, therefore can serve as suitable biomaterial candidates for skin regeneration applications.

A Drop-By-Drop Self-Assembled All-Natural Hydrogel as a Desensitizer for Rapid and Enduring Management of Dentin Hypersensitivity.

Dentin hypersensitivity (DH) is a prevalent dental condition arising from the exposure of dentin tubules (DTs), leading to discomfort upon external stimuli. However, achieving swift and profound occlusion of these exposed DTs for immediate and enduring relief remains challenging due to the intricate dentin structure and oral environment. Herein, a pioneering and facile drop-by-drop strategy involving an in situ generated natural supramolecular hydrogel formed by self-assembling silk fibroin (SF) and tannic acid (TA) within the narrow DT space is proposed. When SF and TA aqueous solutions are applied successively to exposed dentin, they penetrate deeply within DTs and coassemble into compact gels, robustly adhering to DT walls. This yields a rapid and compact occlusion effect with an unprecedented depth exceeding 250µm, maintaining stable occlusion efficacy even under rigorous in vitro and in vivo erosion and friction conditions for no less than 21 days. Furthermore, the biocompatibility and effective occlusion properties are verified through cell studies in simulated oral settings and an in vivo rabbit model. This study, for the first time, demonstrates the translational potential of hydrogel-based desensitizers in treating DH with prompt action, superior occlusion depth and enduring treatment benefits, holding promise as clinical-friendly restorative solutions for delicate-structured biosystems.

Self-assembled silk fibroin injectable hydrogels based on layered double hydroxides for spinal cord injury repair

Self-assembled silk fibroin injectable hydrogels based on layered double hydroxides for spinal cord injury repair

Expanding the Potential of Self-Assembled Silk Fibroin as Aerogel Particles for Tissue Regeneration.

A newly produced silk fibroin (SF) aerogel particulate system using a supercritical carbon dioxide (scCO2)-assisted drying technology is herein proposed for biomedical applications. Different concentrations of silk fibroin (3%, 5%, and 7% (w/v)) were explored to investigate the potential of this technology to produce size- and porosity-controlled particles. Laser diffraction, helium pycnometry, nitrogen adsorption-desorption analysis and Fourier Transform Infrared with Attenuated Total Reflectance (FTIR-ATR) spectroscopy were performed to characterize the physicochemical properties of the material. The enzymatic degradation profile of the SF aerogel particles was evaluated by immersion in protease XIV solution, and the biological properties by cell viability and cell proliferation assays. The obtained aerogel particles were mesoporous with high and concentration dependent specific surface area (203-326 m2/g). They displayed significant antioxidant activity and sustained degradation in the presence of protease XIV enzyme. The in vitro assessment using human dermal fibroblasts (HDF) confirm the particles' biocompatibility, as well as the enhancement in cell viability and proliferation.

Self-assembled silk fibroin cross-linked with genipin supplements microbial carbonate precipitation in building material.

The process of microbially induced carbonate precipitation (MICP) is known to effectively improve engineering properties of building materials and so does silk fibroin (SF). Thus, in this study, an attempt was taken to see the improvement in sand, that is, basic building material coupled with MICP and SF. Urease producing Bacillus megaterium was utilized for MICP in Nutri-Calci medium. To improve the strength of SF itself in bacterial solution, it was cross-linked with genipin at the optimized concentration of 3.12 mg/mL. The Fourier transform infrared (FTIR) spectra confirmed the crosslinking of SF with genipin in bacterial solution. In order to understand how such cross-linking can improve engineering properties, sand moulds of 50 mm3 dimension were prepared that resulted in 35% and 55% more compressive strength than the one prepared with bacterial solution with SF and bacterial solution only, respectively with higher calcite content in former one. The FTIR, SEM, x-ray powder diffraction spectrometry and x-ray photoelectron spectroscopy analyses confirmed higher biomineral precipitation in bacterial solution coupled with genipin cross-linked SF. As the process of MICP is proven to replace cement partially from concrete without negatively influence mechanical properties, SF cross-linked with genipin can provide additional significance in developing low-carbon cement-based composites.

Multi-functional Calotropis gigantea fabric using self-assembly silk fibroin, chitosan and nano-silver microspheres with oxygen low-temperature plasma treatment

The present paper describes a novel multi-functional Calotropis gigantea (CG) fabric with durable antibacterial property and comfortable wearability for various applications. First, antibacterial microspheres (AMs) were prepared by self-assembly of silk fibroin (SF), chitosan (CS) and nano-silver microspheres (NSMs). Oxygen low-temperature plasma (OLTP) treatment was used to improve the adhesion between AMs and CG yarn. The AMs modified CG fabric has soft hand feeling, high moisture regain (10.37 ± 1.87%), good breathability, strong wrinkle resistance, and durable antibacterial properties. The antibacterial rate of the modified CG fabric against Escherichia coli (E. Coil) and Staphylococcus aureus (S. Aureus) is as high as 99.9 ± 0.1%, and it still has an antibacterial activity after washing 20 times (90.24 ± 0.65% and 80.25 ± 1.14%, respectively). The in vivo biocompatibility test showed clear signs of angiogenesis at the implantation site in the rats. Thus, this study offers the foundation for the development of functional CG fiber-based biomedical textiles.

Mineralized self-assembled silk fibroin/cellulose interpenetrating network aerogel for bone tissue engineering

The preparation of bioactive materials with biomolecules as templates to control the nucleation and growth of nano-hydroxyapatite (n-HA) crystals is a vital research field in bone tissue engineering. However, meeting the performance requirements of possessing appropriate surface roughness, high porosity, structural stability, adequate mechanical strength, biodegradability and biocompatibility at the same time is the core issue that restricts the development of these biomimetic materials in biosciences as well as medical clinical translation. In this work, a mineralized self-assembled silk fibroin (SF)/cellulose interpenetrating network composite aerogel (M-S-C) material was prepared by freeze-drying using sol-gel and in situ mineralization strategy. The effects of the main factors, such as the surface properties of SF macromolecules and the change of mineralization time, on the n-HA self-assembly process and the property of M-S-C under defined conditions were explored. The properties of M-S-C, including the physicochemical properties, morphology, mechanical property, degradation behavior and in vitro cytotoxicity, were investigated to evaluate its application prospects in bone tissue engineering. M-S-C exhibits the microstructure required for an ideal cancellous bone repair material, porosity up to 99.2%, high thermal stability, moderately adjustable compressive strength (12.7–22.4 MPa), and appreciable in vitro degradation rate. Moreover, M-S-C extracts can significantly accelerate the proliferation of human embryonic kidney cells. This mineralized interpenetrating polymer network aerogel material with excellent comprehensive performance shows potential for application in bone repair and regeneration.

Self-Assembled Inhalable Immunomodulatory Silk Fibroin Nanocarriers for Enhanced Drug Loading and Intracellular Antibacterial Activity.

In this study, a pH-induced self-assembly-based method has been developed to form silk fibroin nanoparticles (SFN-2) with a higher drug loading capacity (21.0 ± 2.1%) and cellular uptake than that of silk fibroin particles produced by a conventional desolvation method (SFN-1). Using the self-assembly method, rifampicin-encapsulated silk fibroin nanoparticles (R-SFN-2) were prepared with a size of 165 ± 38 nm at an optimum pH of 3.8. In silico analysis reveals that at acidic pH, the amino acid side chain charge neutralization of acidic residues, especially GLU64, promotes the formation of additional favorable interactions between the silk fibroin and the drug. The SFN-2 also possess a good aerosol property with a mass median aerodynamic diameter of 3.82 ± 0.71 μm and fine particle fraction of 64.0 ± 1.4%. These SFN-2 particles were selectively endocytosed by macrophages through clathrin- and caveolae-mediated endocytosis with a higher uptake efficiency (66.2 ± 2.1%) and were found to exhibit a sustained drug release in the presence of macrophage intracellular lysates. The cytokine and biomarker expression analyses revealed that SFN-2 could exhibit an immunomodulatory effect by polarizing the macrophages to an initial M1 phase and later M2 phase. Further, R-SFN-2 also exhibited an enhanced and sustained intracellular antibacterial activity against Mycobacterium smegmatis-infected macrophages than free rifampicin. Thus, the self-assembled silk fibroin particles with immunomodulatory action combined with a good aerosol and intracellular drug release property can be an attractive choice as a carrier for developing pulmonary drug delivery systems.

Self-assembled silk fibroin hydrogels: from preparation to biomedical applications

This review describes the methods to prepare self-assembled silk fibroin hydrogels with a focus on gelation mechanisms and biomedical applications.

Hierarchically Organized Biomimetic Architectured Silk Fibroin-Ceramic-Based Anisotropic Hybrid Aerogels for Thermal Energy Management.

Due to the current energy crises, the search for thermal energy management systems based on thermal insulating porous materials has drawn a significant deal of attention. Herein, we demonstrated the thermal insulation and management capabilities of cuttlefish bone mimetic aerogels with hierarchically organized porous structures directly fabricated from surface-modified and self-assembled silk fibroin (SF) biopolymer extracted from Bombyx mori silkworm cocoon biomass; hereafter, the materials developed referred to as X-AeroSF. Exploiting from creating an interpenetrating network of the secondary ceramic components of various one-, two-, and three-dimensional sepiolite (Mg2H2Si3O9·xH2O), MXene (Ti3C2TX), and silica nanostructures inside the self-assembled SF biopolymer and subsequent uni-directional freeze-casting and drying the resulted hydrogels, composites with aerogel features were obtained. The obtained aerogels possess low bulk density (ρb = 0.059-0.090 g cm-3), low thermal conductivities (λ = 0.035-0.042 W m-1 K-1), and high thermal stability (up to ∼260 °C) with multi-modal lamella-bridge porous microstructures found in the cuttlefish bone structure. In addition, the intriguing anisotropy in the X-AeroSF composite porous structure enables thermal dissipation along with the aligned pore directions, thus decreasing the local overheating on the heated side. As a result, an improvement in thermal insulation in the perpendicular direction with respect to the pore lamellae was obtained. Therefore, the exquisite thermal energy management, biodegradability, low bulk density, fire resistivity, together with possible manufacture scalability of X-AeroSF composite, make this material attractive for future practical applications.

Silk fibroin directs the formation of monetite nanocrystals and their assembly into hierarchical composites.

Natural biominerals are usually composite materials produced through mineralization of inorganic crystals within an organic matrix. Silk fibroin is known to be capable of directing the nucleation and growth of hydroxyapatite crystals. Here, we used silk films as the substrate to induce the mineralization of calcium phosphate. We show that the silk fibroin in solution could induce the formation of monetite crystals with a hierarchical structure, which are assembled by well aligned single crystals of monetite. In addition, we show that silk fibroins are incorporated inside the crystals. Therefore, the self-assembly of silk fibroin during the crystallization process is critical for the formation of such hierarchical structures.

The innate immune response of self-assembling silk fibroin hydrogels.

Silk has a long track record of use in humans, and recent advances in silk fibroin processing have opened up new material formats. However, these new formats and their applications have subsequently created a need to ascertain their biocompatibility. Therefore, the present aim was to quantify the haemocompatibility and inflammatory response of silk fibroin hydrogels. This work demonstrated that self-assembled silk fibroin hydrogels, as one of the most clinically relevant new formats, induced very low blood coagulation and platelet activation but elevated the inflammatory response of human whole blood in vitro. In vivo bioluminescence imaging of neutrophils and macrophages showed an acute, but mild, local inflammatory response which was lower than or similar to that induced by polyethylene glycol, a benchmark material. The time-dependent local immune response in vivo was corroborated by histology, immunofluorescence and murine whole blood analyses. Overall, this study confirms that silk fibroin hydrogels induce a similar immune response to that of PEG hydrogels, while also demonstrating the power of non-invasive bioluminescence imaging for monitoring tissue responses.

Chitosan/polydopamine layer by layer self-assembled silk fibroin nanofibers for biomedical applications

Silk fibroin (SF) is increasingly needed in tissue engineering for its superior biocompatibility. However, the practical applications of pure SF biomaterials confront bacterial infection problems. In this study, chitosan (CS) and polydopamine (PDA) were introduced into electrospun nanofibrous SF mats through layer-by-layer self-assembly (LBL) to obtain enhanced antibacterial ability and cytocompatibility. The surface morphology and composition analysis confirmed the successful deposition. After depositing 15 bilayers, the tensile modulus of the mats in wet condition increased from 2.16 MPa (pristine SF mats) to 4.89 MPa. A trend towards better hydrophilicity performance was also recorded with more bilayers coating on the mats. Besides, LBL structured mats showed improved antibacterial ability of more than 98 % against E. coli and S. aureus. In addition, advancement in biocompatibility was observed during the proliferation experiment of L929 cells. Overall, the deposition of CS and PDA may further expand the use of SF in biomedical field.

RETRACTED ARTICLE: Glyoxal modification mediates conformational alterations in silk fibroin: Induction of fibrillation with amyloidal features

Silkwormsilk protein fibroin is widely exploited to develop novel silk-based biomaterials due to its stable β-sheet structure, providing high crystallinity and tensile strength. The polymorphic behaviour of silk fibroin provides a window to modulate its structural transitions during self-assembly for different functional outcomes. Most studies are therefore mainly focused on formation of well-developed β-sheet structure and self-assembly of silk fibroin which are regulated by many parameters. Glyoxal, a highly reactive α-oxoaldehyde, reacts with different proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. Considering the significance of protein modification by glyoxal-derived AGEs, in the present study the effect of glyoxal (250, 500 and 1000 μM) on the structure of silk fibroin has been investigated. CD and fluorescence studies reveal that higher concentrations of the α-oxoaldehyde induce considerable alterations of secondary and tertiary structure of the protein leading to aggregation following incubation with for 3 weeks. The aggregates exhibit fibrillar morphology with amyloidal nature as evident from SEM, FTIR and XRD experiments. The findings highlight that glycationinduced modification can be a possible approach for modulating the conformation of the silk protein which may be relevant in connection to clinical, biomedical or synthetic biology based applications.

Stabilization of black rice anthocyanins by self-assembled silk fibroin nanofibrils: Morphology, spectroscopy and thermal protection

Nanofibrillated proteins possess diverse techno-functional properties are considered as matrices for stabilizing bioactive agents. In this study, the morphology and interaction of silk fibroin nanofibrils (SNFs) with black rice anthocyanins (ACN) were investigated through multiple microscopy and spectroscopic techniques. The impact of SNFs on the thermal stability of ACN was analyzed. Nanofibrils with diameter less than 20 nm were self-assembled from silk fibroin (SF) lyophilized powder solution at pH 6.0. A static model occurred in the interactions of cyanidin-3-O-glucoside (C3G, the major anthocyanin in black rice) with SF and SNFs. C3G binding altered the secondary structures of SF and SNFs. Moreover, the thermal retention rates of ACN combined with SF and SNFs were significantly (P < 0.05) higher than that of ACN alone followed the order of SNFs-ACN (60.87%) > SF-ACN (57.47%) > ACN alone (40.33%) after heating 120 min at 80 °C and SNFs-ACN (50.42%) > SF-ACN (32.81%) > ACN alone (23.64%) after heating 120 min at 90 °C. The half-life (t1/2) of thermal degradation of ACN was elongated in SNFs-ACN complex compared with that in SF-ACN complex. These results showed that SNFs effectively binding with C3G and exhibit better protective effects on the thermal stability of ACN than SF.

Silk Fibroin for CpG Oligodeoxynucleotide Delivery.

CpG oligodeoxynucleotides (ODNs) have attracted increasing attention as immunotherapeutic agents. However, efficient transfection of CpG ODNs into the immune cells remains a big challenge. In this study, for the first time, we reported that silk fibroin (SF) could function as an efficient carrier for CpG ODNs. A novel strategy was developed to prepare SF-CpG ODNs nanoparticles (NPs) based on self-assembly of SF. The as-prepared SF-CpG NPs were spherical in shape and were uniformly dispersed. SF-CpG NPs exhibited good stability and biocompatibility. SF-CpG NPs possessed significantly enhanced (7 folds) cellular uptake compared with CpG ODNs. Release of CpG ODNs from SF-CpG NPs was accelerated in environment-mimicking TLR9-localized endo/lysosome. SF-CpG NPs stimulated about four folds higher levels of immune cytokines and nitric oxide compared with CpG ODNs. Our results suggested that SF notably improved the CpG ODNs delivery. SF-CpG NPs have strong potential in immunotherapy.

In Vivo Evaluation of Engineered Self-Assembling Silk Fibroin Hydrogels after Intracerebral Injection in a Rat Stroke Model.

Targeting the brain cavity formed by an ischemic stroke is appealing for many regenerative treatment strategies but requires a robust delivery technology. We hypothesized that self-assembling silk fibroin hydrogels could serve as a reliable support matrix for regeneration in the stroke cavity. We therefore performed in vivo evaluation studies of self-assembling silk fibroin hydrogels after intracerebral injection in a rat stroke model. Adult male Sprague-Dawley rats (n = 24) underwent transient middle cerebral artery occlusion (MCAo) 2 weeks before random assignment to either no stereotaxic injection or a stereotaxic injection of either self-assembling silk fibroin hydrogels (4% w/v) or PBS into the lesion cavity. The impact on morbidity and mortality, space conformity, interaction with glial scar, interference with inflammatory response, and cell proliferation in the lesion cavity were examined for up to 7 weeks by a blinded investigator. Self-assembling hydrogels filled the stroke cavity with excellent space conformity and presented neither an overt microglial/macrophage response nor an adverse morbidity or mortality. The relationship between the number of proliferating cells and lesion volume was significantly changed by injection of self-assembling silk hydrogels. This in vivo stroke model confirmed that self-assembling silk fibroin hydrogels provide a favorable microenvironment as a future support matrix in the stroke cavity.

Compressible, Thermally Insulating, and Fire Retardant Aerogels through Self-Assembling Silk Fibroin Biopolymers Inside a Silica Structure-An Approach towards 3D Printing of Aerogels.

Thanks to the exceptional materials properties of silica aerogels, this fascinating highly porous material has found high-performance and real-life applications in various modern industries. However, a requirement for a broadening of these applications is based on the further improvement of the aerogel properties, especially with regard to mechanical strength and postsynthesis processability with minimum compromise to the other physical properties. Here, we report an entirely novel, simple, and aqueous-based synthesis approach to prepare mechanically robust aerogel hybrids by cogelation of silk fibroin (SF) biopolymer extracted from silkworm cocoons. The synthesis is based on sequential processes of acid catalyzed (physical) cross-linking of the SF biopolymer and simultaneous polycondensation of tetramethylorthosilicate (TMOS) in the presence of 5-(trimethoxysilyl)pentanoic acid (TMSPA) as a coupling agent and subsequent solvent exchange and supercritical drying. Extensive characterization by solid-state 1H NMR, 29Si NMR, and 2D 1H-29Si heteronuclear correlation (HETCOR) MAS NMR spectroscopy as well as various microscopic techniques (SEM, TEM) and mechanical assessment confirmed the molecular-level homogeneity of the hybrid nanostructure. The developed silica-SF aerogel hybrids contained an improved set of material properties, such as low density (ρb,average = 0.11-0.2 g cm-3), high porosity (∼90%), high specific surface area (∼400-800 m2 g-1), and excellent flexibility in compression (up to 80% of strain) with three orders of magnitude improvement in the Young's modulus over that of pristine silica aerogels. In addition, the silica-SF hybrid aerogels are fire retardant and demonstrated excellent thermal insulation performance with thermal conductivities (λ) of 0.033-0.039 W m-1 K-1. As a further advantage, the formulated hybrid silica-SF aerogel showed an excellent printability in the wet state using a microextrusion-based 3D printing approach. The printed structures had comparable properties to their monolith counterparts, improving postsynthesis processing or shaping of the silica aerogels significantly. Finally, the hybrid silica-SF aerogels reported here represent significant progress for a mechanically customized and robust aerogel for multipurpose applications, namely, as a customized thermal insulation material or as a dual porous open-cell biomaterial used in regenerative medicine.

Investigate the Effect of Thawing Process on the Self-Assembly of Silk Protein for Tissue Applications.

Biological self-assembly is a process in which building blocks autonomously organize to form stable supermolecules of higher order and complexity through domination of weak, noncovalent interactions. For silk protein, the effect of high incubating temperature on the induction of secondary structure and self-assembly was well investigated. However, the effect of freezing and thawing on silk solution has not been studied. The present work aimed to investigate a new all-aqueous process to form 3D porous silk fibroin matrices using a freezing-assisted self-assembly method. This study proposes an experimental investigation and optimization of environmental parameters for the self-assembly process such as freezing temperature, thawing process, and concentration of silk solution. The optical images demonstrated the possibility and potential of −80ST48 treatment to initialize the self-assembly of silk fibroin as well as controllably fabricate a porous scaffold. Moreover, the micrograph images illustrate the assembly of silk protein chain in 7 days under the treatment of −80ST48 process. The surface morphology characterization proved that this method could control the pore size of porous scaffolds by control of the concentration of silk solution. The animal test showed the support of silk scaffold for cell adhesion and proliferation, as well as the cell migration process in the 3D implantable scaffold.