Silk Fibroin Concentration Research Articles

The influence of plasticizer on the mechanical, structural, thermal and strain recovery properties following stress-relaxation process of silk fibroin/sodium alginate biocomposites for biomedical applications

The influence of plasticizer glycerol (GLY) on the mechanical, structural, and thermal properties of silk fibroin (SF)/sodium alginate (SA) biocomposite films was investigated in detail. As the SF/SA ratio increased up to 65%, the SF content significantly improved the Tensile strength (σT), Young's modulus (Ey) but reduced the elongation at break (εb). To modify and enhance the elasticity and flexibility of the biocomposite films, the GLY as a plasticizer was used at different ratio from 20 to 50% for each SF/SA biocomposite films. Although the extensibility of the films was improved greatly with increasing GLY ratio, σT and Ey reduced significantly. The effect was observed more apparently for the GLY ratio starting from 35%. It was also shown that crystallinity index in the Amide I region increased as the SF/SA ratio increased to 65%. Increasing SF content improved the thermal stability of the SF/SA biocomposites. The XRD results showed that crystallinity was increased as SF/SA ratio increased. Stress-relaxation of SF/SA (30%) biocomposite films plasticized with GLY revealed that each kind of plasticized films showed a viscoelastic behavior and a fast relaxation in the first stage (1–2 min) of the processes and then continued slowly. The GLY increased the extensibility and elasticity limit of the SF/SA (30%) composite films. During the strain recovery processes, the plasticized composite films recovered completely in a quite shorter time than that of unplasticized films. It was observed higher the GLY content, the recovery times became shorter.

Preparation and Characterization of Cellulose/Silk Fibroin Composite Microparticles for Drug-Controlled Release Applications

Microparticles derived from biomaterials are becoming increasingly popular for application in drug delivery systems. In this study, the water-in-oil (W/O) emulsification–diffusion method was used to create cellulose (C), silk fibroin (SF), and C/SF composite microparticles. We then observed the morphology of all obtained microparticles using scanning electron microscopy (SEM), evaluated their functional groups using attenuated total reflection–Fourier transform infrared spectroscopy (ATR-FTIR), and conducted thermogravimetric analysis using a thermogravimetric analyzer (TGA). SEM micrographs indicated that the native SF microparticles have the highest spherical shape with smooth surfaces. With blue dextran, the C microparticle was smaller than the native microparticle, while the drug-loaded SF microparticles were larger than the native microparticle. The morphological surfaces of the C/SF composite microparticles were varied in shape and surface depending on the C/SF ratio used. The spherical shape of the C/SF composite microparticle increased as the SF content increased. Furthermore, the size of the drug-loaded C/SF composite microparticles increased when the SF content gradually increased. The significant functional groups in the C and SF structures were identified based on the ATR-FTIR data, and a suggestion was made regarding the interaction between the functional groups of each polymer. When compared to both native polymers, the C/SF composite microparticles exhibit improved thermal stability. XRD patterns indicated that all prepared particles have crystalline structures and are directly affected by the released profile. The C/SF composite microparticle at a 1:3 ratio had the lowest drug release content, whereas the hydrophilicity of the C microparticle affected the highest drug release content. As a result, one crucial factor affecting the medication released from the microparticle is its structure stability. According to the obtained results, C, SF, and C/SF composite microparticles show promise as delivery systems for drugs with controlled release.

Vancomycin-Loaded Silk Fibroin/Calcium Phosphate/Methylcellulose-Based In Situ Thermosensitive Hydrogel: A Potential Function for Bone Regeneration

This study explores the efficacy of a vancomycin-loaded silk fibroin/calcium phosphate/methylcellulose-based in situ thermosensitive hydrogel (VM-SF/CaP/MC) in promoting the osteogenic differentiation of preosteoblast cells. Three VM-SF/CaP/MC formulations with varying low (L) and high (H) concentrations of silk fibroin (SF) and calcium phosphate (CaP) were prepared: VM-HSF/LCaP/MC, VM-LSF/HCaP/MC, and VM-HSF/HCaP/MC. These hydrogels significantly enhanced MC3T3-E1 cell migration and proliferation in a dose- and time-dependent manner, achieving complete cell migration within 48 h. In addition, they significantly promoted alkaline phosphatase activity, collagen content, and mineralization in MC3T3-E1 cells, indicating their potential for osteogenesis. Among the hydrogel formulations, the VM-HSF/HCaP/MC hydrogel, with high SF and CaP content, demonstrated superior potential in promoting the osteogenic differentiation of MC3T3-E1 cells. It exhibited the highest ALP activity (11.13 ± 0.91 U/mg protein) over 14 days, along with increased collagen content (54.00 ± 1.71 µg/mg protein) and mineralization (15.79 ± 1.48 mM) over 35 days. Therefore, this formulation showed a promising candidate for clinical application in localized bone regeneration, particularly in treating osteomyelitis.

Two-dimensional silk.

Despite the promise of silk-based devices, the inherent disorder of native silk limits performance. Here, we report highly ordered two-dimensional silk fibroin (SF) films grown epitaxially on van der Waals (vdW) substrates. Using atomic force microscopy, nano-Fourier transform infrared spectroscopy, and molecular dynamics, we show that the films consist of lamellae of SF molecules that exhibit the same secondary structure as the nanocrystallites of native silk. Increasing the SF concentration results in multilayers that grow either by direct assembly of SF molecules into the lamellae or, at high concentrations, along a two-step pathway beginning with a disordered monolayer that then crystallizes. Scanning Kelvin probe measurements show that these films substantially alter the surface potential; thus, they provide a platform for silk-based electronics on vdW solids.

Highly Porous, Ultralight, Biocompatible Silk Fibroin Aerogel-Based Triboelectric Nanogenerator.

This study presents the fabrication of an ultralight, porous, and high-performance triboelectric nanogenerator (TENG) utilizing silk fibroin (SF) aerogels and PDMS sponges as the friction layer. The transition from two-dimensional film friction layers to three-dimensional porous aerogels significantly increased the specific surface area, offering an effective strategy for designing high-performance SF aerogel-based TENGs. The TENG incorporating the porous SF aerogel exhibited optimal output performance at a 3% SF concentration, achieving a maximum open circuit voltage of 365 V, a maximum short-circuit current of 11.8 μA, and a maximum power density of 7.52 W/m2. In comparison to SF-film-based TENGs, the SF-aerogel based TENG demonstrated a remarkable 6.5-fold increase in voltage and a 4.5-fold increase in current. Furthermore, the power density of our SF-based TENG surpassed the previously reported optimal values for SF-based TENGs by 2.4 times. Leveraging the excellent mechanical stability and biocompatibility of TENGs, we developed an SF-based TENG self-powered sensor for the real-time monitoring of subtle biological movements. The SF-based TENG exhibits promising potential as a wearable bioelectronic device for health monitoring.

Development of Ultraviolet-Shielding Bamboo/Silk Fibroin Hybrid Films with Good Mechanical Properties: A Proof Study on Human Keratinocyte Cells.

In this study, we report the preparation and characterization of water-stable films with UV-shielding and good mechanical properties, exploiting the synergistic effect of regenerated silk fibroin and bamboo-derived cellulose. Silk fibroin (SF)/bamboo (B) hybrid films are achieved by solubilizing both silk and bamboo fibers in formic acid with added CaCl2. Infrared spectroscopy indicates that SF, when combined with bamboo, undergoes a conformational transition, providing evidence of an increase in SF crystallinity. Exploiting the intrinsic absorption of SF in the ultraviolet region, UV-Vis spectroscopy was used to assess the glass transition temperature (Tg) of SF/B films, showing a decrease in Tg by increasing the SF content. The addition of 10 wt% SF to the B matrix improved the elastic modulus by about 10% while conserving the strain at break with respect to the neat B films, increasing the UV shielding properties, while water absorption suggested the material's hydrophilic and swelling capacity even after one month. The hybrid films showed, under solar irradiation, a photoprotective behavior on keratinocyte human cells by increasing cellular viability. These findings may find potential applications in functional fabrics.

Piezoelectric fibers based on silk fibroin with excellent output performance

Abstract The self-powered tissue engineering scaffold with good biocompatibility is of great significance for stimulating nerve cell growth. In this study, silk fibroin (SF)-based fibers with regulatable structure and piezoelectric performance are fabricated by dry-spinning and post-treatment. The concentration of SF and calcium ion in spinning dope and the post-treatment affect the conformation transition and crystallinity of SF. As a result, the SF fibers exhibit high piezoelectric coefficient d 33 (3.24 pm/V) and output voltage (∼ 27 V). Furthermore, these piezoelectric fibers promote the growth of PC-12 cells, demonstrating the promising potential for nerve repair and other energy harvester.

Silk Fibroin-Enriched Bioink Promotes Cell Proliferation in 3D-Bioprinted Constructs.

Three-dimensional (3D) bioprinting technology enables the controlled deposition of cells and biomaterials (i.e., bioink) to easily create complex 3D biological microenvironments. Silk fibroin (SF) has recently emerged as a compelling bioink component due to its advantageous mechanical and biological properties. This study reports on the development and optimization of a novel bioink for extrusion-based 3D bioprinting and compares different bioink formulations based on mixtures of alginate methacrylate (ALMA), gelatin and SF. The rheological parameters of the bioink were investigated to predict printability and stability, and the optimal concentration of SF was selected. The bioink containing a low amount of SF (0.002% w/V) was found to be the best formulation. Light-assisted gelation of ALMA was exploited to obtain the final hydrogel matrix. Rheological analyses showed that SF-enriched hydrogels exhibited greater elasticity than SF-free hydrogels and were more tolerant to temperature fluctuations. Finally, MG-63 cells were successfully bioprinted and their viability and proliferation over time were analyzed. The SF-enriched bioink represents an excellent biomaterial in terms of printability and allows high cell proliferation over a period of up to 3 weeks. These data confirm the possibility of using the selected formulation for the successful bioprinting of cells into extracellular matrix-like microenvironments.

Silk fibroin/vitreous humor hydrogel scaffold modified by a carbodiimide crosslinker for wound healing.

Natural-derived biomaterials can be used as substrates for the growth, proliferation, and differentiation of cells. In this study, bovine vitreous humor as a biological material was cross-linked to silk fibroin with different concentration ratios to design a suitable substrate for corneal tissue regeneration. The cross-linked samples were evaluated with different analyses such as structural, physical (optical, swelling, and degradation), mechanical, and biological (viability, cell adhesion) assays. The results showed that all samples had excellent transparency, especially those with higher silk fibroin content. Increasing the ratio of vitreous humor to silk fibroin decreased mechanical strength and increased swelling and degradation, respectively. There was no significant difference in the toxicity of the samples, and with the increase in vitreous humor ratio, adhesion and cell proliferation increased. Generally, silk fibroin with vitreous humor can provide desirable characteristics as a transparent film for corneal wound healing.

Mild preparation of hyaluronic acid/silk fibroin sponges by modified crosslinking method

The composites composed of hyaluronic acid (HA) and silk fibroin (SF) exhibit great potential in diverse biomedical applications. However, the utilization of commercial crosslinkers such as 1,4-butanediol diglycidyl ether (BDDE) for crosslinking HA typically necessitates harsh conditions involving strong alkaline, which greatly limits its potential applications. In this study, a mild modified approach was developed to fabricate HA/SF blend sponges crosslinked by BDDE without alkaline conditions. The blend solutions were cryo-concentrated to induce crosslinking reactions. The mechanism of freezing crosslinking was elucidated by investigating the effects of ice crystal growth and HA molecular weight on the degree of crosslinking. The results revealed that HA achieved efficient crosslinking when its molecular weight exceeds 1000 kDa and freezing temperatures ranged from −40 °C to −20 °C. After introducing SF, multiple crosslinks were formed between SF and HA chains, producing water-stable porous sponges. The SEM results demonstrated that the introduction of SF effectively enhanced the interconnectivity between macropores through creating subordinate holes onto the pores wall. Raising the SF content significantly enhanced compression strength, resistance to enzymatic degradation and cell viability of blend sponges. This study provides a novel strategy for designing bioactive HA/SF blend sponges as substitutes for tissue repair and wound dressing.

A Novel Approach towards the Preparation of Silk-Fibroin-Modified Polyethylene Terephthalate with High Hydrophilicity and Stability

Silk fibroin (SF) has been widely used in biomedical applications for the hydrophilicity modification of high molecular polymer materials. However, the challenge remains to immobilize SF with high structure stability and strong adhesion strength between SF and the substrate. Here, we propose an effective two-step process for modifying polyethylene terephthalate (PET) with SF: dipping PET film in SF solution and subsequently carrying out plasma-assisted deposition in SF aerosol. The structure and property analysis revealed that the SF-modified PET (PET-SF) prepared using the two-step method exhibited superior structural stability and stronger adhesion strength compared to the dip-coating method and the plasma-assisted deposition method. In addition, PET-SF prepared using the two-step method resulted in a higher concentration of SF and an increased content of active groups on its surface, enhancing its hydrophilicity compared to the other two methods. Additionally, the influence of dipping time and deposition time in the two-step method was investigated. The results demonstrated that the dipping time for 6 h and the deposition time for 3 min resulted in maximum SF grafting amount with a highly stable structure. Furthermore, the PET-SF exhibited satisfactory hydrophilicity when the deposition time was more than 3 min and showed the most hydrophilicity surface at 8 min.

Silk Fibroin-Laponite Porous Microspheres as Cell Microcarriers for Osteogenic Differentiation.

Silk fibroin (SF) has garnered significant attention as a natural polymer for fabricating porous scaffolds in various engineering applications. However, the limited osteoinductive property of SF has hindered its efficacy in bone repair applications. In this study, we constructed an SF-based injectable porous microcarrier that is doped with laponite (LAP), containing magnesium ions (Mg2+). The influence of freezing temperatures and concentrations of SF and LAP on the structural parameters of SF-LAP microcarriers was investigated. The SF-LAP microcarrier exhibited a porosity of 76.7 ± 1.2% and a controlled pore size of 24.6 ± 4.0 μm. At the 6 weeks of in vitro degradation test, a mild alkaline level in culture medium containing SF-LAP microcarriers was detected. The release of Mg2+ from the SF-LAP microcarrier was maintained at a concentration within the range of 1.2-2.3 mM during the 6 weeks. The seeded human adipose-derived stem cells in the SF-LAP microcarrier demonstrated a significant enhancement in osteogenic differentiation compared with cells seeded in the pure SF microcarrier, as evidenced by quantitative alkaline phosphatase activity and the expression of osteogenic marker genes. These findings underscore the potential of the SF-LAP microcarrier as an ideal cell carrier in the treatment of bone defects.

Analyzing the interactions and miscibility of silk fibroin/mucin blends

AbstractMucin, a glycoprotein with viscoelastic properties, and silk fibroin, a protein excreted from silkworms with properties of thermal and mechanical resistance, have been probed as building blocks in the design of biomaterials. Aiming to evaluate the interaction and miscibility between mucin and fibroin, we synthesized silk fibroin and mucin (SF/MU) blends for biomedical applications. The morphological analysis of the SF/MU blends showed the presence of two phases, suggesting a partial miscibility between the polymers. The degradation temperature of the SF/MU blends increased with an increase in the silk fibroin content, indicating that silk fibroin contributed to the thermal stability of the blends. The glass transition temperature of the SF/MU blends lay between the values of the pure polymers. The Fourier‐transform infrared spectroscopy results pointed out that the interaction between fibroin and mucin occurred between the amine group of silk fibroin and mucin carboxyl and hydroxyl groups. The outcomes of this work provided essential information on the miscibility of the SF/MU blends. These findings will be critical for further studies with fibroin and mucin‐based biomaterials, especially in mucoadhesive systems and wound healing applications.

Pickering emulsions by combining nanocellulose/nanochitin and silk fibroin: Silk fibroin as a stabilizer or an emulsifier

Silk fibroin (SF) was considered as emulsifier due to its inherent amphiphilicity. Nevertheless, SF was also speculated to act as a stabilizer in emulsions because of its self-assembly in the interfaces and formation of viscoelastic surface or interfacial layers. Here, the emulsions were produced by mixing SF with 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized nanocellulose (TOCN) or partially-deacetylated nanochitin (DEChN) as a continuous phase. The performance of emulsions was examined in regard to the concentrations of TOCN, DEChN, and SF. And comparative emulsions stabilized by conventional surfactants alongside TOCN or DEChN were prepared. Emulsion stability and droplet size were influenced by TOCN or DEChN concentration, while SF concentration played a minor role. SF exhibited stability enhancement compared to conventional surfactants. Overall, emulsions remained highly stable over 30 days with substantial TOCN, DEChN, and SF (≥ 0.4%) concentrations. This work provides valuable insights into choosing stabilizers or emulsifiers for use in the emulsion.

Poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) as an insulin carrier in silk fibroin hydrogels for transdermal delivery via iontophoresis.

In this study, silk fibroin (SF) was utilized as the starting material to fabricate physically crosslinked hydrogels. Poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) was synthesized and characterized as a drug carrier, with insulin as the model drug. PEDOT:PSS, with a high electrical conductivity of 1666 ± 49 S cm-1, interacted with insulin molecules via electrostatic interaction by replacing the dopant PSS molecules. Insulin-loaded PEDOT:PSS embedded in the SF hydrogel resulted in an increase in the degree of swelling, pore size, and mesh size of the hydrogel. In the in vitro release and release-permeation experiments, the amounts of insulin release and release-permeation were investigated using a modified Franz diffusion cell, under the effects of SF concentrations, electric fields, and pH values. The amounts of insulin release and release-permeation from the pristine SF hydrogel and the PEDOT:PSS/SF hydrogel followed the power laws with the scaling exponents close to 0.5, indicating the Fickian diffusion or the concentration gradient. Under electric fields, with or without PEDOT:PSS used as the drug carrier, the insulin amount and diffusion coefficient were shown to increase with the increasing electric field due to the electro-repulsive forces between the cathode and insulin molecules and SF chains, electroosmosis, and SF matrix swelling. The SF hydrogel and PEDOT:PSS as the drug carrier are demonstrated herein as new components in the transdermal delivery system for the iontophoretically controlled insulin basal release applicable to diabetes patients.

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.

Effect of cocooning conditions on the structure, carbon and nitrogen isotope ratios of silks.

The stable isotope technique provides the possibility to trace ancient textiles because the technique is associated with advantages such as trace indication, fast detection, and accurate results. Since different cocooning conditions may impact cocoons even under identical habitats, it is important to investigate the effects of different cocooning temperatures and humidity on the isotope incorporation values in the cocoons. In this study, silk fibers were reeled under different conditions of temperature and humidity, followed by analysis of the secondary structure of cocoon proteins and isotope incorporation patterns. We found that the deviations in carbon isotope values of silk under different cocooning conditions could reach up to 0.76‰, while the deviation in carbon isotope values at different locations of a single silk was 2.75‰. Further, during the cocooning process, depletion of the 13C-isotope at different locations of the silk fibers was observed, reducing the δ13C values. We proposed that the changes in carbon isotopes in silk were related to the content of sericin and silk fibroin in silk. Finally, we did not observe a significant difference in isotope ratios in degummed cocoons. In summary, the 13C isotope was enriched in sericin, whereas 15N was enriched in fibroin, and these findings provide basic information for tracing the provenance of silks.

Silk fibroin scaffolds with stable silk I crystal and tunable properties

It is crucial to develop a three-dimensional scaffold with tunable physical properties for the biomedical application of silk fibroin (SF). The crystallization of polymers dictates their bulk properties. The presence of two unique crystal types, silk I and silk II, provides a mechanism for controlling the properties of SF biomaterials. However, it remains challenging to manipulate silk I crystallization. In this study, we demonstrate the stability and tunability of SF scaffolds with silk I structure prepared by a freezing-annealing processing. The porous structure and mechanical properties of the scaffolds can be readily regulated by SF concentration. XRD results show that the typical peaks representing silk I do not shift when subjected to various post-treatments, such as ethanol soaking, heating, water vapor annealing, UV irradiation, and high-temperature/high-pressure, indicating the stability of silk I crystal type. Moreover, the crystallization kinetics can be regulated by changing annealing time. This physical process can regulate the transition from non-crystalline to silk I, in turn controlling the mechanical properties and degradation rate of the SF scaffolds. Our result show that this green, all-aqueous strategy provides new directions for the design of SF-based biomaterials with controllable properties.

Effect of silk fibroin scaffold loaded with 17-β estradiol on the proliferation and differentiation of BMSCs.

In this study, different concentrations of 17-β estradiol silk fibroin (SF)porous scaffolds (SFPS) were prepared using freeze-drying technique, with a hope for optimal concentration and apply it locally to the bone defect area. In this study, the porous scaffold morphology structure was characterized by SEM, FTIR and universal capacity testing machines, and the invitro cytocompatibility and biological activity of scaffold materials were studied by cell adhesion, viability and proliferation experiments. The results showed that SFPS boasts better physicochemical properties, while 17-β estradiol SF scaffolds with low concentrations of 10-10mol/L and 10-12mol/L had more growth and proliferation of SF scaffolds with higher concentrations, and 10-10mol/L was the optimal concentration of 17-β estradiol SFPS, which was more conducive to cell adhesion and proliferation. On the other hand, after osteogenesis induction of BMSCs inoculated on 17-β estradiol SFPS at different concentrations, it was found that the expression of alkaline phosphatase in BMSCs on different concentrations of 17-β estradiol porous scaffolds was not large. No conflict of interest exits in the submission of this manuscript.

Extracted Silk Fibroin‐Cellulose Acetate Nanofibrous Scaffolds for Tissue Engineering Applications

AbstractThe extracellular matrix of cells consists of polysaccharides and proteins which provide support for cell attachment, movement, and proliferation thus regulating cellular activities. After a wound, cells require similar support to rebuild tissues with original characteristics. Silk fibroin and cellulose have thus been used to engineer nanofibrous mats through electrospinning process for tissue engineering applications. Silk fibroin (SF) is extracted from B. mori cocoons and cellulose acetate (CA) is obtained from textile‐based yarn. The resulting SF:CA nanofibrous mats have fiber diameters in the range of 0.26 µm (SF:CA 40:60) to 0.81 µm (SF:CA 10:90), tensile modulus in the range of 74.5 MPa (100% CA) to 458 MPa (SF:CA 80:20) and showed good material miscibility as evidenced through Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) analysis. In vitro, nanofibrous mats with high silk fibroin content maintained murine macrophages in the non‐inflammatory stage and encouraged the movement of fibroblasts cells.