Fibroin Solution Research Articles

Radiation-processed silk fibroin micro- /nano-gels as promising antioxidants: Electron beam treatment and physicochemical characterization

This study explored some aspects of the radiation-induced chemical modification of silk fibroin in pure water solution as a means to synthesize micro- /nano-gels with antioxidant properties and low cytotoxicity. The amino acid composition of the starting SF material was analyzed by NMR spectroscopy. Special attention was paid to the initial content and fate upon irradiation of the tyrosine residues. The effects of electron beam irradiation on aqueous solutions of silk fibroin was assessed by measuring structural and dimensional changes of SF particles in the solutions as a function of dose. This revealed the gradual decrease of the average hydrodynamic dimensions, initially in the 300–500 nm range for the unirradiated samples to values smaller than 100 nm for doses exceeding 20 kGy. Radical scavenging efficiency of the obtained micro- /nano-gels was assessed by DPPH-based spectrophotometric measurements which revealed the gradual enhancement of the antioxidant properties as a function of dose, yet with a correlated reduction of the scavenging kinetics. Cell viability tests have shown that introducing either the unirradiated or 10 kGy-irradiated SF micro-/nano-gels confirmed the low cytotoxicity of SF-based hydrogel materials, opening new perspectives for the radiation-mediated preparation of SF-based bioactive materials for biomedical and cosmetic applications.

3D bioprinted silk-reinforced Alginate-Gellan Gum constructs for cartilage regeneration

Even though a substantial amount of research has been undertaken in the domains of bioprinting over the last few years, various challenges exist concerning printability. One of the grave challenges in developing a bioink with superior printability is the constraint of material availability, cross-linking, and other processing parameters that should warrant adequate functioning post bioprinting. This study demonstrates the development of a multicomponent shear-thinning bioink comprising Alginate and Gellan Gum with good mechanical, biological, and adequate printing properties. The addition of two types of silk nanoparticles (SNP), native and regenerated, acted as a reinforcement to the bioink, enhancing its printability. Additionally, silk fibroin solution was added to the bioink that served as a control apart from SNP. The addition of cationic SNP (native) to the anionic polymer mixture of Alginate-Gellan Gum exhibited a remarkable increase in the viscosity, storage modulus and mechanical property compared to the other experimental sets. Molecular characterization studies involving Real-Time PCR, gene expression, immunofluorescence, and histology analysis depicted enhanced chondrogenesis in the bioprinted construct containing the silk fibroin solution. The incorporated SNPs resulted in extracellular matrix secretion towards chondrogenesis of articular cartilage. Taken together, the method's broad applicability is likely to propel the field closer to the goal of precision bioprinting.

A Biological Study of Composites Based on the Blends of Nanohydroxyapatite, Silk Fibroin and Chitosan.

In this work, the biological properties of three-dimensional scaffolds based on a blend of nanohydroxyapatite (nHA), silk fibroin (SF), and chitosan (CTS), were prepared using a lyophilization technique with various weight ratios: 10:45:45, 15:15:70, 15:70:15, 20:40:40, 40:30:30, and 70:15:15 nHA:SF:CTS, respectively. The basic 3D scaffolds were obtained from 5% (w/w) chitosan and 5% silk fibroin solutions and then nHA was added. The morphology and physicochemical properties of scaffolds were studied and compared. A biological test was performed to study the growth and osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). It was found that the addition of chitosan increases the resistance properties and extends the degradation time of materials. In vitro studies with human mesenchymal stem cells found a high degree of biotolerance for the materials produced, especially for the 20:40:40 and 15:70:15 (nHa:SF:CTS) ratios. The presence of silk fibroin and the elongated shape of the pores positively influenced the differentiation of cells into osteogenic cells. By taking advantage of the differentiation/proliferation cues offered by individual components, the composites based on the nanohydroxyapatite, silk fibroin, and chitosan scaffold may be suitable for bone tissue engineering, and possibly offer an alternative to the widespread use of collagen materials.

Silk fibroin and Nettle extract promote wound healing in a rat model: A histological and morphometrical study

IntroductionConsidering the anti-inflammatory, antimicrobial ability, and antioxidant effects besides stimulating ability of silk fibroin (SF) in cell migration and proliferation of Nettle, the current study aimed to investigate the effect of Nettle leaf extract (NLE) and SF on histology, morphometrical parameters and apoptosis on the wound in the rat model. Materials and methodsWistar rats are divided into 5 groups, including 1-control (rats with healthy skin and no treatment); 2-wound (without any treatment); 3-SF (administration of silk fibroin solution for 14 consecutive days); 4- Nettle (administration of Nettle ointment for 14 consecutive days), and 5- Eucerin group (administration of Eucerin substance for 14 consecutive days) and then assessed wound area by photography, angiogenesis, inflammation, and thickness of epidermis using hematoxylin and eosin (H&E) staining, collagen deposition, and structure of dermis layers evaluated by Masson's trichrome staining and the apoptosis index determined by tunnel assay on days 7, 14 and 21. Resultsphotographic illustrations showed that the wound surface environment on the seventh day in group 4 was significantly different from group 2 (p < 0.002). The rate of wound healing on the fourteenth day was higher in groups 3 and 4 than in group 2 (p < 0.001). Also, at this time, group 4 was significantly different from group 3 and group 5 (p = 0.003 and p = 0.000, respectively). There was a significant difference in epidermal thickness between the wound group and other experimental groups (p < 0.05). The number of apoptotic cells at the wound edges on the seventh day in both group 3 and group 4 had a significant decrease compared to other groups of wounds (p = 0.000), but there was a significant increase on the fourteenth day. Also, on the 21st day, a significant decrease in apoptotic cells was observed in both group 3 and group 4 compared to other wound groups (p = 0.000). Discussion and ConclusionNettle and SF maintain cell homeostasis and accelerate wound closure by reducing cell apoptosis and enhancing cell proliferation on the seventh day, but by increasing the apoptosis of fibroblast cells on the fourteenth day, they lead to remodeling and keratinocytes migration to epidermis formation. Increased apoptosis also seems to be one of the pathophysiological mechanisms to prevent the formation of keloid and hypertrophic scar tissue. SF and Nettle extract, by increasing cell proliferation and migration of different cell types to the site of injury, control the remodeling process by inducing and regulating apoptosis in the first two weeks of wound healing and accelerating the process of collagen deposition and epithelialization.

Self-Assembling Imageable Silk Hydrogels for the Focal Treatment of Osteosarcoma.

Background: The standard treatment for osteosarcoma comprises complete surgical resection and neoadjuvant chemotherapy, which may cause serious side effects and partial or total limb loss. Therefore, to avoid the disadvantages of traditional treatment, we developed self-assembling imageable silk hydrogels for osteosarcoma. Methods: We analysed whether iodine induced apoptosis in MG-63 and Saos-2 cells by using CCK-8 and flow cytometry assays and transmission electron microscopy. Western blotting was used to analyse the pathway of iodine-induced apoptosis in osteosarcoma cells. PEG400, silk fibroin solution, polyvinylpyrrolidone iodine (PVP-I), and meglumine diatrizoate (MD) were mixed to produce an imageable hydrogel. A nude mouse model of osteosarcoma was established, and the hydrogel was injected locally into the interior of the osteosarcoma with X-ray guidance. The therapeutic effect and biosafety of the hydrogel were evaluated. Results: Iodine treatment at 18 and 20 µM for 12 h resulted in cell survival rate reduced to 50 ± 2.1% and 50.5 ± 2.7% for MG-63 and Sao-2 cells, respectively (p < 0.01). The proportion of apoptotic cells was significantly higher in the iodine-treatment group than in the control group (p < 0.05), and apoptotic bodies were observed by transmission electron microscopy. Iodine could regulate the death receptor pathway and induce MG-63 and Saos-2 cell apoptosis. The hydrogels were simple to assemble, and gels could be formed within 38 min. A force of less than 50 N was required to inject the gels with a syringe. The hydrogels were readily loaded and led to sustained iodine release over 1 week. The osteosarcoma volume in the PEG-iodine-silk/MD hydrogel group was significantly smaller than that in the other three groups (p < 0.001). Caspase-3 and poly (ADP-ribose) polymerase (PARP) expression levels were significantly higher in the PEG-iodine-silk/MD hydrogel group than in the other three groups (p < 0.001). Haematoxylin and eosin (H&E) staining showed no abnormalities in the heart, liver, spleen, lung, kidney, pancreas or thyroid in any group. Conclusions: Self-assembling imageable silk hydrogels could be injected locally into osteosarcoma tissues with X-ray assistance. With the advantages of good biosafety, low systemic toxicity and minimal invasiveness, self-assembling imageable silk hydrogels provide a promising approach for improving the locoregional control of osteosarcoma.

Preparation and Characterization of Natural Silk Fibroin Hydrogel for Protein Drug Delivery.

In recent years, hydrogels have been widely used as drug carriers, especially in the area of protein delivery. The natural silk fibroin produced from cocoons of the Bombyx mori silkworm possesses excellent biocompatibility, significant bioactivity, and biodegradability. Therefore, silk fibroin-based hydrogels are arousing widespread interest in biomedical research. In this study, a process for extracting natural silk fibroin from raw silk textile yarns was established, and three aqueous solutions of silk fibroin with different molecular weight distributions were successfully prepared by controlling the degumming time. Silk fibroin was dispersed in the aqueous solution as “spherical” aggregate particles, and the smaller particles continuously accumulated into large particles. Finally, a silk fibroin hydrogel network was formed. A rheological analysis showed that as the concentration of the silk fibroin hydrogel increased its storage modulus increased significantly. The degradation behavior of silk fibroin hydrogel in different media verified its excellent stability, and the prepared silk fibroin hydrogel had good biocompatibility and an excellent drug-loading capacity. After the protein model drug BSA was loaded, the cumulative drug release within 12 h reached 80%. We hope that these investigations will promote the potential utilities of silk fibroin hydrogels in clinical medicine.

Cover Image, Volume 139, Issue 25

This cover image designed by Di Zhao and Huiqin Liu delineates the preparation of functional silk fibers (SFs) by microencapsulation technology. This review describes the principles and techniques for the preparation of functional microcapsules. Functional SFs can be obtained by microcapsule finishing and printing technology, and even by electrospinning based on silk fibroin solution. Functional silk fibroin nanofibers have great promise for biomedical applications. DOI: 10.1002/app.52351

3D Printing of Monolithic Proteinaceous Cantilevers Using Regenerated Silk Fibroin.

Silk fibroin, regenerated from Bombyx mori, has shown considerable promise as a printable, aqueous-based ink using a bioinspired salt-bath system in our previous work. Here, we further developed and characterized silk fibroin inks that exhibit concentration-dependent fluorescence spectra at the molecular level. These insights supported extrusion-based 3D printing using concentrated silk fibroin solutions as printing inks. 3D monolithic proteinaceous structures with high aspect ratios were successfully printed using these approaches, including cantilevers only supported at one end. This work provides further insight and broadens the utility of 3D printing with silk fibroin inks for the microfabrication of proteinaceous structures.

Effect of silk fibroin microcarrier loaded with clematis total saponins and chondrocytes on promoting rabbit knee articular cartilage defects repair

To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups ( n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 μm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes ( P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers ( P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B ( P<0.05), and the level of TIMP-1 in group C was significantly higher ( P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B ( P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B ( P<0.05). CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.

Fabrication of silk fibroin film enhanced by acid hydrolyzed silk fibroin nanowhiskers to improve bacterial inhibition and biocompatibility efficacy

In this study, silk fibroin nanowhiskers (SNWs) were extracted from natural silk fiber by sulfuric acid hydrolysis with the assistance of ultrasonic wave treatment. The obtained SNWs were mixed with regenerated silk fibroin (RSF) solution to fabricate the SNWs/RSF films. The fabricating SNWs were systematically characterized by using SEM, FTIR, and the SNWs/RSF films were observed by digital camera, PM, etc. The results show that the monodisperse SNWs are evenly distributed in the RSF film. The presence of SNWs in RSF film significantly improves the performances of the film, including the swelling ability, mechanical properties, hydrophilicity, antibacterial efficacy, cytocompatibility. Meanwhile, the SNWs/RSF film can endorse the wound healing efficiency in vivo mice wound site. The proposed techniques for extracting SNWs and fabricating silk fibroin composite film may provide a valuable method for creating an ideal silk-based material for biomedical applications.

Silk fibroin microfiber‐reinforced polycaprolactone composites with enhanced biodegradation and biological characteristics

There is an enormous demand for bone graft biomaterials to treat developmental and acquired bony defects arising from infections, trauma, tumor, and other conditions. Polycaprolactone (PCL) has been extensively utilized for bone tissue engineering but limited cellular interaction and tissue integration are the primary concerns. PCL-based composites with different biomaterials have been attempted to improve the mechanical and biological response. Interestingly, a few studies have tried to blend PCL with aqueous silk fibroin solution, but the structures prepared with the blend were mechanically weak due to phase mismatch. As a result, silk microparticle-based PCL composites have been prepared, but the microfibers-reinforced composites could be superior to them due to significant fiber-matrix interaction. This study aims at developing a unique composite by incorporating 100-150μm long (aspect ratio; 8:1-5:1) silk-fibroin microfibers into the PCL matrix for superior biological and mechanical properties. Two silk variants were used, that is, Bombyx mori and a wild variant, Antheraea mylitta, reported to have cell recognizable Arginine-Glycine-Aspartic acid (RGD) sequences. A.mylitta silk fibroin microfibers were produced, and composites were made with PCL for the first time. The morphological, tensile, thermal, biodegradation, and biological properties of the composites were evaluated. Importantly, we tried to optimize the silk concentration within the composite to strike a balance among the cellular response, biodegradation, and mechanical strength of the composites. The results indicate that the PCL-silk fibroin microfiber composite could be an efficient biomaterial for bone tissue engineering.

Physico-chemical properties of Artificial tear ducts from Fractionated Thai silk fibroin

Artificial tear ducts (ATD) or Jones tubes, have been used in patients with tear ducts obstruction for draining tears into the nose. Commercial ATDs are generally made of glass but they have slippery surfaces causing the ducts pushed out and easily broken. Polymer-based ATDs, are more flexible and adhesive to tissues. However, the users could encounter material-related problems due to allergic reactions, low tissue adhesion and complications. We designed artificial tear ducts from Thai silk fibroin (SF) solution. The SF was fractionated using freeze-thaw cycles (-4°C and 25°C for 5 cycles) into SF-P (precipitants, 63.53±4.58% yields) and SF-S (soluble, 34.05±5.76% yields (w/v)). The concentrated solutions (20%W/W) of SF, SF-P and SF-S were dip-coated into tubes. The leaking test was performed using simulated natural tear flow rate of 0.0022 mL/min for 6 h. SF-S duct lost its shape and leaked. Absorption of the balanced salt solution (BSS) of the SF and SF-P were at 5.67±0.76% and 8.05±1.28 wt.% respectively, giving their wet inner and outer diameters at 2 and 2.5 mm. and the thickness 500 microns. Crystallinities of SF and SF-P analyzed using ATR-FTIR, were at 42.27 and 44.51% respectively. The thermal decomposition temperature of SF and SF-P analyzed using TGA, were at 277 and 280°C. Degradability in BSS containing lysozyme 1.69 U/mg to mimic tears at 37°C in vitro showing that both ATDs are stabilized for at least 4 weeks. BSS height obtained capillary test that compared with glass ducts, SF and SF-P at 7.0±0.0, 6.9±0.4 and 7.1±0.4 mm, respectively, while glass ducts were 34.81±0.0. The SF and SF-P have flexural stress were 55.24±7.68 MPa and 81.42±2.71 MPa, %flexural strain at max stress was 5.43±0.46% and 2.54±0.17% and flexural Modulus was 1.82±0.29 GPa and 6.13±0.51GPa respectively. The results of all experiments indicate that SF-P had the highest potential for further development into natural polymer-based ATDs.

Preparation and evaluation of antibacterial wound dressing based on vancomycin-loaded silk/dialdehyde starch nanoparticles.

One of the main reasons infected wounds go untreated is that antibiotic-resistant bacteria mainly cause infection. Vancomycin is an antibiotic used against Gram-positive bacteria, such as MRSA, but it has limited intravenous use due to its toxicity. This study describes using a local drug delivery approach at the wound site. The aim is to prepare a silk dressing containing dialdehyde starch nanoparticles loaded with vancomycin that can cure infection through the controlled release of antibiotics. First, the starch was oxidized by sodium periodate solution and converted to dialdehyde starch. Dialdehyde starch was converted into nanoparticles by the microemulsion method. Simultaneously, with nanoparticle formation, the antibiotic vancomycin (VAN), added to the solution, was loaded into the dialdehyde starch nanoparticles (DASNP). The wound dressing (SF/DASNP/VAN) was prepared by adding nanoparticles containing antibiotics to the silk fibroin (SF) solution, and then, the solution containing the nanoparticles was freeze-dried, and the nanoparticles were placed inside the silk matrix. Drug release of dressings was performed by immersion in phosphate-buffered saline, and cytotoxicity by MTT assay and antibacterial properties of dressings were investigated by the inhibition zone method. The morphology of the SF/DASNP/VAN dressing, its biocompatibility, antibacterial efficiency, and antibiotic release kinetics were assessed. The synthesized dressing has the desired biocompatibility with 69% cell viability and shows antibacterial properties against MRSA with a growth inhibition zone diameter of 12mm. Also, VAN was successfully incorporated into the dressing, resulting in a 144-h continuous release profile. It may be concluded that the fabricated dressing based on silk and dialdehyde starch nanoparticles opens up a new option for topical administration of antibiotics. We believe its properties can be considered a new dressing for infectious wounds by reducing infection associated with controlled drug delivery.

Restorative and pain-relieving effects of fibroin in preclinical models of tendinopathy

The term tendinopathy indicates a wide spectrum of conditions characterized by alterations in tendon tissue homeostatic response and damage to the extracellular matrix. The current pharmacological approach involves the use of nonsteroidal anti-inflammatory drugs and corticosteroids often with unsatisfactory results, making essential the identification of new treatments. In this study, the pro-regenerative and protective effects of an aqueous fibroin solution (0.5–500 μg/mL) against glucose oxidase (GOx)-induced damage in rat tenocytes were investigated. Then, fibroin anti-hyperalgesic and protective actions were evaluated in two models of tendinopathy induced in rats by collagenase or carrageenan injection, respectively. In vitro, 5–10 μg/mL fibroin per se increased cell viability and reverted the morphological alterations caused by GOx (0.1 U/mL). Fibroin 10 μg/mL evoked proliferative signaling upregulating the expression of decorin, scleraxin, tenomodulin (p < 0.001), FGF-2, and tenascin-C (p < 0.01) genes. Fibroin enhanced the basal FGF-2 and MMP-9 protein concentrations and prevented their GOx-mediated decrease. Furthermore, fibroin positively modulated the production of collagen type I. In vivo, the peri-tendinous injection of fibroin (5 mg) reduced the development of spontaneous pain and hypersensitivity (p < 0.01) induced by the intra-tendinous injection of collagenase; the efficacy was comparable to that of triamcinolone. The pain-relieving action of fibroin (peri-tendinous) was confirmed in the model of tendinopathy induced by carrageenan (intra-tendinous) where this fibrous protein was also able to improve tendon matrix organization, normalizing the orientation of collagen fibers. In conclusion, the use of fibroin in tendinopathies is suggested taking advantage of its excellent mechanical properties, pain-relieving effects, and ability to promote tissue regeneration processes.

Toughening robocast chitosan/biphasic calcium phosphate composite scaffolds with silk fibroin: Tuning printable inks and scaffold structure for bone regeneration

The present work aims the production of composite bioceramic scaffolds by robocasting suppressing sintering as post printing process. To achieve this purpose, extrudable ink compositions containing a high concentration of bioceramic powders (hydroxyapatite and β-tricalcium phosphate) embedded in aqueous polymeric solutions of chitosan and silk fibroin were fine-tuned. Polymeric solutions of chitosan/silk fibroin with different ratios were tested, maintaining the total amount of bioceramic solids at 30 vol%. The inks were characterized by rheological studies in viscometry and oscillatory modes, being the printable ones selected to produce scaffolds with different macropore sizes (300 μm and 500 μm). The scaffolds were characterized by mechanical properties (dry and wet conditions) and morphological features, as well as its degradability. In vitro studies were also evaluated in the scaffolds that presented the best structural performance.The addition of 2 wt% silk fibroin to a 5 wt% chitosan matrix allows to significantly improve the mechanical performance of the printed composite scaffolds, reflected in high values for Young's modulus and maximum compressive strength. This trend was continued in wet scaffolds with a concomitant reduction of mechanical properties. Regarding degradability, the scaffolds in general presented a weight loss in the range of 14–18% after 28 days incubation in HEPES solution at two different pH values at 37 °C, with an associated release of calcium and phosphorus ions. The scaffold with 300 μm porosity comprising the both polymers in its composition presented the less rate degradation when compared to the scaffolds with similar porosity and containing only chitosan as base matrix. Moreover, the combined natural polymers gave rise to a significant increase in the metabolic activity of human osteoblasts grown on the scaffolds with both macropore' size, being in line with the full cellular filling of their surfaces, demonstrated by SEM and confocal imaging. The advances presented in this work are a promising path in the ink's development for extrusion-based additive manufacturing techniques and subsequent biomaterials, encompassing suitable physical and chemical characteristics with high potential to be used as bone substitutes.

Seeking Solvation: Exploring the Role of Protein Hydration in Silk Gelation.

The mechanism by which arthropods (e.g., spiders and many insects) can produce silk fibres from an aqueous protein (fibroin) solution has remained elusive, despite much scientific investigation. In this work, we used several techniques to explore the role of a hydration shell bound to the fibroin in native silk feedstock (NSF) from Bombyx mori silkworms. Small angle X-ray and dynamic light scattering (SAXS and DLS) revealed a coil size (radius of gyration or hydrodynamic radius) around 12 nm, providing considerable scope for hydration. Aggregation in dilute aqueous solution was observed above 65 °C, matching the gelation temperature of more concentrated solutions and suggesting that the strength of interaction with the solvent (i.e., water) was the dominant factor. Infrared (IR) spectroscopy indicated decreasing hydration as the temperature was raised, with similar changes in hydration following gelation by freezing or heating. It was found that the solubility of fibroin in water or aqueous salt solutions could be described well by a relatively simple thermodynamic model for the stability of the protein hydration shell, which suggests that the affected water is enthalpically favoured but entropically penalised, due to its reduced (vibrational or translational) dynamics. Moreover, while the majority of this investigation used fibroin from B. mori, comparisons with published work on silk proteins from other silkworms and spiders, globular proteins and peptide model systems suggest that our findings may be of much wider significance.

Structural stability of biofilms produced from silkworm cocoon fibers

Biofilms were obtained from cocoons of the silkworm, Bombyx mori, involving the removal of sericin, extraction and solubilization of fibroin fibers, dialysis of fibroin dispersions and preparation of biofilms by the casting process. Biofilm transparency was verified by UV?Vis spectroscopy and thermal stability by thermogravimetric/differential scanning calorimetry (TG/DSC). Soon after preparation, the solidification of the fibroin solution prepared from the cocoons and extracted by the Ajisawa method was monitored until the biofilm stabilized, using attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) as a function of time. The results showed that there was a change in the conformation from the silk I structure (?-helix) to silk II (?-sheet). In order to improve the characterization of the biofilms obtained by the Ajisawa method and LiBr solubilization of fibroin fibers, Raman spectroscopy was used to verify the stabilization of the different possible molecular conformations for the fibers in these materials, by comparison with the cocoon spectra and those of the solid (freeze-dried precipitated by dialysis for 72 h. By comparing the Raman spectra of the biofilms in terms of the intensities of the broadened band characteristic of amide I, it was possible to assess the conformational changes in both materials based on the possible transitions between ?-sheet conformations and flexible ?-helix and ?-turn structures. The results showed a dispersion of these conformations in the biofilms generated and in the solid freeze-dried hydrogel spectrum, and the ?-sheet conformation was found to be predominant. The TG and DSC curves showed that the materials with higher ?-sheet content exhibited higher thermal stability. Thus, the data obtained further elucidated the properties of these materials that are widely used in various processes.

Multifunctional silicon calcium phosphate composite scaffolds promote stem cell recruitment and bone regeneration.

A scaffold is one of the most significant implants for treating bone injury, while the precise control of stem cell proliferation and differentiation within a scaffold is still challenging. In this work, a composite scaffold was designed to be capable of recruiting endogenous stem cells, stimulating osteogenic differentiation and achieving significant bone repair function. The designed SiCP + SF@PFS silica-calcium phosphate composite scaffold was obtained by mixing the peptide PFS containing silk fibroin solution with the SiCP scaffold, and treating with horseradish peroxidase and H2O2. The results showed that the composite scaffold was able to release the PFS peptide continuously to induce the migration of mesenchymal stem cells. Meanwhile, cell proliferation and osteogenic differentiation were also improved after being seeded on the scaffold. In the cranial defect rat model, the composite scaffold was able to recruit CD29+ and CD90+ cells one week after implantation around the injury sites. The results of Micro-CT, H&E staining, Masson's staining and immunohistochemical staining indicated that the composite scaffold was able to promote new bone formation significantly.

Development of Silk Fibroin Scaffolds by Using Indirect 3D-Bioprinting Technology.

Due to the excellent biocompatibility of natural polymers, a variety of natural polymers have been widely used as biomaterials for manufacturing tissue engineered scaffolds. Despite the excellent biological activity of natural polymers, there have been obstacles in using them on their own to prepare 3D scaffolds with sufficient mechanical strength. Although multiple 3D-bioprinting technologies have recently emerged as effective manufacturing tools for scaffold preparation, scaffold preparation using only natural polymers with tunable mechanical properties is still difficult. Herein, we introduce novel scaffold fabrication methods using the natural polymer silk fibroin via indirect 3D-bioprinting technology. The developed silk fibroin scaffolds showed biocompatibility and tunable mechanical strength by changing the concentration of the silk fibroin. Furthermore, controlling the flexibility of the silk fibroin scaffolds was made possible by changing the solvent for the silk fibroin solution used to fabricate the scaffold. Consequently, silk fibroin scaffolds fabricated via our method can be considered for various applications in the bioengineering of either soft or musculoskeletal tissues.

PH-responsive discoloration silk fibroin films based on prodigiosin from microbial fermentation

Smart color-changing films are emerging as promising functional materials having multiple applications. pH-responsive discoloration film can present optical response to outer pH value, which has diverse functions like indicating, packaging and monitoring, etc. Prodigiosin possesses biodegradability and biocompatibility, which is a natural red pigment derived primarily from secondary metabolites of microorganisms. Based on these properties, an effective approach to develop pH-responsive discoloration film with biodegradability is proposed. Prodigiosins from microbial fermentation were evenly dispersed in silk fibroin solution to form a well-developed composite film via the casting method, exhibiting switchable pH-responsive color-changing performance. The film showed reddish-purple (λ max = 530 nm) at pH below 8, and turned to orange and yellow (λ max = 480 nm) as the pH increased from 8 to 10. The glycerol and 1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydro (EDC) were incorporated as plasticizing and cross-linking agents, respectively. Glycerol and EDC significantly reduced the water contact angle from 83.4° to 4.7°and improved the mechanical properties of pH-responsive silk fibroin film. In addition, this pH-responsive silk fibroin-based film is biodegradable and can be attached to the skin for long-term colorimetric indicating. The design of color-changing silk fibroin film presented here provides a new direction of thinking for the preparation of substrate materials for stimulation-responsive biosensors. Smart color-changing films are emerging as promising functional materials having multiple applications. pH-responsive discoloration film can present optical response to outer pH value, which has diverse functions like indicating, packaging and monitoring, etc. Prodigiosin possesses not only pH-responsive discoloration property but biodegradability and biocompatibility. Based on these properties, an effective approach to develop pH-responsive discoloration film with biodegradability is proposed. Prodigiosins from microbial fermentation were evenly dispersed in silk fibroin solution to form a well-developed composite film via the casting method, exhibiting switchable pH-responsive color-changing performance. The film showed reddish-purple (λ max = 535 nm) at pH below 8, and turned orange and yellow (λ max = 480 nm) as the pH increased from 8 to 10. The glycerol and 1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydro (EDC) were incorporated as plasticizing and cross-linking agents, respectively. Glycerol and EDC significantly reduced the water contact angle from 83.4° to 4.7°and improved the mechanical properties of pH-responsive silk fibroin film. In addition, this pH-responsive silk fibroin-based film is biodegradable and can be attached to the skin for long-term colorimetric indicating. The design of color-changing silk fibroin film presented here provides a new direction of thinking for the preparation of substrate materials for stimulation-responsive biosensors.