The influence of UV radiation on silk fibroin

An investigation into the influence of UV-irradiation on regenerated silk fibroin dissolved in water was carried out using UV-Vis and fluorescence spectroscopy. It was found that the absorption of regenerated silk fibroin in solution increased during UV-irradiation of the sample, most notably between 250 and 400 nm. Moreover, after UV-irradiation a wide peak emerged between 290 and 340 nm with maximum at about 305 nm. The new peak suggests that new photoproducts are formed during UV-irradiation of regenerated silk fibroin.The fluorescence of regenerated silk fibroin was observed at 305 nm, at 480 nm and at 601 nm after excitation at 275 nm. UV-irradiation caused fluorescence fading at 305 nm and at 601 nm. The increase of fluorescence was observed at 480 nm, probably due to formation of new photoproducts. After excitation at 305 nm the fluorescence of regenerated silk fibroin was observed at 340 nm and at 400 nm. UV-irradiation caused fluorescence fading at 340 nm. FTIR spectroscopy showed that primary structure of regenerated silk fibroin was not significantly affected by UV radiation. SDS-PAGE chromatography showed alterations of molecular weight of silk after UV exposure.     

Preparation and characterization of chitin whisker-reinforced silk fibroin nanocomposite sponges

For highly porous form such as sponges or scaffolds, the induction of the β-sheet formation of silk fibroin to make the water-stable materials usually results in their high shrinkage leading to a difficulty in controlling shape and size of materials. Thus, the objective of this study was to improve dimensional stability of silk fibroin sponge by incorporating chitin whiskers as nanofiller. Chitin whiskers exhibited the average length and width of 427 and 43 nm, respectively. Nanocomposite sponges at chitin whiskers to silk fibroin weight ratio (C/S ratio) of 0, 1/8, 2/8, or 4/8 were prepared by using a freeze-drying technique. The dispersion of chitin whiskers embedded in the silk fibroin matrix was found to be homogeneous. The presence of chitin whiskers embedded into silk fibroin sponge not only improved its dimensional stability but also enhanced its compression strength. Regardless of the chitin whisker content, SEM micrographs showed that all samples possessed an interconnected pore network with an average pore size of 150 μm. To investigate the feasibility of the nanocomposites for tissue engineering applications, L929 cells were seeded onto their surfaces, the results indicated that silk fibroin sponges both with and without chitin whiskers were cytocompatible. Moreover, when compared to the neat silk fibroin sponge, the incorporation of chitin whiskers into the silk fibroin matrix was found to promote cell spreading.     

Mineralization of HA crystals regulated by terephthaloyl chloride-modified silk fibroin films

Terephthaloyl chloride(DB)-modified silk fibroin(SF) films were immersed into 1.5 times simulated body fluid(1.5 SBF) to regulate the mineralization of hydroxyapatite(HA) crystals at about 36.5℃for 24 h.UV was used to prove that the new bonds form between the DB and SF.The structure and morphology of the SF/HA were investigated by FTIR,ICP,XRD and SEM.The results showed that the apatite deposited on the matrix of SF mainly was HA.HA was self-assembled on the matrix of SF and formed three-dimensional framework when the weight ratio of DB/SF was 0.30.The content of DB affected the structure and morphology of the apatite composites deposited on the SF films.     

In vitro and in vivo degradation behavior of aqueous-derived electrospun silk fibroin scaffolds

We investigated the in vitro degradation behavior of the electrospun silk fibroin (SF) scaffolds by protease XIV. Phosphate-buffered saline (PBS) without enzyme was used as a control. About 65% of the electrospun SF scaffolds were degraded within 24 d in protease XIV, while almost no scaffolds were degraded in PBS. A great deal of fragments was visible in protease XIV solution. SEM indicated surface erosion of the scaffolds increased during protease degradation with increasing exposure time. FTIR and XRD indicated the crystalline structure of the scaffolds decreased after protease degradation for 24 d, and a degradation mechanism was proposed. Furthermore, the results of the in vivo degradation by implantation in rats showed that the scaffolds were completely degraded in vivo after implantation for 8 weeks and well tolerated by the host animals. The insights gained in this study can serve as a guide to match desired degradation behavior with specific applications for the electrospun SF scaffolds, such as tissue engineering and drug delivery.     

Preparation and characterization of new materials based on silk fibroin,chitosan and nanohydroxyapatite

Abstract

In this paper, a series of porous nanohydroxyapatite/silk fibroin/chitosan (nHA/SF/CTS) scaffolds were successfully prepared using the freeze-drying method. The biomaterials were characterized by attenuated total reflection Fourier transform infrared spectroscopy, and mechanical testing and thermogravimetric analysis. Moreover, studies of porosity, pore size, swelling properties and in vitro degradation test were performed. Research has proved that micro-structure, porosity, water adsorption and compressive strength were greatly affected by the components’ concentration, in particular the content of silk fibroin. SEM observations showed that the scaffolds of nHA/SF/CTS are highly porous, with pore size in wide range from 25 to 300?µm which is suitable for cell growth. nHA/SF/CTS scaffolds have sufficient mechanical integrity to resist handling during implantation and in vivo loading. Both, the compressive modulus and compressive strength of the scaffold, decrease with the increase in silk fibroin content.     

Synthesis,characterization, and biological evaluation of doxorubicin containing silk fibroin micro- and nanoparticles

The aim of this study was biological evaluation of doxorubicin containing silk fibroin micro- and nanoparticles (Dox-MF and Dox-NF). Dox-MF and Dox-NF were synthesized. Cell toxicity on MCF-7, Saos2, and HFF cells was assessed using MTT assay. Induced apoptosis was assessed using flow cytometry and staining with PI/annexin V. Production of reactive oxygen species (ROS) was measured. Gene expression of p53 was evaluated by real-time PCR. FTIR, SEM, and DLS confirmed the accurate synthesis. Cytotoxicity of Dox-MF and Dox-NF showed significant inhibition of cell growth compared with the controls. Regarding Dox-NF, a significant increase was seen in mRNA level of P53 in MCF-7 and SAOS-2 ​cells and a significant decrease in HFF cells compared to the controls. There was a significantly higher expression of P53 gene in MCF-7 and HFF cells treated by Dox-MF. However, a significant decrease in P53 gene expression was detected in SAOS-2 ​cells. Significant apoptotic induction of cell lines by Dox-MF and Dox-NF was observed in both early and late stages. Dox-MF and Dox-NF acted in the direction of cell death through the apoptotic pathway and changing p53 gene expression. So, Dox-MF and Dox-NF can be considered as a candidate for new anticancer agents.     

Dual-functional composite with anticoagulant and antibacterial properties based on heparinized silk fibroin and chitosan

Heparinized biomaterials exhibit great anticoagulant properties. However, they promote proliferation of Staphylococcus aureus (S. aureus) and therefore cause infection within the bloodstream upon implantation in vivo. In the present study, an interesting dual-functional composite with anticoagulant and antibacterial properties based on heparinized silk fibroin and chitosan was synthesized. First, heparin was grafted onto the silk fibroin by covalent immobilization with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). All data gathered from Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and elemental analysis (EA) indicated that the heparin was successfully immobilized onto the silk fibroin. The dual-functional composite of heparinized silk fibroin and chitosan was then fabricated by a blending method. The anticoagulant activity of the heparinized materials was evaluated using the prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT). The results showed that both heparinized silk fibroin and the composite material exhibited better hemocompatibility in comparison with single silk fibroin or chitosan. The antibacterial property of the materials was investigated by the pour-plate method. Results further suggested that the composite antibacterial property with respect to S. aureus was significantly enhanced. The dual-functionality of the composite material may supply a potential choice in blood contact devices.     

Magnesium-containing silk fibroin/polycaprolactone electrospun nanofibrous scaffolds for accelerating bone regeneration

Bone tissue engineering has become one of the most effective methods for treating bone defects. In this study, an electrospun tissue engineering membrane containing magnesium was successfully fabricated by incorporating magnesium oxide (MgO) nanoparticles into silk fibroin and polycaprolactone (SF/PCL)-blend scaffolds. The release kinetics of Mg²⁺ and the effects of magnesium on scaffold morphology, and cellular behavior were investigated. The obtained Mg-functionalized nanofibrous scaffolds displayed controlled release of Mg²⁺, satisfactory biocompatibility and osteogenic capability. The in vivo implantation of magnesium-containing electrospun nanofibrous membrane in a rat calvarial defect resulted in the significant enhancement of bone regeneration twelve weeks post-surgery. This work represents a valuable strategy for fabricating functional magnesium-containing electrospun scaffolds that show potential in craniofacial and orthopedic applications.     

Nanofibers of silk fibroin controlled by the crystallization of polyethylene glycol in frozen solution

The conformation of silk fibroin(SF) frozen with polyethylene glycol(PEG) at a molecular weight from 2kDa to 20kDa and a mass ratio of PEG:SF from 1:5 to 10:1 was studied by spectral and microscopic methods. It is found that the conformation transition of SF from random coil to b-sheet could be induced by the stress resulting from PEG crystallization at-20 °C, and greatly depended on the cooling rate, PEG:SF mass ratio and PEG molecular weight. These findings provide a new method for the preparation of desired SF nanofibers.     

Degradation behaviors of nerve guidance conduits made up of silk fibroin in vitro and in vivo

We have developed a new design of nerve guidance conduits (NGCs) made up of silk fibroin (SF), referred to as SF-NGCs, by a well-established method. The present study aimed to comprehensively investigate the degradation behaviors of SF-NGCs versus SF fibers. After they were allowed to incubate in the protease XIV solution or to be subcutaneously implanted in rabbits, SF-NGCs and SF fibers were subjected to degradation level measurement, mass loss assessment and gel electrophoresis, or light and electron microscopy and mass loss assessment for testing the dynamic course of in vitro or in vivo degradation. The results collectively indicated that SF-NGCs were able to degrade at a significantly increasing rate as compared to SF fibers, thus meeting the requirements of peripheral nerve regeneration. Furthermore, based on the possible involvement pathway in the in vivo degradation of SF-NGCs, the time-dependent changes in the mRNA level of lysosome-related genes (Hip1R, cathepsin D, and tPA) in subcutaneous implantation site within 24-week period post-implantation was determined by real-time RT-PCR, and the resulting data might contribute to our understanding of the molecular aspects that affect in vivo degradation and absorption of SF-NGCs.     

Preparation and in vitro degradation of porous three-dimensional silk fibroin/chitosan scaffold

Degradation behaviors of porous scaffolds play an important role in the engineering process of a new tissue. In this study, three-dimensional porous silk fibroin/chitosan (SFCS) scaffolds were successfully prepared by freeze-drying method. In vitro degradation behaviors of SFCS scaffolds have been systematically investigated up to 8 weeks in phosphate buffer saline (PBS) solution at 37 °C. The following properties of the scaffolds were measured as a function of degradation time: pore morphology, structure, weight loss, and wet/dry weight value. The pH value of the PBS solution during degradation was also detected. SFCS scaffolds maintained its porous structure till 6 weeks of degradation. During the first 2 weeks, the pH value fluctuated in a narrow range from 6.53 to 6.93. SFCS scaffolds degraded much more quickly during the first 2 weeks, and the weight loss reached 19.28 wt% after 8 weeks of degradation. The degradation process affects little SFCS scaffolds' swelling properties.     

Gelation behavior of Antheraea pernyi silk fibroin

The sol-gel transition behavior of Antherae pernyi silk fibroin(Ap-SF) has not been systematically investigated.In this work,the influence of environmental temperature,pH,the concentration of Ap-SF,K+ and Ca2+ on the gelation time,and the structural changes of Ap-SF in sol-gel transformation were studied.The results indicated that the gelation time of the Ap-SF aqueous solution decreased with the increase of the Ap-SF concentration and environmental temperature.The sol-gel transformation of Ap-SF was much m...     

Physical characteristics and properties of waterborne polyurethane materials reinforced with silk fibroin powder

Degummed silk filament was pulverized with a home‐made machine to obtain silk fibroin (SF) powder, and the structure, morphology, and particle size of the SF powder were investigated. The individual spherical particles and aggregates with different morphology of silk fibroin coexisted in water. A waterborne polyurethane (WPU) aqueous dispersion was blended with the SF powder to prepare novel blended materials with improved physical properties. The average particle size and zeta potential of the WPU/SF aqueous dispersions were characterized. The result showed that the WPU/SF dispersion with higher SF content exhibited a less negative zeta potential and a larger average particle size. Furthermore, the effect of SF content on the morphology, miscibility, and mechanical properties of the resulting blended films was studied by scanning electron microscopy, wide‐angle X‐ray diffraction, dynamic mechanical thermal analysis, and tensile testing. The films showed an improved Young's modulus and tensile strength from 0.3 to 33.8 MPa, and 0.6 to 5.2 MPa, respectively, with the increasing of SF up to a content of 26 wt %. The negative charges in the periphery and the small particle size made a good effort on dispersing SF powder into the WPU matrix as small aggregates, and the SF powder led to the efficient strengthening of WPU materials. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 940–950, 2010     

Physical and chemical properties of tussah silk fibroin films

Physical and chemical structure, as well as thermal behavior of solution-cast regenerated films, prepared from tussah (Antheraea pernyi) silk fibroin, were compared with those of solution-cast native films, in order to ascertain whether treatment (degumming, dissolution) used for preparation affected their properties. Regenerated fibroin films exhibited a higher thermal stability than native ones, as shown by differential scanning calorimetry, thermomechanical analysis, and dynamic mechanical behavior. Glass transition temperature and other relevant thermal transitions of the regenerated silk specimen shifted to higher temperatures compared with those of native specimen. Molecular conformation and crystalline structure did not show significant differences between the two kinds of silk films. Amino acid composition and molecular weight, however, distribution changed markedly after dissolving tussah silk fibroin fiber in concentrated LiSCN in polypeptide size was the main features for the regenerated silk fibroin. © 1994 John Wiley & Sons, Inc.     

Conformation transition of silk fibroin induced by blending chitosan

The conformation of silk fibroin in silk fibroin/chitosan (SF/CS) blend membrane was analyzed by infrared spectrum, X-ray diffractometry, and Raman spectrum. The results demonstrated that the SF could show β-sheet conformation when the SF content in blend membranes was 10% (w/w) and 60–80% (w/w), while the pure SF membrane showed random coil conformation. A mechanism of the conformation transition was suggested in that the SF chain could use the rigid CS chain as a mold plate to stretch itself to form a β-sheet structure according to the strong hydrogen bond between CS and SF. Therefore, a new concept, named “Polymer-Induced Conformation Transition,” was proposed. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35 : 2293–2296, 1997     

1H pulsed NMR study of bombyx mori silk fibroin: Dynamics of fibroin and of absorbed water

The dynamical behavior of the Bombyx mori silk fibroin chain and of absorbed water in silk fiber, film, and powder has been studied by ¹H pulsed nuclear magnetic resonance (NMR). Segmental motions do not occur and only the rapid rotation of the methyl groups of alanine residues is observed from ?120 to 130°C. This is independent of the conformation or form of the silk fibroin samples. Magnetization of dry silk fibroin by the solid-echo method shows a single Gaussian decay, while two components are observed in the solid-echo signals of films containing 6–10 w/w% water. An immobile component with a T₂ value of 11 μs is attributed to silk fibroin, and the mobile component to bound water. The T₂ of the latter varies from 50 to 200 μs, depending on the sample. The dynamical behavior of water trapped in the film is discussed on the basis of these T₂ values.     

环氧化合物与丝素蛋白化学交联凝胶的结构

对丝素蛋白水溶液与环氧化合物交联剂(PGDE)反应制备的丝素凝胶(CFG)的结构进行了研究.用位相差显微镜观察到CFG具有整体均一的形态结构,而未经PGDE交联的纯丝素凝胶(FG)为颗粒聚集结构.CFG的红外吸收光谱在1104cm^-1处出现PGDE分子中烷醚的吸收峰,而没有出现PGDE两端环醚的特征吸收峰,显示PGDE已经开环交联在丝素蛋白上.氨基酸分析结果显示,酪氨酸、组氨酸和赖氨酸的含量与反应前相比明显减少,PGDE在这些位点与丝素蛋白发生了交联反应.固体¹³CNMR谱观察到酪氨酸羟苯基上的碳发生了化学位移,进一步证实了酪氨酸残基与PGDE发生了化学交联.研究表明,PGDE与丝素蛋白交联形成了分子间交联网络结构,从而使CFG成为较为匀质的凝胶,并有较好的透明度和柔韧性.     

Structure and microporous formation of cellulose/silk fibroin blend membranes: Part II. Effect of post-treatment by alkali

Blend membranes (RCF1) were prepared from mixture solution of cellulose and silk fibroin (SF) in cuoxam by coagulating with acetone–acetic acid (4:1 by volume). The blend membranes were subjected to post-treatment with 10% NaOH aqueous solution, and their structure and properties were characterized by FT-IR, X-ray diffraction, DSC, SEM and DMTA. In previous work, cellulose/SF blend membranes (RCF2) prepared by coagulating with 10% NaOH aqueous solution formed a microporous structure, in which the SF as a pore former was almost completely removed from the membrane. However, when the blend membranes RCF1 were immersed in 10% NaOH aqueous solution for post-treatment, a strong hydrogen bonding between cellulose and SF inhibited the removal of SF. Although alkali is a good solvent for SF, the blend membranes RCF1 such obtained from cellulose and SF were alkali resistant. The crystallinity and the mean pore size of the blend membranes slightly decreased with increasing post-treatment time. This work provided a cellulose/silk blend membrane, which can be used under alkaline medium.     

Surface properties of silk fibroin films and their interaction with fibroblasts

There is a growing interest in the use of silk as a biomaterial for tissue engineering. Silk threads from Bombyx mori have a fibrous core of fibroin, the protein responsible for biocompatibility and bioactivity, which is surrounded by a family of "gummy" proteins, called sericins, which are almost completely removed during silk degumming. Three different methanol treatments on regenerated fibroin films were used to convert viscous solutions of Silk I to an insoluble crystalline form (Silk II), in an attempt to devise new processing protocols for the creation of a cell guiding fibroin surface. Human fibroblasts (MRC5 line) were used as probes of the cell-biomaterial interaction in the early stages of the process (1 h, 3 h, 6 h and 4 d after seeding). The effect of each treatment on cell adhesion, spreading and distribution was monitored by scanning electron microscopy (SEM) and was correlated to superficial properties (like roughness and crystallinity) and fibroin conformation by means of atomic force microscopy (AFM), used in both topographical and acoustic mode, and attenuated total internal reflection infrared spectroscopy (FTIR-ATR). It was found that traditional methanol treatments where fibroin films were soaked in methanol solution produced roughness patterns that affected only the very early stages of fibroblast adhesion (until 3 h from seeding), while the new treatment proposed could really dialogue with the cells. Its non-homogeneous surface can explain the existence of cells spreading in specific directions and the presence of cell repellent areas even 4 d after seeding.     

Miscibility and biodegradability of silk fibroin/carboxymethyl chitin blend films

Blend films of silk fibroin and carboxymethyl chitin were prepared by solution casting using water as a cosolvent. The blend films were subjected to post-treatment with an aqueous methanol solution to induce beta-sheet formation of silk fibroin. The miscibility of the blend films both before and after methanol treatments was investigated in terms of chemical interactions, morphologies, thermal properties, and crystal structures by using FTIR spectroscopy, SEM, DSC, and XRD. The results indicate that the blend between silk fibroin and CM-chitin was semi-miscible because only the amorphous parts of the polymers were compatible with each other. The enzymatic degradation showed that the incorporation of CM-chitin enhanced biodegradability and swelling ability of silk fibroin.