Porous 3-D scaffolds from regenerated silk fibroin

Three fabrication techniques, freeze-drying, salt leaching and gas foaming, were used to form porous three-dimensional silk biomaterial matrixes. Matrixes were characterized for morphological and functional properties related to processing method and conditions. The porosity of the salt leached scaffolds varied between 84 and 98% with a compressive strength up to 175 +/- 3 KPa, and the gas foamed scaffolds had porosities of 87-97% and compressive strength up to 280 +/- 4 KPa. The freeze-dried scaffolds were prepared at different freezing temperatures (-80 and -20 degrees C) and subsequently treated with different concentrations (15 and 25%) and hydrophilicity alcohols. The porosity of these scaffolds was up to 99%, and the maximum compressive strength was 30 +/- 2 KPa. Changes in silk fibroin structure during processing to form the 3D matrixes were determined by FT-IR and XrD. The salt leached and gas foaming techniques produced scaffolds with a useful combination of high compressive strength, interconnected pores, and pore sizes greater than 100 microns in diameter. The results suggest that silk-based 3D matrixes can be formed for utility in biomaterial applications.     

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, ¹³C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol–gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were -helix and random coil in solution. There also existed a little β-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no -helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol–gel transformation. The velocity increased obviously when the temperature was above 30 °C. During the sol–gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to β-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.     

Study on the structure and properties of wool keratin regenerated from formic acid

Structural characteristics of keratin regenerated from water (KW) and from formic (KF) acid solutions were compared. Amino acid composition and molecular weight distribution of KW and KF samples were studied by high performance liquid chromatography (HPLC) and SDS-PAGE electrophoresis. Turbidity measurement showed that keratin dissolved in formic acid forms transparent and stable solutions and no flocculation occurs. In addition, because of its good solvation properties, studied by viscosity measurements, formic acid can be used as a co-solvent to prepare keratin-based blend solutions. Structural studies carried out by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) and near infrared (NIR) suggest that formic acid stabilizes the beta-sheet structure. Thermogravimetric analysis (TGA) reveals a higher thermal stability of keratin regenerated from formic acid with respect to keratin regenerated from water.     

Structural analysis of Bombyx mori silk fibroin peptides with formic acid treatment using high-resolution solid-state 13C NMR spectroscopy

Bombyx mori silk fibroin fiber is a fibrous protein produced by the silkworm at room temperature and from an aqueous solution whose primary structure is highly repetitive. In this study we analyzed the structural characteristics of native peptides, derived from B. mori silk fibroin, with formic acid treatment using high-resolution solid-state 13C NMR. We establish that the Ser residue bearing a short polar side chain has the ability to stabilize the conformation formed in the model peptides due to its ability to form intermolecular hydrogen bonds involving its hydroxyl group as a donor and the carbonyl groups of other residues as acceptors. On the other hand, insertion of Tyr residue in the basic (AG)n and (AGSGAG)n sequence motifs usually exhibited disruptive effects on the preferred conformations. Moreover, the environmental effect was investigated by mixing the native Cp fraction with the model peptides, showing that there is no significant structural difference on the Ser-containing peptides, while structural transformation was observed on the peptides containing the GAAS unit. This may be attributed to the fact that the Cp fraction promotes the formation of an antiparallel beta-sheet in the Ala-Ala unit. Such periodically disrupted ordered structures in the semicrystalline region of B. mori silk fibroin may be critical not only for facilitating the conformational transformation from silk I to silk II structural form but also for having some correlation with the unique properties of the silk materials.     

Effect of shearing on formation of silk fibers from regenerated Bombyx mori silk fibroin aqueous solution

In this paper, the spinnable regenerated silk fibroin aqueous solution with high concentration was prepared and the regenerated silk fibers were obtained from the aqueous solution by two different spinning processes at ambient temperature. The orientation of these fibers was characterized by polarizing microscope. Their secondary structure was investigated by Raman spectroscopy and related mechanical properties were also measured. These data showed that shearing is an important step for increasing orientation and silk II (β-sheet) structure, and the mechanical properties of the regenerated silk fibers can also be improved by shearing.     

Correlation between fibroin amino acid sequence and physical silk properties

The fiber properties of lepidopteran silk depend on the amino acid repeats that interact during H-fibroin polymerization. The aim of our research was to relate repeat composition to insect biology and fiber strength. Representative regions of the H-fibroin genes were sequenced and analyzed in three pyralid species: wax moth (Galleria mellonella), European flour moth (Ephestia kuehniella), and Indian meal moth (Plodia interpunctella). The amino acid repeats are species-specific, evidently a diversification of an ancestral region of 43 residues, and include three types of regularly dispersed motifs: modifications of GSSAASAA sequence, stretches of tripeptides GXZ where X and Z represent bulky residues, and sequences similar to PVIVIEE. No concatenations of GX dipeptide or alanine, which are typical for Bombyx silkworms and Antheraea silk moths, respectively, were found. Despite different repeat structure, the silks of G. mellonella and E. kuehniella exhibit similar tensile strength as the Bombyx and Antheraea silks. We suggest that in these latter two species, variations in the repeat length obstruct repeat alignment, but sufficiently long stretches of iterated residues get superposed to interact. In the pyralid H-fibroins, interactions of the widely separated and diverse motifs depend on the precision of repeat matching; silk is strong in G. mellonella and E. kuehniella, with 2-3 types of long homogeneous repeats, and nearly 10 times weaker in P. interpunctella, with seven types of shorter erratic repeats. The high proportion of large amino acids in the H-fibroin of pyralids has probably evolved in connection with the spinning habit of caterpillars that live in protective silk tubes and spin continuously, enlarging the tubes on one end and partly devouring the other one. The silk serves as a depot of energetically rich and essential amino acids that may be scarce in the diet.     

X-ray structural study of noncrystalline regenerated Bombyx mori silk fibroin

X-ray diffraction measurements of regenerated Bombyx mori silk fibroin were carried out to determine its structural characteristic from an analysis of differential radial distribution functions (DRDFs). The temperature dependence of X-ray diffraction patterns from noncrystalline and crystal structures of regenerated silk fibroin was investigated using a high temperature furnace. Time resolved X-ray diffraction profiles were also obtained to construct kinematical models of structural changes caused by the addition of water. DRDFs, calculated from the experimental data, were compared with the DRDFs simulated on the basis of the Monte Carlo method. In order to model the noncrystalline structures, structural units were assumed to be parts of the crystalline structure of silk and those with appropriate structural defects reported previously. From the comparison of experimental and simulated DRDFs, it was determined that noncrystalline regenerated silk consisted of locally ordered atomic sheets similar to the atomic arrangement in the silk I crystal (Type-I sheets), and the final state of the structural change was noncrystalline, consisting of small crystallites, the structure of which is similar to that of silk II (Type-II crystallites). Time resolved DRDFs were also qualitatively interpreted by both the ordering of Type-I sheets and structural changes from Type-I to Type-II. The formation of the small Type-II crystallites obtained in this study was consistent with the nucleation of silk II by birefringence measurements of silk glands and the spinneret of Bombyx mori silkworm reported previously. X-ray diffraction should be a useful technique to understand the structural characteristics of noncrystalline organic materials.     

Structural characteristics and biological performance of silk fibroin nanofiber containing microalgae spirulina extract

Silk fibroin (SF) nanofiber scaffold containing microalgae Spirulina extract were prepared by electrospinning and the performance and functionality of the scaffold were evaluated. The viscosity and conductivity of the dope solution of Spirulina containing SF were examined for electrospinability and we found that the morphological structure of SF nanofiber is affected by the concentration of Spirulina extract added. The platelet adhesion and coagulation time test confirmed that the Spirulina containing SF nanofiber scaffold had excellent ability to prevent blood clotting or antithrombogenicity that is comparable to heparin. Low cytotoxicity and excellent cell adhesion and proliferation were also observed for Sprulina containing SF nanofiber scaffold by methylthiazolyldiphenyl‐tetrazolium bromide assay and confocal fluorescence microscope using fibroblast and human umbilical vein endothelial cells. Based on these results, we believe SF nanofiber scaffold containing Spirulina extract has the potential to be used as tissue engineering scaffold that requires high hemocompatibility. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 307–318, 2014.     

A novel three-dimensional tubular scaffold prepared from silk fibroin by electrospinning

Effects of electrospinning parameters (including voltage, collection distance, solution concentration and flow rate) on the morphology and diameter distribution of regenerated SF (silk fibroin) fiber were investigated. Afterward, SF tubular scaffold composed of homogenous fibers was fabricated at voltage of 18 kV, collection distance of 18 cm, concentration of 37%, and flow rate of 0.15 mL/min. After methanol treatment, SF tubular scaffold showed tensile strength of 3.57 MPa and porosity of 80.85%. It is satisfied that our work offers a simple method to fabricate seamless and porous tubular scaffold from SF without any additives and organic solvents. Furthermore, the results suggest that this tubular scaffold shows promising applications in small-diameter vascular graft.     

A study on the flow stability of regenerated silk fibroin aqueous solution

The flow stability of silk fibroin (SF) aqueous solutions with different concentrations under different temperatures was investigated. It was found that the flow stability decreased quickly with the increase of solution concentration and temperature. X-ray diffraction, Fourier transform infrared (FTIR) and Raman spectroscopy analysis showed that silk fibroin in aqueous solution was mainly in random coil and alpha-helix conformation. However, it turned into alpha-helix and beta-sheet conformation after gelation, and both silk I and silk II crystalline structures appeared accordingly. The investigation implies that the original dilute regenerated SF aqueous solution should be stored under low temperature and concentrated just before spinning.     

Morphology and structure of electrospun mats from regenerated silk fibroin aqueous solutions with adjusting pH

In this paper, regenerated silk fibroin (SF) aqueous solutions were adjusted to a pH of 6.9 by mimicing the condition in the posterior division of silkworm's gland and rheological behavior of solutions was investigated. The electrospinning technique was used to prepare fibers, and non-woven mats of regenerated B. mori silk fibroin were successfully obtained. The effects of electrospinning parameters on the morphology and diameter of regenerated silk fibers were investigated by orthogonal design. Statistical analysis showed that voltage, the concentration of regenerated SF solutions and the distance between tip and collection plate were the most dominant parameters to fiber morphology, diameter and diameter distribution, respectively. An optimal electrospinning condition was obtained in producing uniform cylindrical fibers with an average diameter of 1300nm. It was as follows: the concentration 30%, voltage 40kV, distance 20cm. The structure of electrospun mats was characterized by Raman spectroscopy (RS), wide-angle X-ray diffraction (WAXD) and modulated differential scanning calorimetry (MDSC). It was found that electrospun mats were predominantly random coil/silk I structure, and the transition to silk II (beta-sheet) rich structure should be further explored.     

Compliant film of regenerated Antheraea pernyi silk fibroin by chemical crosslinking

A compliant film was prepared by chemical crosslinking of fibroin from silk fiber of wild silkworm, Antheraea pernyi. The silk fiber was dissolved in concentrated aqueous lithium thiocyanate and desalinated by dialysis. The film was cast from the regenerated aqueous solution, and crosslinked by polyethylene glycol diglycidyl ether (PEG-DE). This film showed high water resistively while maintaining random coil and -helix structure, unlike films prepared by organic solvent treatment that causes β-sheet formation. The films containing about 20 wt.% crosslinker were remarkably compliant and tenacious. These features, combined with the living-cell affinity of the wild silkworm fibroin, are expected to be useful in biomedical applications.     

Wet spinning of Bombyx mori silk fibroin dissolved in N-methyl morpholine N-oxide and properties of regenerated fibres

Silk fibroin (SF) was dissolved in N-methyl morpholine N-oxide (NMMO) at a polymer concentration of 13% (w/w); thermal and rheological solution properties were characterized. The melting/crystallization behaviour of NMMO was influenced by SF presence. Melting of NMMO hydrate decreased to 71 degrees C and a cold crystallization peak appeared at 35 degrees C on heating. None crystallization occurred on cooling. Quenching at a temperature of 50 degrees C or higher did not induce any crystallization on heating. Viscosity of SF-NMMO solutions decreased as a function of temperature. At 75 degrees C, viscosity remained constant for 360 min. SF-NMMO dope was spun by using a lab-scale wet spinning line. The extruded filament was coagulated in an ethanol bath. Regenerated SF fibres were collected at different draw ratios and their morphological, physical, and mechanical properties were characterized. Fibre diameters ranged from 133 to 19mum, cross-section was regularly circular, and surface was generally smooth, with a very fine granular aspect. Birefringence increased with increasing the draw ratio, especially when take up and post-spinning draw were coupled. FT-IR spectra and DSC thermograms confirmed that SF fibres crystallized into Silk II structure. The IR crystallinity index did not change as a function of drawing. Regenerated SF fibres undrawn or drawn only during the coagulation step showed the mechanical behaviour typical of a brittle material. However, when both take up and post-spinning draw were applied, fibres displayed a ductile-stable behaviour. Typical values of the mechanical parameters of regenerated SF fibres were: E=8.7 GPa, sigma(b)=120 MPa and epsilon(b)=35%.     

Conformational transition and liquid crystalline state of regenerated silk fibroin in water

The conformational transition of molecular chains of regenerated silk fibroin (SF) aqueous solution is systematically investigated by circular dichroism, Raman, IR, and UV-vis spectroscopies. It is found that an initial random coil conformation of the SF can be readily changed into an ordered beta-sheet structure by optimizing the solution conditions, such as the SF concentration, pH, temperature, or metal-ion content. Circular dichroic spectra quantitatively confirm a steadily decreased content of the random coil conformation but a significantly increased beta-sheet content after an ultrasonic or extruding treatment. Furthermore, the extrusion is more powerful to achieve high beta-sheet content than the ultrasonic. It is interesting that the polarized optical micrographs of the SF aqueous solution extruded by injection illustrate the formation and existence of liquid crystalline state. A study of extrusion in vitro could be used as a model system to understand the natural silk spinning process in silkworm.     

Structural evolution of regenerated silk fibroin under shear: combined wide- and small-angle x-ray scattering experiments using synchrotron radiation

The structural evolution of regenerated Bombyx mori silk fibroin during shearing with a Couette cell has been studied in situ by synchrotron radiation small- and wide-angle x-ray scattering techniques. An elongation of fibroin molecules was observed with increasing shear rate, followed by an aggregation phase. The aggregates were found to be amorphous with beta-conformation according to infrared spectroscopy. Scanning x-ray microdiffraction with a 5 microm beam on aggregated material, which had solidified in air, showed silk II reflections and a material with equatorial reflections close to the silk I structure reflections, but with strong differences in reflection intensities. This silk I type material shows up to two low-angle peaks suggesting the presence of water molecules that might be intercalated between hydrogen-bonded sheets.     

Time-resolved structural investigation of regenerated silk fibroin nanofibers treated with solvent vapor

Nonwoven matrices of silk fibroin (SF) nanofibers were prepared by electrospinning a regenerated SF solution, followed by treatment with solvent vapor including water, methanol, ethanol, and propanol. Structural changes of solvent vapor-treated SF nanofibers were investigated in a time-resolved manner using IR spectroscopy. Conformational transitions of SF nanofibers from random coil to beta-sheet forms were dependent on the type of solvent vapor used, and their transition rates were strongly influenced by treatment temperatures. Consistent with previous findings, methanol vapor treatment provided a fast and effective means by which to alter the secondary structure of SF nanofibers. However, treatment with water vapor, as compared to treatment with alcohol vapor, was also useful for inducing structural changes in SF nanofibers. As demonstrated in the present study, our approach of controlling secondary structure formation of proteins by solvent vapor treatment and monitoring real-time conformational changes may be useful for the design and tailoring of materials for biomedical applications.     

X-ray structural study of noncrystalline regenerated Bombyx mori silk fibroin

X-ray diffraction measurements of regenerated Bombyx mori silk fibroin were carried out to determine its structural characteristic from an analysis of differential radial distribution functions (DRDFs). The temperature dependence of X-ray diffraction patterns from noncrystalline and crystal structures of regenerated silk fibroin was investigated using a high temperature furnace. Time resolved X-ray diffraction profiles were also obtained to construct kinematical models of structural changes caused by the addition of water. DRDFs, calculated from the experimental data, were compared with the DRDFs simulated on the basis of the Monte Carlo method. In order to model the noncrystalline structures, structural units were assumed to be parts of the crystalline structure of silk and those with appropriate structural defects reported previously. From the comparison of experimental and simulated DRDFs, it was determined that noncrystalline regenerated silk consisted of locally ordered atomic sheets similar to the atomic arrangement in the silk I crystal (Type-I sheets), and the final state of the structural change was noncrystalline, consisting of small crystallites, the structure of which is similar to that of silk II (Type-II crystallites). Time resolved DRDFs were also qualitatively interpreted by both the ordering of Type-I sheets and structural changes from Type-I to Type-II. The formation of the small Type-II crystallites obtained in this study was consistent with the nucleation of silk II by birefringence measurements of silk glands and the spinneret of Bombyx mori silkworm reported previously. X-ray diffraction should be a useful technique to understand the structural characteristics of noncrystalline organic materials.     

纳米丝素蛋白微球的制备及性能研究

以家蚕丝素蛋白为原料,基于丝素自组装理论,通过酶解-干燥-溶解法制备不同尺寸的丝素蛋白微球,制备出的微球具有良好的水不溶性和稳定的分散性。对微球的形貌和结构表征结果表明,用该方法制备的丝素蛋白微球为纳米微球,当酶的添加量为2%且蛋白自组装时间为4 h时,丝素蛋白微球的平均粒径最小,仅为(32±11)nm。红外光谱(FT-IR)和X射线衍射(XRD)结果显示,微球中β-折叠结构的多少决定了微球晶体的大小,β-折叠越多,微球中晶体的体积越大。通过调控丝素蛋白自组装过程,可以制备平均粒径在30～140 nm之间的纳米丝素蛋白微球,且不引入任何有机溶剂和无机溶剂,制备过程绿色环保,制备出的丝素蛋白微球安全无毒。