Silk Solution Research Articles

Simulation of Flow in the Silk Gland

Spiders and silkworms employ the complex flow of highly concentrated silk solution as part of silk fiber spinning. To understand the role of fluidic forces in this process, the flow of silk solution in the spider major ampullate and silkworm silk glands was investigated using numerical simulation. Our simulations demonstrate significant differences between flow in the spider and silkworm silk glands. In particular, shear flow effects are shown to be much greater in the spider than the silkworm, the silkworm gland exhibits a much different flow extension profile than the spider gland, and the residence time within the spider gland is eight times greater than in the silkworm gland. Lastly, simulations on the effect of spinning speed on the flow of silk solution suggest that a critical extension rate is the initiating factor for fiber formation from silk solution. These results provide new insight into silk spinning processes and will guide the future development of novel fiber spinning technologies.

Conformational and Orientational Transformation of Silk Proteins in the Major Ampullate Gland of Nephila clavipes Spiders

The orientational and conformational transformation of the native liquid silk into a solid fiber in the major ampullate gland of the spider Nephila clavipes has been studied by Raman spectromicroscopy. The spectra show that the conformation of silk proteins in the glandular sac contains several secondary structure elements, which is consistent with intrinsically unfolded proteins. A few alpha-helices are also present and involve some alanine residues located in the polyalanine segments of the spidroin sequence. The conversion of the silk solution in the major ampullate gland appears to be a two-state process without intermediate states. In the first and second limbs of the duct, silk is isotropic and spidroins are generally native-like. beta-Sheets start to develop between the second and the third limb of the duct, suggesting that early beta-sheets are generated by shear forces. However, most of the beta-sheets are formed between the draw down taper and the valve. The early beta-sheets formed upward of the draw down taper might play the role of nucleation sites for the subsequent beta-sheet aggregation. The alignment of the polypeptides chains occurs near the valve, revealing that orientational and conformational changes do not occur simultaneously. Extensional flow seems to be the driving force to produce the orientational order, which in turn is associated with the formation of the major part of the beta-sheets. The slow evolution of the spidroin conformation up to the draw down taper followed by the rapid transformation between the drawn down taper and the valve may be important to achieve the optimal structure of the final fiber.

A microfluidic cell for studying the formation of regenerated silk by synchrotron radiation small- and wide-angle X-ray scattering

A tube-in-square-pipe microfluidic glass cell has been developed for studying the aggregation and fiber formation from regenerated silk solution by in-situ small-angle X-ray scattering using synchrotron radiation. Acidification-induced aggregation has been observed close to the mixing point of the fibroin and buffer solution. The fibrous, amorphous material is collected in a water bath. Micro-wide-angle X-ray scattering of the dried material confirms its beta-sheet nature.

Silk Fibroin Microfluidic Devices.

Silk Fibroin Microfluidic Devices.

Intrinsic fluorescence changes associated with the conformational state of silk fibroin in biomaterial matrices

Silk fibroin is emerging as an important biomaterial for tissue engineering applications. The ability to monitor non-invasively the structural conformation of silk matrices prior to and following cell seeding could provide important insights with regards to matrix remodeling and cell-matrix interactions that are critical for the functional development of silk-based engineered tissues. Thus, we examined the potential of intrinsic fluorescence as a tool for assessing the structural conformation of silk proteins. Specifically, we characterized the intrinsic fluorescence spectra of silk in solution, gel and scaffold configurations for excitation in the 250 to 335 nm range and emission from 265 to 600 nm. We have identified spectral components that are attributed to tyrosine, tryptophan and crosslinks based on their excitation-emission profiles. We have discovered significant spectral shifts in the emission profiles and relative contributions of these components among the silk solution, gel and scaffold samples that represent enhancements in the levels of crosslinking, hydrophobic and intermolecular interactions that are consistent with an increase in the levels of ss-sheet formation and stacking. This information can be easily utilized for the development of simple, non-invasive, ratiometric methods to assess and monitor the structural conformation of silk in engineered tissues.

HOW SPIDERS SPIN

There are almost as many anecdotes about man trying to harvest spider silk as web styles; even Napoléon tried farming the territorial arachnids in a bid to harness the robust material. However, despite decades of intense effort and millions of research dollars, no one has successfully spun synthetic spider silk to the specifications that spiders achieve effortlessly. Gareth McKinley realised that although a great deal was known about the spun thread's composition and mechanical properties, few had studied the silk solution's behaviour as it is extruded from the animal's major ampullate gland. Knowing that polymer solutions often behave in strange and unexpected ways when squeezed thorough microscopic apertures, McKinley decided to study the material properties of natural spider silk solutions on a microscopic scale. When biological engineer Nikola Kojiç joined the MIT lab,McKinley knew he'd found the right person to begin unlocking the spinner's secrets (p. 4355).But before Kojiç could begin testing the fluid's flow properties, he had to learn how to extract the microscopic volume of silk protein solution stored in the golden orb spider's major ampullate gland. Kojiç visited Marian Goldsmith's lab at the University of Rhode Island where he learned to extract silk protein solutions from silk worms before he tried his hand on the rarer golden orb spider. Having successfully extracted a few microlitres of the scarce solution Kojiç explains that he had to work fast, keeping the gel-like solution under water to prevent evaporation, to measure the fluid's flow properties.First Kojiç measured the thick solution's viscosity using Christian Clasen's flexure-based micro-rheometer. Shearing 0.7 μl of the sample between two glass plates, the pair found that the silk solution's viscosity decreased dramatically as the plates moved faster. The silk solution was becoming increasingly slippery until it was even slipperier than silicone oil. McKinley admits that he was surprised at how slippery the material became, but adds that it explains how spiders extrude the thick gel to produce the delicate thread. According to McKinley, the shearing process aligns the long protein polymers in the silk solution until the molecules begin slipping easily past each other and the viscosity drops dramatically.Next the team tested the protein solution's stickiness by placing 1 μl of the sample between two small plates on an extensional rheometer, built by José Bico. Pulling the rheometer's plates 5 mm apart to produce a thin filament of the gooey solution, Kojiç measured the filament's diameter as it thinned and found that the silk solution was as sticky as egg white,becoming stiffer as the thread dried.McKinley explains that many of the solution's unique properties boil down to the protein's long molecules, which are relatively entangled at high concentrations making the solution highly viscous. However, as the solution is squeezed through the spider's ampullate gland the molecules become aligned,sliding past each other easily as the viscosity drops before the sticky silk is extruded into a fine filament, drying to form one of the toughest materials known to man.

Electrospun Bombyx mori gland silk

Solutions of Bombyx mori gland silk can be electrospun with the addition of some polyethylene oxide (PEO). Green fluorescent protein (GFP) can also be incorporated and electrospun without apparent phase separation from the silk. The dimensions of the fibers with and without the GFP are qualitatively similar. The results indicate the possibility of making fibers with uniform non-linear optical properties.

The spinning processes for spider silk.

This paper summarizes recent work in our groups on the factors that influence the formation of spider silks during the spinning process. The review encompasses: (a) extrusion variables that greatly affect the mechanical properties of the silk filaments; such as rate and temperature at spinning as well as the post-drawn treatment and (b) other factors affecting the conformation transition of the spider silk proteins (spidroin) such as pH and metallic ions. The observations taken together imply that the spinning process is at least as central as, and probably more important than, the composition of the 'raw' protein spinning solution. This conclusion leads us to suggest that in the future high-performance, artificial 'spider' silks may be spun from a range of solutions of silk and synthetic proteins.

Differentiation of Bone Marrow Stem Cells on Inkjet Printed Silk Lines

Water based silk solutions were successfully inkjet printed for the first time into patterns of parallel lines onto vinyl plastic substrates. Human bone marrow stromal cells (hMSCs) were seeded on the silk printed patterns and cultured in the presence of 100 ng/ml of bone morphogenic protein (BMP-2). After one week of culture cell growth and attachment showed site specificity on the silk printed lines. Both alkaline phosphatase activity and cell morphology indicated hBMSCs differentiation into osteogenic cells along the silk printed lines. After 4 week of culture, the cellular bridging of adjacent silk printed lines took place for all interline distances lower than 1.25 mm. Therefore, commercial inkjet printing technology can produce complex viable cellular patterns with 111 ± 24 μm lateral resolution, through the deposition of bioactive materials. The results provide a first step toward cell specific control using 3D inkjet printing techniques using biocompatible gel systems to regulate cell functions.

Differentiation of Bone Marrow Stem Cells on Inkjet Printed Silk Lines

ABSTRACTWater based silk solutions were successfully inkjet printed for the first time into patterns of parallel lines onto vinyl plastic substrates. Human bone marrow stromal cells (hMSCs) were seeded on the silk printed patterns and cultured in the presence of 100 ng/ml of bone morphogenic protein (BMP-2). After one week of culture cell growth and attachment showed site specificity on the silk printed lines. Both alkaline phosphatase activity and cell morphology indicated hBMSCs differentiation into osteogenic cells along the silk printed lines. After 4 week of culture, the cellular bridging of adjacent silk printed lines took place for all interline distances lower than 1.25 mm. Therefore, commercial inkjet printing technology can produce complex viable cellular patterns with 111 ± 24 μm lateral resolution, through the deposition of bioactive materials. The results provide a first step toward cell specific control using 3D inkjet printing techniques using biocompatible gel systems to regulate cell functions.

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.

Thermally Induced α-Helix to β-Sheet Transition in Regenerated Silk Fibers and Films

The structure of thin films cast from regenerated solutions of Bombyx mori cocoon silk in hexafluoroisopropyl alcohol (HFIP) was studied by synchrotron X-ray diffraction during heating. A solid-state conformational transition from an alpha-helical structure to the well-known beta-sheet silk II structure occurred at a temperature of approximately 140 degrees C. The transition appeared to be homogeneous, as both phases do not coexist within the resolution of the current study. Modulated differential scanning calorimetry (DSC) of the films showed an endothermic melting peak followed by an exothermic crystallization peak, both occurring near 140 degrees C. Oriented fibers were also produced that displayed this helical molecular conformation. Subsequent heating above the structural transition temperature produced oriented beta-sheet fibers very similar in structure to B. mori cocoon fibers. Heat treatment of silk films at temperatures well below their degradation temperature offers a controllable route to materials with well-defined structures and mechanical behavior.

Acid extraction and purification of recombinant spider silk proteins.

A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers.

PH induced changes in the rheology of silk fibroin solution from the middle division of Bombyx mori silkworm.

The rheological properties of fibroin silk solutions extracted from the middle division of Bombyx mori silkworms were examined. Acidification of the solutions with acetic acid vapor gelled the material, a process which at short time scales could be reversed by exposure to ammonia vapor. The solution could also be converted to sol from the gel state by the addition of EDTA. The possible mechanisms for gel formation in fibroin solutions is discussed as are the implications for the process of spinning silk fibers.

Structural conformation of spidroin in solution: a synchrotron radiation circular dichroism study.

Spider silk is made and spun in a complex process that tightly controls the conversion from soluble protein to insoluble fiber. The mechanical properties of the silk fiber are modulated to suit the needs of the spider by various factors in the animal's spinning process. In the major ampullate (MA) gland, the silk proteins are secreted and stored in the lumen of the ampulla. A particular structural fold and functional activity is determined by the spidroins' amino acid sequences as well as the gland's environment. The transition from this liquid stage to the solid fiber is thought to involve the conversion of a predominantly unordered structure to a structure rich in beta-sheet as well as the extraction of water. Circular dichroism provides a quick and versatile method for examining the secondary structure of silk solutions and studying the effects of various conditions. Here we present the relatively novel technique of synchrotron radiation based circular dichroism as a tool for investigating biomolecular structures. Specifically we analyze, in a series of example studies on structural transitions induced in liquid silk, the type of information accessible from this technique and any artifacts that might arise in studying self-assembling systems.

Human bone marrow stromal cell responses on electrospun silk fibroin mats

Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700±50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (∼15 μm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.

Aqueous Processing and Fiber Spinning of Recombinant Spider Silks

Spiders have captured the interest of scientists for many years because spider silks are among the toughest materials, having properties that surpass some man-made synthetic materials. Spinning recombinant silk to duplicate those properties has proved to be extremely difficult. This is the first known report of spinning recombinant silk fibers in an aqueous environment. The method seeks to keep the protein soluble throughout the process, not unlike the way the spider stores and spins silk. Recombinant silk proteins were produced by bacterial fermentation in which the cell pellets were lyophilized and lysed with organic acid. Silk protein was purified from the lysate by chromatography and processed in dilute denaturing buffer into a fiber spinning solution. Circular dichroism measurements of the silk solutions revealed an increase in β-sheet content as a function of time. Time-dependent self-association of silk protein was monitored in solution by dynamic light scattering. Furthermore, the observed increas...

Regenerated Bombyx silk solutions studied with rheometry and FTIR

Several different solvent systems are commonly used to dissolve Bombyx mori silk fibroin to prepare regenerated silk membranes and fibers though differences in the behavior of these solvents have not been fully investigated. Here we compare the effects of four of these on the rheology of silk fibroin solutions and on protein secondary structure as revealed by FTIR spectroscopy of cast membranes. The results demonstrated that Ca(NO 3) 2–MeOH–H 2O and LiBr–EtOH–H 2O had the strongest solvation on the silk fibroin chains, which showed an almost constant viscosity (Newtonian behavior) over most of the shear rate range (0.1–500 s −1). In contrast, the 9.5 M aqueous LiBr appeared to have the weakest solvation with similar effects on the silk fibroin molecules to pure water as indicated by rheological behavior. It was also found that the silk fibroin membranes prepared using all four solvent system showed mainly random coil conformation with a small proportion of β-sheet by FTIR spectroscopy. We discuss the implications of these findings for the preparation of regenerated silk for different applications.

From Natural Silks to New Polymer Fibres

The future of silk as an engineering fibre is considered, taking into account the significant shortcomings of this material as well as its much-publicised attractions. Attention is focused on issues that remain unsolved or unresolved — either reflecting the difficulty of characterising the complex hierarchical structure in silk solutions and fibres, or arising from the poor reproducibility of silk tensile property measurements. Advantages and attendant practical limitations of harnessing biotechnology to synthesise silk and silk-like proteins in transgenic hosts are addressed.

Optical Rheometry of Silk in Aqueous LiBr Solution

The rheo-optical response of solutions of silk in aqueous 9 M LiBr of concentrations ranging from 7.5 to 15 wt % was investigated. These samples were found to be birefringent under flow, with the steady state birefringence having a concentration scaling of c3.9. This scaling behavior suggests a wormlike conformation of the silk molecules in solution. The relaxation was fast, and Maxwellian behavior was observed at low shear rates. No dichroism was detected suggesting that electrostatic screening suppresses structure formation under shear.