Materials fabrication from Bombyx mori silk fibroin

Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery.     

Degradation mechanism and control of silk fibroin

Controlling the degradation process of silk is an important and interesting subject in the field of biomaterials. In the present study, silk fibroin films with different secondary conformations and nanostructures were used to study degradation behavior in buffered protease XIV solution. Different from previous studies, silk fibroin films with highest β-sheet content achieved the highest degradation rate in our research. A new degradation mechanism revealed that degradation behavior of silk fibroin was related to not only crystal content but also hydrophilic interaction and then crystal-noncrystal alternate nanostructures. First, hydrophilic blocks of silk fibroin were degraded. Then, hydrophobic crystal blocks that were formerly surrounded and immobilized by hydrophilic blocks became free particles and moved into solution. Therefore, on the basis of the mechanism, which enables the process to be more controllable and flexible, controlling the degradation behavior of silk fibroin without affecting other performances such as its mechanical or hydrophilic properties becomes feasible, and this would greatly expand the applications of silk as a biomedical material.     

Electrospinning Bombyx mori silk with poly(ethylene oxide)

Electrospinning for the formation of nanoscale diameter fibers has been explored for high-performance filters and biomaterial scaffolds for vascular grafts or wound dressings. Fibers with nanoscale diameters provide benefits due to high surface area. In the present study we explore electrospinning for protein-based biomaterials to fabricate scaffolds and membranes from regenerated silkworm silk, Bombyx mori, solutions. To improve processability of the protein solution, poly(ethylene oxide) (PEO) with molecular weight of 900,000 was blended with the silk fibroin. A variety of compositions of the silk/PEO aqueous blends were successfully electrospun. The morphology of the fibers was characterized using high-resolution scanning electron microscopy. Fiber diameters were uniform and less than 800 nm. The composition was estimated by X-ray photoelectron spectroscopy to characterize silk/PEO surface content. Aqueous-based electrospining of silk and silk/PEO blends provides potentially useful options for the fabrication of biomaterial scaffolds based on this unique fibrous protein.     

Phosphorylation of silk fibroins improves the cytocompatibility of silk fibroin derived materials: A platform for the production of tuneable material

Silk fibroin demonstrates great biocompatibility and is suitable for many biomedical applications, including tissue engineering and regenerative medicine. Current research focuses on manipulating the physico‐chemical properties of fibroin, and examining the effect of this manipulation on firobin's biocompatibility. Regenerated silk fibroin was modified by in vitro enzymatic phosphorylation and cast into films. Films were produced by blending, at several ratios, the phosphorylated and un‐phosphorylated fibroin solutions. Fourier transform infra‐red spectroscopy was used to determine the specific P–OH vibration peak, confirming the phosphorylation of the regenerated silk fibroin solution. Differential scanning calorimetry showed that phosphorylation altered the intra‐ and inter‐molecular interactions. Further experiments demonstrated that phosphorylation can be used to tailor the hydrophylicity/hydrophobicity ratio as well as the crystalinity of silk fibroin films. Release profiling of a model drug was highly dependent on silk modification level. Cytotoxicity assays showed that exposure to lixiviates of phosphorylated films only slightly affected cellular metabolism and proliferation, although direct contact resulted in a strong direct correlation between phosphorylation level and cell proliferation. This new method for tuning silk biomaterials to obtain specific structural and biochemical features can be adapted for a wide range of applications. Phosphorylation of silk fibroins may be applied to improve the cytocompatibility of any silk‐based device that is considered to be in contact with live animals or human tissues.     

Fabrication and characterization of biomaterial film from gland silk of muga and eri silkworms

This study discusses the possibilities of liquid silk (Silk gland silk) of Muga and Eri silk, the indigenous non mulberry silkworms of North Eastern region of India, as potential biomaterials. Silk protein fibroin of Bombyx mori, commonly known as mulberry silkworm, has been extensively studied as a versatile biomaterial. As properties of different silk‐based biomaterials vary significantly, it is important to characterize the non mulberry silkworms also in this aspect. Fibroin was extracted from the posterior silk gland of full grown fifth instars larvae, and 2D film was fabricated using standard methods. The films were characterized using SEM, Dynamic contact angle test, FTIR, XRD, DSC, and TGA and compared with respective silk fibers. SEM images of films reveal presence of some globules and filamentous structure. Films of both the silkworms were found to be amorphous with random coil conformation, hydrophobic in nature, and resistant to organic solvents. Non mulberry silk films had higher thermal resistance than mulberry silk. Fibers were thermally more stable than the films. This study provides insight into the new arena of research in application of liquid silk of non mulberry silkworms as biomaterials. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 292–333, 2013.     

Improving cell-adhesive properties of recombinant Bombyx mori silk by incorporation of collagen or fibronectin derived peptides produced by transgenic silkworms

Silk of Bombyx mori can be used as various biomaterials. Especially, it is useful as a protein for coating the surface of cell culture plates since the silk possesses a biocompatibility to the cultured cells. However, the cell-adhesive ability is weaker than collagen or fibronectin, which are used for coating the plate more frequently (Yao et al. J. Biochem., 2004, 136, 643-649). To increase the biocompatibility of the silk, we constructed transgenic silkworms, inserting the modified fibroin light-chain genes for making recombinant silks that possessed partial collagen or fibronectin sequences, that is, [GERGDLGPQGIAGQRGVV(GER)3GAS]8GPPGPCCGGG or [TGRGDSPAS]8, respectively. Films were made from the recombinant silks, and the cell-adhesive activity for cultured mammalian cells was observed. The results showed that the two types of recombinant silk films possessed a much higher cell-adhesive activity as compared to the original unmodified silk. Especially, the recombinant silk with the sequence [TGRGDSPAS]8, produced by a transgenic Nd-sD mutant, gave a 6 times higher activity than the original unmodified silk.     

Structure and properties of silk hydrogels

Control of silk fibroin concentration in aqueous solutions via osmotic stress was studied to assess relationships to gel formation and structural, morphological, and functional (mechanical) changes associated with this process. Environmental factors potentially important in the in vivo processing of aqueous silk fibroin were also studied to determine their contributions to this process. Gelation of silk fibroin aqueous solutions was affected by temperature, Ca(2+), pH, and poly(ethylene oxide) (PEO). Gelation time decreased with increase in protein concentration, decrease in pH, increase in temperature, addition of Ca(2+), and addition of PEO. No change of gelation time was observed with the addition of K(+). Upon gelation, a random coil structure of the silk fibroin was transformed into a beta-sheet structure. Hydrogels with fibroin concentrations >4 wt % exhibited network and spongelike structures on the basis of scanning electron microscopy. Pore sizes of the freeze-dried hydrogels were smaller as the silk fibroin concentration or gelation temperature was increased. Freeze-dried hydrogels formed in the presence of Ca(2+) exhibited larger pores as the concentration of this ion was increased. Mechanical compressive strength and modulus of the hydrogels increased with increase in protein concentration and gelation temperature. The results of these studies provide insight into the sol-gel transitions that silk fibroin undergoes in glands during aqueous processing while also providing important insight in the in vitro processing of these proteins into useful new materials.     

Production of scFv-conjugated affinity silk powder by transgenic silkworm technology

Bombyx mori (silkworm) silk proteins are being utilized as unique biomaterials for medical applications. Chemical modification or post-conjugation of bioactive ligands expand the applicability of silk proteins; however, the processes are elaborate and costly. In this study, we used transgenic silkworm technology to develop single-chain variable fragment (scFv)-conjugated silk fibroin. The cocoons of the transgenic silkworm contain fibroin L-chain linked with scFv as a fusion protein. After dissolving the cocoons in lithium bromide, the silk solution was dialyzed, concentrated, freeze-dried, and crushed into powder. Immunoprecipitation analyses demonstrate that the scFv domain retains its specific binding activity to the target molecule after multiple processing steps. These results strongly suggest the promise of scFv-conjugated silk fibroin as an alternative affinity reagent, which can be manufactured using transgenic silkworm technology at lower cost than traditional affinity carriers.     

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ^1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications ^3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications ^9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment ^12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.     

Silk fibroin film from golden‐yellow Bombyx mori is a biocomposite that contains lutein and promotes axonal growth of primary neurons

The use of doped silk fibroin (SF) films and substrates from Bombyx mori cocoons for green nanotechnology and biomedical applications has been recently highlighted. Cocoons from coloured strains of B. mori, such as Golden‐Yellow, contain high levels of pigments that could have a huge potential for the fabrication of SF based biomaterials targeted to photonics, optoelectronics and neuroregenerative medicine. However, the features of extracted and regenerated SF from cocoons of B. mori Golden‐Yellow strain have never been reported. Here we provide a chemophysical characterization of regenerated silk fibroin (RSF) fibers, solution, and films obtained from cocoons of a Golden‐Yellow strain of B. mori, by SEM, ¹H‐NMR, HPLC, FT‐IR, Raman and UV‐Vis spectroscopy. We found that the extracted solution and films from B. mori Golden‐Yellow fibroin displayed typical Raman spectroscopic and optical features of carotenoids. HPLC‐analyses revealed that lutein was the carotenoid contained in the fiber and RSF biopolymer from yellow cocoons. Notably, primary neurons cultured on yellow SF displayed a threefold higher neurite length than those grown of white SF films. The results we report pave the way to expand the potential use of yellow SF in the field of neuroregenerative medicine and provide green chemistry approaches in biomedicine. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 287–299, 2016.     

Surface Modification and Characterisation of Silk Fibroin Fabric Produced by the Layer-by-Layer Self-Assembly of Multilayer Alginate/Regenerated Silk Fibroin

Silk-based medical products have a long history of use as a material for surgical sutures because of their desirable mechanical properties. However, silk fibroin fabric has been reported to be haemolytic when in direct contact with blood. The layer-by-layer self-assembly technique provides a method for surface modification to improve the biocompatibility of silk fibroin fabrics. Regenerated silk fibroin and alginate, which have excellent biocompatibility and low immunogenicity, are outstanding candidates for polyelectrolyte deposition. In this study, silk fabric was degummed and positively charged to create a silk fibroin fabric that could undergo self-assembly. The multilayer self-assembly of the silk fibroin fabric was achieved by alternating the polyelectrolyte deposition of a negatively charged alginate solution (pH = 8) and a positively charged regenerated silk fibroin solution (pH = 2). Finally, the negatively charged regenerated silk fibroin solution (pH = 8) was used to assemble the outermost layer of the fabric so that the surface would be negatively charged. A stable structural transition was induced using 75% ethanol. The thickness and morphology were characterised using atomic force microscopy. The properties of the self-assembled silk fibroin fabric, such as the bursting strength, thermal stability and flushing stability, indicated that the fabric was stable. In addition, the cytocompatibility and haemocompatibility of the self-assembled silk fibroin fabrics were evaluated. The results indicated that the biocompatibility of the self-assembled multilayers was acceptable and that it improved markedly. In particular, after the self-assembly, the fabric was able to prevent platelet adhesion. Furthermore, other non-haemolytic biomaterials can be created through self-assembly of more than 1.5 bilayers, and we propose that self-assembled silk fibroin fabric may be an attractive candidate for anticoagulation applications and for promoting endothelial cell adhesion for vascular prostheses.     

Flexibility regeneration of silk fibroin in vitro

Although natural silk fibers have excellent strength and flexibility, the regenerated silk materials generally become brittle in the dry state. How to reconstruct the flexibility for silk fibroin has bewildered scientists for many years. In the present study, the flexible regenerated silk fibroin films were achieved by simulating the natural forming and spinning process. Silk fibroin films composed of silk I structure were first prepared by slow drying process. Then, the silk fibroin films were stretched in the wet state, following the structural transition from silk I to silk II. The difference between the flexible film and different brittle regenerated films was investigated to reveal the critical factors in regulating the flexibility of regenerated silk materials. Compared with the methanol-treated silk films, although having similar silk II structure and water content, the flexible silk films contained more bound water rather than free water, implying the great influence of bound water on the flexibility. Then, further studies revealed that the distribution of bound water was also a critical factor in improving silk flexibility in the dry state, which could be regulated by the nanoassembly of silk fibroin. Importantly, the results further elucidate the relation between mechanical properties and silk fibroin structures, pointing to a new mode of generating new types of silk materials with enhanced mechanical properties in the dry state, which would facilitate the fabrication and application of regenerated silk fibroin materials in different fields.     

Bombyx mori silk fibroin liquid crystallinity and crystallization at aqueous fibroin-organic solvent interfaces.

A banded morphology has been observed for Bombyx mori silk fibroin films obtained from an aqueous hexane interface; the period of the banding is approximately 1 microm. Morphology and diffraction from different regions of the banded structure suggest that it is a free surface formed by a cholesteric liquid crystal. Truncated hexagonal lamellar crystallites of B. mori silk fibroin have been observed in films formed in the surface excess layer of fibroin at the interface between aqueous fibroin and hexane or chloroform. Based on initial crystallographic evidence, a three-fold helical conformation has been ascribed to the fibroin chains within the crystals. The chain conformation and crystalline habit appear to be similar to the silk III structure previously observed at the air-water interface (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Valluzzi R, Gido S, Zhang W, Muller W, Kaplan D. Macromolecules 1996;29:8606-8614) but the crystalline packing is different. Diffraction data obtained for the crystallites are similar to diffraction behavior for a collagen-like model peptide. Diffraction patterns obtained from crystallized regions of the banded morphology can be indexed using the same unit cell as the hexagonal lamellar crystallites. Surfactancy of fibroin and subsequent aggregation and mesophase formation may help to explain the liquid crystallinity reported for silk, which is long suspected to play a role in the biological silk spinning process (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Willcox, P. J.; Gido, SP, Muller W, Kaplan DL. Macromolecules 1996:29:5106-5110; Magoshi J, Magoshi Y, Nakamura S. In: Kaplan D, Adams W, Farmer B, Viney C, editors, Mechanism of Fiber Formation of Silkworm. Washington, DC: American Chemical Society 1994:292-310; Magoshi J, Magoshi Y, Nakamura S. J Appl Polym Sci Appl Polym Symp 1985;41:187-204; Magoshi J, Magoshi Y, Nakamura S. Polym Commun 1985;26:309.).     

Non-bioengineered silk gland fibroin protein: characterization and evaluation of matrices for potential tissue engineering applications

The possibility of using wild non-mulberry silk protein as a biopolymer remains unexplored compared to domesticated mulberry silk protein. One of the main reasons for this was for not having any suitable method of extraction of silk protein fibroin from cocoons and silk glands. In this study non-bioengineered non-mulberry silk gland fibroin protein from tropical tasar silkworm Antheraea mylitta, is regenerated and characterized using 1% (w/v) sodium dodecyl sulfate (SDS). The new technique is important and unique because it uses a mild surfactant for fibroin dissolution and is advantageous over other previous reported techniques using chaotropic salts. Fabricated fibroin films are smooth as confirmed by atomic force microscopy. Circular dichroism spectrometry along with Fourier transformed infrared spectroscopy and X-ray diffraction reveal random coil/alpha-helix conformations in regenerated fibroin which transform to beta-sheets, resulting in crystalline structure and protein insolubility through ethanol treatment. Differential scanning calorimetry shows an increase in glass transition (Tg) temperature and enhanced degradation temperature on alcohol treatment. Enhanced cell attachment and viability of AH927 feline fibroblasts were observed on fibroin matrices. Higher mechanical strength along with controllable water stability of regenerated gland fibroin films make non-mulberry Indian tropical tasar silk gland fibroin protein a promising biomaterial for tissue engineering applications.     

Biodegradable materials based on silk fibroin and keratin

Wool and silk were dissolved and used for the preparation of blended films. Two systems are proposed: (1) blend films of silk fibroin and keratin aqueous solutions and (2) silk fibroin and keratin dissolved in formic acid. The FTIR spectra of pure films cast from aqueous solutions indicated that the keratin secondary structure mainly consists of alpha-helix and random coil conformations. The IR spectrum of pure SF is characteristic of films with prevalently amorphous structure (random coil conformation). Pure keratin film cast from formic acid shows an increase in the amount of beta-sheet and disordered keratin structures. The FTIR pattern of SF dissolved in formic acid is characteristic of films with prevalently beta-sheet conformations with beta-sheet crystallites embedded in an amorphous matrix. The thermal behavior of the blends confirmed the FTIR results. DSC curve of pure SF is typical of amorphous SF and the curve of pure keratin show the characteristic melting peak of alpha-helices for the aqueous system. These patterns are no longer observed in the films cast from formic acid due to the ability of formic acid to induce crystallization of SF and to increase the amount of beta-sheet structures on keratin. The nonlinear trend of the different parameters obtained from FTIR analysis and DSC curves of both SF/keratin systems indicate that when proteins are mixed they do not follow additives rules but are able to establish intermolecular interactions. Degradable polymeric biomaterials are preferred candidates for medical applications. It was investigated the degradation behavior of both SF/keratin systems by in vitro enzymatic incubation with trypsin. The SF/keratin films cast from water underwent a slower biological degradation than the films cast from formic acid. The weight loss obtained is a function of the amount of keratin in the blend. This study encourages the further investigation of the type of matrices presented here to be applied whether in scaffolds for tissue engineering or as controlled release drug delivery vehicles.     

Tubular silk scaffolds for small diameter vascular grafts

Vascular surgeries such as coronary artery bypass require small diameter vascular grafts with properties that are not available at this time. Approaches using synthetic biomaterials have been not completely successful in producing non-thrombogenic grafts with inner diameters less than 6 mm, and there is a need for new biomaterials and graft designs. We propose silk fibroin as a microvascular graft material and describe tubular silk scaffolds that demonstrate improved properties over existing vascular graft materials. Silk tubes produced using an aqueous gel spinning technique were first assessed in vitro in terms of thrombogenicity (thrombin and fibrinogen adsorption, platelet adhesion) and vascular cell responses (endothelial and smooth muscle cell attachment and proliferation) in comparison with polytetrafluoroethylene (PTFE), a synthetic material most frequently used for vascular grafts. Silk tubes were then implanted into the abdominal aortas of Sprague-Dawley rats. At time points of 2 weeks and 4 weeks post implantation, tissue outcomes were assessed through gross observation (acute thrombosis, patency) and histological staining (H&E, Factor VIII, smooth muscle actin). Over the 4-week time period, we observed graft patency and endothelial cell lining of the lumen surfaces. These results demonstrate the feasibility of using silk fibroin as a vascular graft material and some advantages of silk tubes over the currently used synthetic grafts.     

Freeze-gelled silk fibroin protein scaffolds for potential applications in soft tissue engineering

Recently tissue engineering has escalated much interest in biomedical and biotechnological applications. In this regard, exploration of new and suitable biomaterials is needed. Silk fibroin protein is used as one of the most preferable biomaterials for fabrication of scaffolds and several new techniques are being adopted to fabricate silk scaffolds with greater ease, efficiency and perfection. In this study, a freeze gelation technique is used for fabrication of silk fibroin protein 3D scaffolds, which is both time and energy efficient as compared to the conventional freeze drying technique. The fabricated silk fibroin freeze-gelled scaffolds are evaluated micro structurally for morphology with scanning electron microscopy which reveals relatively homogeneous pore structure and good interconnectivity. The pore sizes and porosity of these scaffolds ranges between 60-110 μm and 90-95%, respectively. Mechanical test shows that the compressive strength of the scaffolds is in the range of 20-40 kPa. The applicability to cell culture of the freeze gelled scaffolds has been examined with human keratinocytes HaCat cells which show the good cell viability and proliferation of cells after 5 days of culture suggesting the cytocompatibility. The freeze-gelled 3D scaffolds show comparable results with the conventionally prepared freeze dried 3D scaffolds. Thus, this technique may be used as an alternative method for 3D scaffolds preparation and may also be utilized for tissue engineering applications.     

An investigation into the effect of potassium ions on the folding of silk fibroin studied by generalized two-dimensional NMR-NMR correlation and Raman spectroscopy

We used generalized two-dimensional NMR-NMR correlation to examine the effect of potassium ions on the conformation transition in silk fibroin to investigate the possibility that the fairly high K+ ion content found in the distal end of silk-secreting ducts in the silkworms could have a bearing on natural formation of the silk fiber. This has enabled us to propose a detailed mechanism for the transition process. Our evidence indicates that increasing the [K+] from 0 to 3.7 mg.g(-1) in the silk fibroin, as is thought to occur as the silk fibroin moves through the secretory pathway to the spigot, produces a sequence of secondary structural changes: helix and/or random coil-->helix-like-->beta-sheet-like-->beta-sheet. The sequence is the same as that produced in silk fibroin films by decreasing the pH of fibroin from 6.8 to 4.8. In addition, we used Raman spectroscopy to study the effect of K+ ions on the Fermi doublet resonance of the tyrosyl phenolic ring at 850 and 830 cm(-1). The intensity ratio I(850)/I(830) at these wave numbers indicated that the hydrogen bonding formed by the tyrosyl phenolic-OH becomes more stable with an increase in the K+ ion concentration as above. Our investigation on the effect of K+ ions on fibroin may help provide a theoretical basis for understanding the natural silk-spinning process and the conditions required for biomimetic spinning. It may also have relevance to the aggregation of other beta-sheet proteins, including prion proteins, neurofibrillary proteins and amyloid plaques.     

Conformation transition kinetics of Bombyx mori silk protein

Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which beta-sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of beta-sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations > 40% in films or > 50% in silk fibroin solutions. Our results indicate that the ethanol-induced conformation transition of silk fibroin in films and solutions is a three-phase process. The first phase is the initiation of beta-sheet structure (nucleation), the second is a fast phase of beta-sheet growth while the third phase represents a slow perfection of previously formed beta-sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation-dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber.     

Orientation of silk III at the air-water interface.

A threefold helical crystal structure of Bombyx mori silk fibroin has been observed in films prepared from aqueous silk fibroin solutions using the Langmuir Blodgett (LB) technique. The films were studied using a combination of transmission electron microscopy and electron diffraction techniques. Films prepared at a surface pressure of 16.7 mN/m have a uniaxially oriented crystalline texture, with the helical axis oriented perpendicular to the plane of the LB film. Films obtained from the air-water interface without compression have a different orientation, with the helical axes lying roughly in the plane of the film. In both cases the d-spacings observed in electron diffraction are the same and match a threefold helical model crystal structure, silk III, described in previous publications. Differences in the relative intensities of the observed reflections in both types of oriented samples, as compared to unoriented samples, allows estimations of orientation distributions and the calculations of orientation parameters. The orientation of the fibroin chain axis in the plane of the interfacial film for uncompressed samples is consistent with the amphiphilic behavior previously postulated to drive the formation of the threefold helical silk III conformation.