Bombyx Mori Silk Fibroin Films Research Articles

Electrospun Silk Fibroin Scaffolds for Tissue Regeneration: Chemical, Structural, and Toxicological Implications of the Formic Acid-Silk Fibroin Interaction.

The dissolution of Bombyx mori silk fibroin (SF) films in formic acid (FA) for the preparation of electrospinning dopes is widely exploited to produce electrospun SF scaffolds. The SILKBridge® nerve conduit is an example of medical device having in its wall structure an electrospun component produced from an FA spinning dope. Though highly volatile, residual FA remains trapped into the bulk of the SF nanofibers. The purpose of this work is to investigate the type and strength of the interaction between FA and SF in electrospun mats, to quantify its amount and to evaluate its possible toxicological impact on human health. The presence of residual FA in SF mats was detected by FTIR and Raman spectroscopy (new carbonyl peak at about 1,725 cm−1) and by solid state NMR, which revealed a new carbonyl signal at about 164.3 ppm, attributed to FA by isotopic 13C substitution. Changes occurred also in the spectral ranges of hydroxylated amino acids (Ser and Thr), demonstrating that FA interacted with SF by forming formyl esters. The total amount of FA was determined by HS-GC/MS analysis and accounted for 247 ± 20 μmol/g. The greatest part was present as formyl ester, a small part (about 3%) as free FA. Approximately 17% of the 1,500 μmol/g of hydroxy amino acids (Ser and Thr) theoretically available were involved in the formation of formyl esters. Treatment with alkali (Na2CO3) succeeded to remove the greatest part of FA, but not all. Alkali-treated electrospun SF mats underwent morphological, physical, and mechanical changes. The average diameter of the fibers increased from about 440 nm to about 480 nm, the mat shrunk, became stiffer (the modulus increased from about 5.5 MPa to about 7 MPa), and lost elasticity (the strain decreased from about 1 mm/mm to about 0.8 mm/mm). Biocompatibility studies with human adult dermal fibroblasts did not show significant difference in cell proliferation (313 ± 18 and 309 ± 23 cells/mm2 for untreated and alkali-treated SF mat, respectively) and metabolic activity. An in-depth evaluation of the possible toxicological impact of residual FA was made using the SILKBridge® nerve conduit as case study, following the provisions of the ISO 10993-1 standard. The Potential Patient Daily Intake, calculated from the total amount of FA determined by HS-GC/MS, was 2.4 mg/day and the Tolerable Exposure level was set to 35.4 mg/day. This allowed to obtain a value of the Margin of Safety of 15, indicating that the amount of FA left on SF mats after electrospinning does not raise concerns for human health.

An Insulin-like Growth Factor-1 Conjugated Bombyx mori Silk Fibroin Film for Diabetic Wound Healing: Fabrication, Physicochemical Property Characterization, and Dosage Optimization In Vitro and In Vivo.

This study aimed to develop a silk fibroin (SF)-film for the treatment of chronic diabetic wounds. Silk fibroin was purified through a newly developed heating degumming (HD) process and casted on a hydrophobic surface to form SF-films. The process allowed the fabricated film to achieve a 42% increase in transparency and a 32% higher proliferation rate for BALB/3T3 fibroblasts compared to that obtained by conventional alkaline degumming treatment. Fourier transform infrared analysis demonstrated that secondary structure was retained in both HD- and alkaline degumming-derived SF preparations, although the crystallinity of beta-sheet in SF-film after the HD processing was slightly increased. This study also investigated whether conjugating insulin-like growth factor-1 (IGF-1) would promote diabetic wound healing and what the optimal dosage is. Using BALB/3T3 cells grown in hyperglycemic medium as a model, it was demonstrated that the optimal IGF-1 dosage to promote the cell growth was approximately 0.65 pmol. Further analysis of wound healing in a diabetic mouse model indicated that SF-film loaded with 3.25 pmol of IGF-1 showed significantly superior wound closure, a 13% increase at the 13th day after treatment relative to treatment with 65 pmol of free IGF-1. Improvement in diabetic wound healing was exerted synergistically by SF-film and IGF-1, as reflected by parameters including levels of re-epithelialization, epithelial tissue area, and angiogenesis. Finally, IGF-1 increased the epithelial tissue area and micro-vessel formation in a dose-dependent manner in a low dosage range (3.25 pmol) when loaded to SF-films. Together, these results strongly suggest that SF-film produced using HD and loaded with a low dosage of IGF-1 is a promising dressing for diabetic wound therapy.

Comparison of Native and Regenerated Antheraea Pernyi Silk Fibroin

The regenerated Antheraea pernyi silk fibroin (RASF) was harvested by dissolving the silk fibers in a calcium nitrate solution. XRD result demonstrated that both native and regenerated Antheraea pernyi silk fibroin involved the α-helix conformation, and DTG curves showed that their thermal decompositions were quite similar and proceeded three steps. However, rheological measurements figured out that the molecule weight of RASF decreased from 246 kDa to 199 kDa, comparing with native Antheraea pernyi silk fibroin. Also, the tensile properties of the RASF film, which were the same to those of regenerated Bombyx mori silk fibroin film, were observed.

Surface Modification of Bombyx mori Silk Fibroin Film via Thiol-ene Click Chemistry

The purpose of this work is to develop a strategy for the surface modification of Bombyx mori silk fibroin (SF) film, via a thiol-ene click reaction, in order to expand its potential applicability. To achieve this goal, terminal thiol groups, from reduced glutathione, were introduced onto the surface of the SF film via a carbodiimide coupling reaction. These immobilized thiol groups act as robust crosslinkers and allow us to rapidly functionalize the surface with alkene group-containing molecules via ultraviolet (UV) light-initiated thiol-ene click chemistry. The X-ray photoelectron spectroscopy results specifically determined that alkene group-containing molecules and UV illumination are required for the thiol-ene click modification to take place on thiolated SF films. Fourier-transform infrared spectroscopy characterization of SF films indicated that the β-sheet conformation of SF was hampered throughout the modification process. Furthermore, it was found that the processes for thiol-ene click chemistry that took place on the SF film surface did not affect the thermal stability of SF films significantly, indicating the developed strategy preserved the intrinsic properties of the SF film. Taking into account that many alkene-containing molecules are suitable for thiol-ene click chemistry, we see immense potential for the incorporation of SF films into various applications, using this methodology.

Thiolation and characterization of regenerated Bombyx mori silk fibroin films with reduced glutathione

Bombyxmori silk fibroin-based materials have good biocompatibility and biodegradability. In order to maximize their utility while maintain appropriate features, silk fibroin (SF) films were modified with reduced glutathione (GSH) (NH2)–ECG–(COOH), using the carbodiimide chemistry method, for the introduction of thiol groups onto surfaces. The effects of this modification on SF films’ chemical and physical properties, and cytotoxicity were assessed. The chemical and elemental composition analysis results suggested that reduced glutathione (GSH) was covalently coupled onto the surface of silk fibroin films. Atomic force microscopy (AFM) results indicated the surface roughness of silk fibroin film was increased after the modification by GSH. The GSH-modified silk fibroin films also showed the smaller contact angle due to the hydrophilic peptides coupled on the film surface. Through MTT assay, it was shown that the chemically modified SF film was not cytotoxic to HEK293 cells, and it had no adverse influence on the growth of HEK293 cells. Our approach provides a new option to engineer SF-based material surface with thiol groups in order to allow for secondary reactions and holds great promise for applications of SF-based materials in the biomedical field.

Structural and characterization studies of insoluble thai bombyx mori silk fibroin films

Bombyx Mori fiber consists of two major proteins which are fibroin and sericin. The silk fibroin (SF) is the core structural protein of silk fiber. SF protein structures comprise of primary and secondary structures; where the primary structure contains series of amino acid and secondary structure with Silk I refers to the water-soluble and Silk II, high β sheet extent which is insoluble. This study was conducted to compare the structural and characterization of insoluble Thai Bombyx Mori SF with different types of post-treatement. Thai silk cocoons, which were degummed and dissolved in 9.3 M LiBr solution at 60 °C. The obtained SF solutions were dialyzed and purified. SF films were prepared by solution casting and immersing in methanol and ethanol, followed by water annealing in water saturated vacuum. Post-treatment was purposely done to regenerate and induce of the β sheet structure to enhance the insolubilities and the stabilities properties of the SF films. The SF films structural conformation, characterization and thermal stability were characterized. Attenuated total reflectance-Fourier transformed infrared spectroscopy (ATR-FTIR) showed that SF films were presented in a more stable form after ethanol post treatment, which also supporting by X-ray diffraction (XRD) analysis which indicated the tendency to higher structural organization. Thermal analysis resutls showed that SF was thermally stable and improved after post treatment. The contact angle of post treated SF increased the hydrophobicity of the films. The thai SF films could be the promising candidate for applications in tissue regeneration, optical devices, and flexible electronic displays with the possibility to control the SF structure and properties.

Characterization of water in hydrated Bombyx mori silk fibroin fiber and films by 2H NMR relaxation and 13C solid state NMR.

The mechanical properties of Bombyx mori silk fibroin (SF) change remarkably upon hydration. However, the microscopic interaction between SF and water is not currently well understood on a molecular level. We were able to identify four distinct components in the relaxation times for water in SF fiber by 2H solution NMR relaxation and exchange measurements. In addition, the effects of hydration on the conformation of Ser and Tyr residues in the site-specific crystalline and non-crystalline domains of 13C selectively labeled SF, respectively, could be determined independently. Thus, our measurements provide new insight relating the characteristics of water and the hydration structure of silk, which are relevant in light of current interest in the design of novel silk-based biomaterials.

Glycerin-Induced Conformational Changes in Bombyx mori Silk Fibroin Film Monitored by (13)C CP/MAS NMR and ¹H DQMAS NMR.

In order to improve the stiff and brittle characteristics of pure Bombyx mori (B. mori) silk fibroin (SF) film in the dry state, glycerin (Glyc) has been used as a plasticizer. However, there have been very limited studies on the structural characterization of the Glyc-blended SF film. In this study, 13C Cross Polarization/Magic Angle Spinning nuclear magnetic resonance (CP/MAS NMR) was used to monitor the conformational changes in the films by changing the Glyc concentration. The presence of only 5 wt % Glyc in the film induced a significant conformational change in SF where Silk I* (repeated type II β-turn and no α-helix) newly appeared. Upon further increase in Glyc concentration, the percentage of Silk I* increased linearly up to 9 wt % Glyc and then tended to be almost constant (30%). This value (30%) was the same as the fraction of Ala residue within the Silk I* form out of all Ala residues of SF present in B. mori mature silkworm. The 1H DQMAS NMR spectra of Glyc-blended SF films confirmed the appearance of Silk I* in the Glyc-blended SF film. A structural model of Glyc-SF complex including the Silk I* form was proposed with the guidance of the Molecular Dynamics (MD) simulation using 1H–1H distance constraints obtained from the 1H Double-Quantum Magic Angle Spinning (DQMAS) NMR spectra.

Potentially functional polypeptides for modification of biomaterials

Event Abstract Back to Event Potentially functional polypeptides for modification of biomaterials Jiannan Wang1, Mingyang Wu1, Yunfei Liu1 and Fangfang Tu1 1 Soochow University, National Engineering Laboratory for Modern Silk, China Potentially functional polypeptides for modification of biomaterials Mingyang Wu, Yunfei Liu, Fangfang Tu, Jiannan Wang* National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province, 215123, China *Corresponding author: Jiannan Wang, wangjn@suda.edu.cn Silk fibroin is a native protein with regular amino acid sequence. Such as, Bombyx mori silk fibroin is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. The RGD tripeptides distribute in Antheraea yamamai and Antheraea pernyi silk fibroins up to 14 and 12 repeats, respectively. In order to determine the biomedical function of these typical peptides or potentially use them to modify materials, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain of Bombyx mori silk fibroin and a gene motif (-rgd-) encoding RGD-contained domain of Antheraea yamamai or Antheraea pernyi silk fibroins. These motif and their multimers were inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif (f(1)) and multimers (f(4), f(8), (-rgd-)4, and (-rgd-)8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography. All express products (GST-F(1), GST-F(4), GST-F(8), GST-(-RGD-)4, and GST-(-RGD-)8) were successfully expressed confirmed by SDS-PAGE, mass spectrometry (MS), amino acid composition and charge assay. The peptides F(1), F(4), F(8), (-RGD-)4, and (-RGD-)8 were all cleaved clearly from the GST-fusion tag following thrombin digestion. The molecular weight by MS, amino acid composition and isoelectric points of released peptides were all consistent with the predicted values. The peptides F(1), F(4), and F(8) were used to modify dacron membrane. Results showed F(1), F(4), and F(8) significantly improved water contact angle of dacron, and increased rapid adhesion and proliferation ability of cells. Cell adhesion and cell proliferation on (-RGD-)4 and (-RGD-)8 modified Bombyx mori silk fibroin films were better than that on unmodified film. The study indicated that the express products of F(1), F(4), and F(8) could improve the hydrophilicity of hydrophobe materials for better application in biomaterials. F(1), F(4), and F(8) exhibited a negative ζ-potential and the measured pI was about 3.3, 3.2 and 3.0, respectively, expecting to be used in vascular tissue engineering. The express products (-RGD-)4 and (-RGD-)8 could be used to modify cellular interface materials to improve cell adhesion and growth on materials surface. Acknowledgements This work was supported by National Natural Science Foundation of China (Nos. 51173125 and 51473108), Natural Science Foundation of Jiangsu Province of China (No. BK2012633), College Natural Science Research Project of Jiangsu Province of China (No. 12KJA43004). Refences [1] Y. X. Yang, H. Y. Huang, Z. F. Tian, et al. Textile Bioengineering and Informatics Symposium Proceedings, 2012, 70-75. [2] H. Y. Huang, Z. F. Tian, Y. X. Yang, et al. Journal of Donghua University (English Edition ), 2012, 29: 26-29. [3] H. R. Zhao, Y. X. Yang, H. G. Yi, et al. Bio-Medical Materials and Engineering, 2014, 24: 2057–2064. Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Adhesive biomaterials Citation: Wang J, Wu M, Liu Y and Tu F (2016). Potentially functional polypeptides for modification of biomaterials. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02647 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jiannan Wang Mingyang Wu Yunfei Liu Fangfang Tu Google Jiannan Wang Mingyang Wu Yunfei Liu Fangfang Tu Google Scholar Jiannan Wang Mingyang Wu Yunfei Liu Fangfang Tu PubMed Jiannan Wang Mingyang Wu Yunfei Liu Fangfang Tu Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Optical properties of γ-irradiated Bombyx mori silk fibroin films

In the present work the Bombyx mori silk fibroin (SF) films were prepared by the solution casting method and effects of γ-irradiation on the optical properties and optical constants of the films have been studied by using Ultra Violet–Visible (UV–Vis) spectrophotometer. The recorded UV–Vis absorption and transmission spectra have been used to determine the optical band gap (Eg), refractive index (n), extinction coefficient (k), optical conductivity (σopt) and dielectric constants (ε*) of virgin and γ-irradiated films. Reduction in optical band gap and increase in refractive index with increasing radiation dosage were observed. It is also found that there is an increase in dielectric constants with increasing photon energy. The obtained results reveal that the refractive index of the SF films may be efficiently changed by γ-irradiation.

Optical properties of electron irradiated Bombyx mori silk fibroin films

Energetic electron irradiation effects on the optical properties of Bombyx mori silk fibroin (SF) films are studied using UV–visible spectrophotometer. The recorded UV–visible absorption and transmission graphs have been used to determine the optical band gap (E g ), refractive index (n), extinction coefficient (k), optical conductivity (σ opt ) and dielectric constants (e *) of the films. The studies have shown a reduction in optical band gap and increase in refractive index with increasing electron dosage. It is also observed that dielectric constants increase with increasing photon energy. The observed optical changes have been tried to be correlated with the structural changes, revealed through FT-IR spectroscopy. These results indicate that the refractive index of SF films can be changed efficiently by electron irradiation.

Effect of mechanical deformation on the structure of regenerated Bombyx mori silk fibroin films as revealed using Raman and infrared spectroscopy.

To better understand the effect of mechanical stress during the spinning of silk, the protein orientation and conformation of Bombyx mori regenerated silk fibroin (RSF) films have been studied as a function of deformation in a static mode or in real time by tensile-Raman experiments and polarization modulation infrared linear dichroism (PM-IRLD), respectively. The data show that either for step-by-step or continuous stretching, elongation induces the progressive formation of β-sheets that align along the drawing axis, in particular above a draw ratio of ~2. The formation of β-sheets begins before their alignment during a continuous drawing. Unordered chains were, however, never found to be oriented, which explains the very low level of orientation of the amorphous phase of the natural fiber. Stress-perturbed unordered chains readily convert into β-sheets, the strain-induced transformation following a two-state process. The final level of orientation and β-sheet content are lower than those found in the native fiber, indicating that various parameters have to be optimized in order to implement a spinning process as efficient as the natural one. Finally, during the stress relaxation period in a step-by-step drawing, there is essentially no change of the content and orientation of the β-sheets, suggesting that only unordered structures tend to reorganize.

Self-assembly of monolayered lipid membranes for surface-coating of a nanoconfined Bombyx mori silk fibroin film

A self-assembled lipid membrane provides a smooth, hydrophilic and biocompatible surface coating film for materials.

Controlling the Cell Adhesion Property of Silk Films by Graft Polymerization

We report here a graft polymerization method to improve the cell adhesion property of Bombyx mori silk fibroin films. B. mori silk has evolved as a promising material for tissue engineering because of its biocompatibility and biodegradability. However, silk's hydrophobic character makes cell adhesion and proliferation difficult. Also, the lack of sufficient reactive amino acid residues makes biofunctionalization via chemical modification challenging. Our study describes a simple method that provides increased chemical handles for tuning of the surface chemistry of regenerated silk films (SFs), thus allowing manipulation of their bioactivity. By grafting pAAc and pHEMA via plasma etching, we have increased carboxylic acid and hydroxyl groups on silk, respectively. These modifications allowed us to tune the hydrophilicity of SFs and provide functional groups for bioconjugation. Our strategy also allowed us to develop silk-based surface coatings, where spatial control over cell adhesion can be achieved. This control over cell adhesion in a particular region of the SFs is difficult to obtain via existing methods of modifying the silk fibroin instead of the SF surface. Thus, our strategy will be a valuable addition to the toolkit of biofunctionalization for enhancing SFs' tissue engineering applications.

Effect of the sterilization method on the properties of Bombyx mori silk fibroin films

We have compared the effects of different sterilization techniques on the properties of Bombyx mori silk fibroin thin films with the view to subsequent use for corneal tissue engineering. The transparency, tensile properties, corneal epithelial cell attachment and degradation of the films were used to evaluate the suitability of certain sterilization techniques including gamma-irradiation (in air or nitrogen), steam treatment and immersion in aqueous ethanol. The investigations showed that gamma-irradiation, performed either in air or in a nitrogen atmosphere, did not significantly alter the properties of films. The films sterilized by gamma-irradiation or by immersion in ethanol had a transparency greater than 98% and tensile properties comparable to human cornea and amniotic membrane, the materials of choice in the reconstruction of ocular surface. Although steam-sterilization produced stronger, stiffer films, they were less transparent, and cell attachment was affected by the variable topography of these films. It was concluded that gamma-irradiation should be considered to be the most suitable method for the sterilization of silk fibroin films, however, the treatment with ethanol is also an acceptable method.

Effects of Gamma Irradiation on the Structure and Mechanical Properties of Wild Silkworms and Bombyx Mori Silk Fibroin Films

The structure and mechanical properties of A. yamamai, A. perny and B. mori silk fibroin films irradiated by gamma ray with various doses of 0, 25, 50, 100 and 200 kGy, respectively were determined by XRD, FT-IR, DSC and Instron 3365 equipment. Results showed that the aggregation structure and molecular conformation of A. yamamai, A. perny and B. mori silk fibroin films irradiated by gamma ray with those doses mentioned above were not significantly changed. However, with the increase of radiation intensity, the thermal stability of silk fibroin films declined slightly, and the breaking strength and extensibility reduced significantly, due to the breakdown of parts of secondary bonds and covalent bonds. These results suggested that, when these silk fibroin materials were sterilized by gamma irradiation, smaller radiation doses should be used, otherwise irreversible damages on these materials would be caused.

Novel silk fibroin/hydroxyapatite composite films: Structure and properties

Novel composite films of Bombyx mori silk fibroin (SF) and hydroxyapatite (HA) composite films, with glycerin as an additive, were fabricated by means of co-precipitation, where the theoretical HA content was varied from 2 (w/w)% to 31 (w/w)%. The structure and properties of the composite films were investigated by SEM, XRD, AFM, TGA and tensile testing. The results showed that the composite films were smooth and transparent with the uniform distribution of HA into the composites when the final HA content was lower than 21 (w/w)%. XRD and TGA data showed that the silk fibroin in the composites was predominantly in a β-sheet crystalline structure, which was induced not only by the addition of glycerin, also by the HA crystal growth during the composite fabrication, leading to the thermal stable composite films. On the other hand, the HA crystals had the anisotropic growth with high extent of lattice imperfection and the preferential orientation along c-axis, probably promoted by the silk fibroin. The mechanical testing results showed that both break strain and stress were declined with the increase of HA content in the composites, presumably due to the original brittleness of HA compound.

Surface modification and properties of Bombyx mori silk fibroin films by antimicrobial peptide

The Bombyx mori silk fibroin films (SFFs) were modified by a Cecropin B ( CB) antimicrobial peptide, (NH 2)-NGIVKAGPAIAVLGEAAL-CONH 2, using the carbodiimide chemistry method. In order to avoid the dissolution of films during the modification procedure, the SFFs were first treated with 60% (v/v) ethanol aqueous solution, resulting a structural transition from unstable silk I to silk II. The investigation of modification conditions showed that the surface-modified SFFs had the satisfied antimicrobial activity and durability when they were activated by EDC·HCl/NHS solution followed by a treatment in CB peptide/PBS buffer (pH 6.5 or 8) solution at ambient temperature for 2 h. Moreover, the surface-modified SFFs showed the smaller contact angle due to the hydrophilic antimicrobial peptides coupled on the film surface, which is essential for the cell adhesion and proliferation. AFM results indicated that the surface roughness of SFFs was considerably increased after the modification by the peptides. The elemental composition analysis results also suggested that the peptides were tightly coupled to the surface of SFFs. This approach may provide a new option to engineer the surface-modified implanted materials preventing the biomaterial-centered infection (BCI).

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.

Comparative Raman spectroscopic analysis of orientation in fibers and regenerated films of Bombyx mori silk fibroin

AbstractThis study is focused on the Raman spectroscopic analysis of degummed silk fibroin (SF) fibers and regenerated Bombyx mori silk fibroin films: a correlation was found between some spectral features related to the methylene deformation modes and the molecular orientation of the samples.Polarized Raman spectra on SF fibers were used to obtain the orientation distribution function of carbonyl groups along the protein backbone. The variation of the intensity ratio of 1400/1450 cm−1 for the peaks attributed to the wagging and bending deformation modes of CH2 groups with respect to the angular orientation of the fiber was measured and quantitatively correlated with the orientation distribution function of the carbonyl groups.Unpolarized Raman spectra were measured for regenerated silk fibroin films and lyophilized solutions. The variation of the intensity ratio of 1415/1455 cm−1, which is related to the deformation modes of CH2 groups in SF regenerated materials, was qualitatively related to the microstructural orientation of the samples observed by scanning electron microscopy (SEM), and to the presence of Silk I phase as suggested by the analysis of samples obtained in different casting conditions and also by the measurements on mechanically deformed films.The results obtained showed the utility of the spectroscopic intensity ratio of 1400/1450 cm−1 for the rapid assessment of molecular orientation in silk fibers, which could be useful for quality and process control of regenerated silk‐based textiles. Moreover, the qualitative dependence of the intensity ratio of 1415/1455 cm−1 was found to be sensitive to both the microstructural orientation and Silk I content of regenerated silk fibroin films, suggesting a possible correlation of this Raman marker of the Silk I phase with the degree of molecular order brought about by this polymorph. Copyright © 2006 John Wiley & Sons, Ltd.