Chemical,Thermal, Time,and Enzymatic Stability of Silk Materials with Silk I Structure

The crystalline structure of silk fibroin Silk I is generally considered to be a metastable structure; however, there is no definite conclusion under what circumstances this crystalline structure is stable or the crystal form will change. In this study, silk fibroin solution was prepared from B. Mori silkworm cocoons, and a combined method of freeze-crystallization and freeze-drying at different temperatures was used to obtain stable Silk I crystalline material and uncrystallized silk material, respectively. Different concentrations of methanol and ethanol were used to soak the two materials with different time periods to investigate the effect of immersion treatments on the crystalline structure of silk fibroin materials. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman scattering spectroscopy (Raman), Scanning electron microscope (SEM), and Thermogravimetric analysis (TGA) were used to characterize the structure of silk fibroin before and after the treatments. The results showed that, after immersion treatments, uncrystallized silk fibroin material with random coil structure was transformed into Silk II crystal structure, while the silk material with dominated Silk I crystal structure showed good long-term stability without obvious transition to Silk II crystal structure. α-chymotrypsin biodegradation study showed that the crystalline structure of silk fibroin Silk I materials is enzymatically degradable with a much lower rate compared to uncrystallized silk materials. The crystalline structure of Silk I materials demonstrate a good long-term stability, endurance to alcohol sterilization without structural changes, and can be applied to many emerging fields, such as biomedical materials, sustainable materials, and biosensors.     

Self-assembled silk fibroin particles: Tunable size and appearance

Silk fibroin had various applications especially outstanding for drug delivery due to its protein component, biocompatibility and biodegradability. In this paper, silk fibroin particles were prepared via self-assembly. Their sizes and appearances could be modified by adjusting of volume ratios among poly vinyl alcohol (PVA), silk fibroin and ethanol. Regular silk particles were formed in PVA solution when the volume ratio of silk to ethanol ranged from 2 to 20. Preparation pathways could be concluded as 1) mixing ethanol with silk fibroin solution, 2) blending the silk fibroin/ethanol solution with PVA, 3) freezing the ternary solution for 48 h and collection of silk fibroin particles via thaw and centrifugation. Silk particles with various appearances were also obtained by addition of concentrated PVA solution. Silk particles reported have potential as drug delivery carriers in a variety of biomedical applications.     

Study on the properties of nano‐TiO2 particles modified silk fibroin porous films

Using the freeze‐drying method, Nano‐TiO₂/silk fibroin porous films were synthesized with different ratios of TiO₂ to silk fibroin solution. Through scanning electron microscopy (SEM), X‐ray diffraction (XRD), thermogravimetric analysis (TGA), tensile strain, and water‐solubility tests, the structures and properties of these porous films were characterized. The SEM results indicated that the pores of the nano‐TiO₂/silk fibroin porous films were uniformly distributed by the freeze‐drying method. The XRD analysis indicated that the formation of nano‐TiO₂ particles might induce a conformational transition of silk fibroin from the typical Silk I to the typical Silk II structure partly with an increase in the crystallinity of the porous films. Compared with the pure silk fibroin porous films, the mechanical properties of nano‐TiO₂/silk fibroin porous films were improved, and its heat transition temperature was also enhanced; however, the water‐solubility of this material was diminished. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Study on porous silk fibroin materials. I. Fine structure of freeze dried silk fibroin

Silk fibroin solution was prepared by dissolving the silk fibroin in triad solvent CaCl₂ · CH₃CH₂OH · H₂O. In this article we tested and analyzed the state of frozen silk fibroin solution and fine structure of freeze dried porous silk fibroin materials. The results indicated that the glass transition temperature of frozen silk fibroin solution ranges from −34 to −20°C, and the initial melting temperature of ice in frozen solution is about −8.5°C. When porous silk fibroin materials are prepared by means of freeze drying, if freezing temperature is below −20°C, the structure of silk fibroin is mainly amorphous with a little silk II crystal structure, and if freezing temperature is above −20°C, quite a lot of silk I crystal structure forms. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 79: 2185–2191, 2001     

Study on secondary structural transition of nano‐TiO2 modified silk fibroin composite films by two‐dimensional Raman correlation spectroscopy and solid‐state 13C‐NMR spectroscopy

By a sol–gel processing, the nano‐TiO₂/silk fibroin (SF) composite films were prepared. One‐dimensional (1D) Raman, two‐dimensional (2D) correlation Raman spectroscopy, and ¹³C cross‐polarization magic‐angle‐spinning nuclear magnetic resonance (¹³C CP‐MAS NMR) were used to characterize the structural evolution of SF as the nano‐TiO₂ content increased from 0 to 0.4 wt%. The experimental data demonstrated that the secondary structures in the pure SF film and nano‐TiO₂/silk fibroin (SF) composite films were random coil, α‐helix and β‐sheet structures. The nano‐TiO₂ particles formed in the SF films might induce partial structural transitions from random coil and Silk I (α‐helix) to Silk II (β‐sheet). The transition identified by 2D‐Raman correlation spectra was the following order: silk I‐like structure, silk I (α‐helical structure), Silk II‐like structure, and Silk II (β‐sheet structure). POLYM. COMPOS., 36:121–127, 2015. © 2014 Society of Plastics Engineers     

Mechanical properties of silk fibroin–microcrystalline cellulose composite films

Silk fibroin–microcrystalline cellulose (cellulose whisker) composite films with varied compositions were prepared by casting mixed aqueous solution/suspensions of the two components. Silk fibroin was dissolved in 10M LiSCN followed by dialysis; a cellulose whisker suspension was prepared by sulfuric acid hydrolysis of tunicate cellulose. Macroscopically homogeneous films were obtained at all mixing ratios. While the Young's modulus of the composite films showed a linear, additive dependence on the mixing ratio, the tensile strength and ultimate strain showed a maximum at a 70–80% cellulose content, reaching five times those of fibroin‐alone or cellulose‐alone films. At the same mixing ratio, infrared spectra of the composite films showed a shift of the amide I peak from 1654 to 1625 cm^?1, indicating the conformational change of fibroin from a random coil to a β structure (silk II) at the whisker–matrix interface. This change seems to be induced by contact of fibroin molecules with a highly ordered surface of cellulose whisker. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 3425–3429, 2002     

用二维FTIR研究纳米TiO2改性丝素蛋白聚集态结构转变

采用溶胶-凝胶方法制备纳米TiO₂复合丝素膜。UV和SEM测试结果表明,该丝素膜中纳米TiO₂均匀分散在丝素中,TiO₂粒径约为80 nm;同时采用一维红外光谱、二维红外相关光谱对纯丝素膜及复合丝素膜结构进行表征。结果表明,随着纳米TiO₂的生成,丝素蛋白中弱氢键缔合的N—H键以及自由的N—H键发生断裂及重组,生成了强氢键;丝素分子从无序状态转变为有序排列,同时无规线团构象及α螺旋构象向β折叠构象发生转变,最后促使丝素蛋白的结晶构象从Silk Ⅰ转变为Silk Ⅱ。     

Self‐Expansion Behavior of Silk Fibroin Film

Summary: Silk fibroin cast film was prepared using a ternary solvent system of CaCl₂/CH₃CH₂OH/H₂O (1/2/8 in mole ratio). A drying temperature at casting influenced crystal structure of fibroin. When a drying temperature was set lower than 9 °C, the cast film became amorphous. When a drying temperature was set higher than 40 °C, a fibroin film of silk‐II structure was obtained. In order to produce a fibroin film of silk‐I structure, a preferable temperature range was from 20 to 26 °C. The crystal transformation from random coil structure into silk‐I could be made through exposure of an amorphous film to water vapor. As for the crystal transformation from silk‐I into silk‐II, the treatment with a glycerin solution was effective. In the course of the treatment a film showed self‐thinning and self‐expanding. The expansion ratio exceeded 40% at maximum. The film produced accompanying self‐expansion was ductile in nature.

The apparent self‐expansion percentage as a function of initial thickness of the film. The ductility of the film was classified into four stages from the observation of recovery behavior after folding: ?, very soft; ?, soft; ?, middle; ?, hard (see Figure 5 ).     

Structural changes and their effect on mechanical properties of silk fibroin/chitosan blends

Silk fibroin/chitosan blend films were prepared by the solvent casting method. Miscibility between silk fibroin and chitosan was examined by dynamic mechanical thermal analysis. Structural changes of silk fibroin by the addition of chitosan were investigated by IR spectroscopy. The conformational transition of silk fibroin from random coil form to β‐sheet structure induced by blending with chitosan resulted in the increase of crystallinity and density of the blend films. The blend film containing 30 wt % chitosan exhibited a maximum increase in crystallinity and density. It was found that the tensile strength and initial tensile modulus of blend films were greatly enhanced with increasing the chitosan content and showed a maximum value at the composition of 30 wt % chitosan. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2571–2575, 1999     

新型丝素复合膜的微结构表征及热稳定性

基于丝素的高分子复合材料可以广泛地应用于组织工程、生物医药和半导体材料等领域。通过物理-共混技术制备了一种新型生物高分子丝素/聚乳酸复合膜。利用扫描电镜、傅里叶红外光谱、拉曼光谱、X射线衍射和热分析技术对其形貌、结构和相态组分以及热稳定性进行了表征,探究了不同比例复合膜的微结构、相互作用机理和热稳定性。结果表明：随着丝素含量的增加,复合膜中的β-折叠含量增多,α-螺旋和无规卷曲含量减少,玻璃化转变温度提高;由于丝素与聚乳酸间的相互作用,提高了复合膜的热稳定性。     

Preparation of insoluble fibroin films without methanol treatment

Insoluble fibroin films were prepared without methanol treatment. A 15 wt % fibroin solution was obtained through concentration, and then, the insoluble film was achieved by the adjustment of the drying temperature and rate. These films were examined through Fourier transform infrared–attenuated total reflection, X‐ray diffraction, and scanning electron microscopy to determine the structure of the silk fibroin. The physical and mechanical properties were investigated to discover the feasibility of using these films as biomedical materials. When fibroin films were dried above 60°C, abundant β‐sheet crystals existed in the fibroin films, and many globule‐containing micelles aggregated and interacted with each other, which resulted in excellent mechanical properties of the regenerated fibroin films in the wet state. Interestingly, the amide III band of the random coil structure in the fibroin films dried at 70 and 80°C was shifted to a lower frequency, 1228 cm^?1, which meant that a partly orientated structure formed. This may have also affected the mechanical properties of the fibroin films. The tensile strength and breaking elongation of the films dried at 70°C were 29.8 MPa and 59.6%, which is distinctly superior to fibroin films treated with methanol. If the drying temperature was raised to 60°C, the stability of the films in water was also excellent. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 2168–2173, 2005     

Silk fibroin/cellulose blend films: Preparation,structure, and physical properties

This article deals with the preparation and characterization of silk fibroin (Bombyx mori)/cellulose blend films. Following dissolution with a metal complex solution, the average molecular weight of silk fibroin slightly decreased. While cellulose was almost unaffected. After coagulation and washing, transparent films were obtained by blending fibroin and cellulose in all proportions. The crystalline structures of regenerated fibroin and cellulose were β-form and cellulose II, respectively, as shown by the characteristic x-ray diffraction profiles. Density values increased with cellulose content, though less than expected from a pure additive behavior. Moisture regain increased following the addition of a small amount of cellulose to silk fibroin. The mechanical properties showed that both strength and elongation at break of silk fibroin films were improved by blending with cellulose. IR spectra exhibited changes in the skeletal frequences of silk fibroin, suggesting the occurrence of intermolecular interactions between fibroin and cellulose through hydrogen bond formation. © 1995 John Wiley & Sons, Inc.     

Facile preparation for robust and freestanding silk fibroin films in a 1‐butyl‐3‐methyl imidazolium acetate ionic liquid system

Silk fibroin film (SFF) with excellent mechanical properties was prepared for the first time with Bombyx mori silk fibroin as the material and 1‐butyl‐3‐methyl imidazolium acetate ([Bmim]OAc) ionic liquid (IL) as the solvent. The aim was to understand whether the microstructure of SFF could be modified and whether the mechanical properties were improved when [Bmim]OAc IL was used as a solution. With this new system, the obtained SFF was easily peeled off of the substrate, and the silk fibroin proteins retained the α‐helix secondary structure (silk I). Further test results show that the tensile strength (126.8–129.7 MPa) and anti‐UV performance were stronger than the silk fibroin regenerated by traditional ways. Therefore, this study provided and identified a new method with [Bmim]OAc to obtain SFF with strong mechanical properties. This facile preparation and related SFF with excellent mechanical strength could have potential applications in biocompatible implants, synthetic coatings for artificial skin, and many other areas. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42822.     

Production of fine powder from silk by radiation

Silk fine powder was prepared directly from silk fibers irradiated with an accelerated electron beam (EB). Irradiated silk fibers were well pulverized only by physical crushing using a ball mill without any chemical pretreatment. Silk fibroin fibers were irradiated at ambient temperature in the dose range of 250–1 000 kGy. Although unirradiated silk fibers were not pulverized at all, irradiated fibers were easily pulverized and showed a high conversion efficiency from fiber to powder at high irradiation doses. The presence of oxygen in the irradiation atmosphere enhanced pulverization of the silk fibers. The electron microscopic observation showed that the minimum particle size of silk powder obtained from fibers irradiated at 1 000 kGy in oxygen was less than 10 μm. It was found that fibroin powder obtained in this work dissolved remarkably in water, thought unirradiated fibroin fibers were insoluble even in hot water. The soluble fraction was about 60% of fibroin powder for 1 000 kGy irradiation in oxygen.     

A Fast and Reliable Process to Fabricate Regenerated Silk Fibroin Solution from Degummed Silk in 4 Hours

Silk fibroin has a high potential for use in several approaches for technological and biomedical applications. However, industrial production has been difficult to date due to the lengthy manufacturing process. Thus, this work investigates a novel procedure for the isolation of non-degraded regenerated silk fibroin that significantly reduces the processing time from 52 h for the standard methods to only 4 h. The replacement of the standard degumming protocol by repeated short-term microwave treatments enabled the generation of non-degraded degummed silk fibroin. Subsequently, a ZnCl₂ solution was used to completely solubilize the degummed fibroin at only 45 °C with an incubation time of only 1 h. Desalting was performed by gel filtration. Based on these modifications, it was possible to generate a cytocompatible aqueous silk fibroin solution from degummed silk within only 4 h, thus shortening the total process time by 48 h without degrading the quality of the isolated silk fibroin solution.     

Deposition of bone‐like apatite on modified silk fibroin films from simulated body fluid

Silk fibroin films with various calcium chloride contents were prepared by a cast film method and subsequent MeOH treatment. The conformational changes from α‐helix to β‐sheet structure were analyzed by Fourier transformed infrared attenuated total reflection spectroscopy measurements. The films obtained were soaked in 1.5‐times simulated body fluid (1.5 SBF) and the deposition of hydroxyapatite crystals were observed by scanning electron microscopy. The films with calcium chloride contents lower than 3 wt % relative to the silk fibroin were not mineralized under these conditions, while those with calcium chloride contents more than 5 wt % mineralized after 6 h. The X‐ray diffraction patterns and inductive coupled plasma spectroscopy analyses indicated that the hydroxyapatite crystals were grown by hydrolysis of octacalcium phosphate, as indicated by differences in diffraction intensities and changes in concentrations of calcium and phosphate ions in 1.5 SBF. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci, 2006     

Deduction of a facile method to construct Antheraea mylitta silk fibroin/gelatin blend films for prospective biomedical applications

Blend films of two types (I and II) were prepared by mixing Antheraea mylitta silk fibroin (AMF) and gelatin solution in various blend ratios via the solution casting method. Two different crosslinkers, namely glutaraldehyde and genipin, were used during blend preparation. The structural characteristics and thermal properties of the blend films were examined by Fourier transform infrared (FTIR) spectroscopy, X‐ray diffraction (XRD), Thermogravimetric analysis (TGA) and Diffrential scanning calorimetery (DSC). The FTIR spectra showed conformational alterations in type I blend films while type II films attained high β‐sheet crystallinity. The XRD diffractograms presented a high degree of crystallinity in type II blend films compared to type I, which showed an almost amorphous structure. Further, thermal and biological studies were conducted on type II films. According to the TGA thermograms, the degradation temperature of the crosslinked blend films shifted compared to pure gelatin and pure AMF films. Partial miscibility of the two components was indicated by DSC thermograms of the blends. The high water uptake capacity of type II blend films was found to imitate hydrogel behaviour. The blend films did not show any toxicity in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay and supported L929 fibroblast cell spreading and proliferation. The biodegradation of the blend films was significantly faster than the pure silk film. © 2020 Society of Industrial Chemistry     

从丝素水溶液中再生的丝纤维的结构与性能

通过使用表面皿直接拉伸、毛细管重力纺丝和人工拉伸3种不同的成丝方法,从高浓度再生丝素水溶液中制得了丝纤维。用偏光显微镜观察了丝纤维的取向,用拉曼光谱仪和Instron拉力仪表征了丝纤维的结构和力学性能。结果发现,经毛细管剪切流动后再拉伸有利于再生丝性能的提高,所得的丝有较好的取向和较多的β折叠结构,力学性能也相对较好。剪切在丝纤维的成形过程中起重要的作用。     

Bioengineered Silkworm for Producing Cocoons with High Fibroin Content for Regenerated Fibroin Biomaterial-Based Applications

Silk fibroin exhibits high biocompatibility and biodegradability, making it a versatile biomaterial for medical applications. However, contaminated silkworm-derived substances in remnant sericin from the filature and degumming process can result in undesired immune reactions and silk allergy, limiting the widespread use of fibroin. Here, we established transgenic silkworms with modified middle silk glands, in which sericin expression was repressed by the ectopic expression of cabbage butterfly-derived cytotoxin pierisin-1A, to produce cocoons composed solely of fibroin. Intact, nondegraded fibroin can be prepared from the transgenic cocoons without the need for sericin removal by the filature and degumming steps that cause fibroin degradation. A wide-angle X-ray diffraction analysis revealed low crystallinity in the transgenic cocoons. However, nondegraded fibroin obtained from transgenic cocoons enabled the formation of fibroin sponges with varying densities by using 1–5% (v/v) alcohol. The effective chondrogenic differentiation of ATDC5 cells was induced following their cultivation on substrates coated with intact fibroin. Our results showed that intact, allergen-free fibroin can be obtained from transgenic cocoons without the need for sericin removal, providing a method to produce fibroin-based materials with high biocompatibility for biomedical uses.     

丝素/羧甲基壳聚糖共混膜的结构性能探讨

将含有甘油和戊二醛的丝素与羧甲基壳聚糖按一定比例混合,制得丝素/羧甲基壳聚糖共混膜,对共混膜的结构与性能进行了探讨。结果表明:随着羧甲基壳聚糖含量的增加,共混膜的透气率增大,加入交联剂戊二醛有效地改善了共混膜的力学性能,但其透气率有所降低;当丝素与羧甲基壳聚糖的质量比为4/1时,共混膜的断裂强度最大,力学性能较好,共混膜相容性较好,其断面光滑、致密。制备丝素/羧甲基壳聚糖共混膜的较佳条件为:丝素中的甘油质量分数为15%,戊二醛质量分数为0.075%,丝素与羧甲基壳聚糖质量比为4/1。