Morphology and structure of silkworm cocoons

Silkworm cocoons are natural polymer fibre composites made from silk fibres and sericin binder. While silk is an interesting natural material per se, an understanding of the role of silk within one of its main functional applications in silkworm cocoons will provide inspiration and tools for the design of new artificial silk composites. Here, we describe in some detail the structure and morphology of the cocoons of 27 different species of silkworm. While cocoon morphology can be described very broadly as a nonwoven fibre composite, we demonstrate a diversity of structural features such as: the number and connectivity of layers through the cocoon wall thickness, the amount and distribution of sericin binder, the diameter and packing density of the silk fibres, the degree of orientation of the nonwoven structure, the distribution of larger holes within that structure, and the presence of calcium oxalate crystals.     

In vitro and in vivo research on using Antheraea pernyi silk fibroin as tissue engineering tendon scaffolds

In this paper, the feasibility of using Antheraea pernyi silk fibroin as tissue engineering tendon scaffold was investigated in vitro and in vivo, respectively, utilizing tenocytes and animal model. The animal model used here was an adult New Zealand White rabbit with a 15-mm gap defect in both sides of the Achilles tendon. The Achilles tendon defects in one side of hind legs were repaired using the braided A. pernyi silk fibroin scaffold in experimental group (n = 24), while the other side left untreated as negative group (n = 24). The recovery of the defect tendons were evaluated postoperatively at the 2nd, 6th, 12th, and 16th week using macroscopic, histological, immunohistochemical, scanning electron micrograph and biomechanical test techniques. In vitro results examined by scanning electron micrograph showed that A. pernyi silk fibroin promote the adhesion and propagation of the tenocytes. In vivo, at 16 weeks after implantation, morphological results showed that neo-tendons were formed, and bundles of collagen fibers in the neo-tendons were uniform and well oriented. Immunohistochemical results showed that collagen type in the regenerated tendons was predominantly type I. The maximum load of regenerated tendon at 16 weeks reached 55.46% of the normal tendon values. Preliminary, we concluded that A. pernyi silk fibroin promoted the recovery of Achilles tendon defect of rabbit and the application of A. pernyi silk fibroin as tissue engineering tendon scaffold is feasible.     

Crystals in Antheraea assamensis silkworm cocoon: Their removal,recovery and roles

Brick shaped mineral deposits or crystals are found in the shell of semi-domestic silk cocoon of Antheraea assamensis (A. assamensis). Effective removal and recovery of these crystals are important to understand their roles in the cocoon's protective function towards pupae. In this study, chemical and physical (ultrasonication) demineralisation methods were investigated for A. assamensis. It was found that the physical demineralisation method could effectively separate crystals without changing their shape and size and not effecting other components of the silk cocoon. The efficient recovery of the crystals, without any change in their chemical composition was confirmed based on FTIR, XRD and EDX techniques. Chemical demineralisation method was optimised and performed under milder conditions than reported in the past. It helped reeling of silk without much loss of strength or natural colour of silk fibre.     

Microstructure and mechanical properties of silk from different components of the Antheraea pernyi cocoon

Silk fibres from different components of the Antheraea pernyi silkworm cocoon, namely peduncle, outer floss, and cocoon shells (outermost layer and pelade layer) were studied in detail to gain insights into the structure–property–function relationship. Among the fibres from different components, peduncle fibres are the softest with the largest viscoelastic lag, which may reduce the oscillation amplitude when a cocoon hangs on a twig. Fibres from the outermost layer are the toughest and have the largest breaking energy. Outer floss fibres have the highest content of sericin (about 11.98%) but their hardness and elasticity are intermediate. Pelade fibres are shape – preservable and stable with superior hardness and elasticity. The understanding of the properties of different silk fibres is essential for understanding their respective roles in the function of a silk cocoon and will also inspire new designs of protective materials under stringent environmental conditions.     

Mapping Nanostructural Variations in Silk by Secondary Electron Hyperspectral Imaging

Nanostructures underpin the excellent properties of silk. Although the bulk nanocomposition of silks is well studied, direct evidence of the spatial variation of nanocrystalline (ordered) and amorphous (disordered) structures remains elusive. Here, secondary electron hyperspectral imaging can be exploited for direct imaging of hierarchical structures in carbon‐based materials, which cannot be revealed by any other standard characterization methods. Through applying this technique to silks from domesticated (Bombyx mori) and wild (Antheraea mylitta) silkworms, a variety of previously unseen features are reported, highlighting the local interplay between ordered and disordered structures. This technique is able to differentiate composition on the nanoscale and enables in‐depth studies into the relationship between morphology and performance of these complex biopolymer systems.     

Structure and physical properties of silkworm cocoons

Silkworm cocoons have evolved a wide range of different structures and combinations of physical and chemical properties in order to cope with different threats and environmental conditions. We present our observations and measurements on 25 diverse types of cocoons in a first attempt to correlate physical properties with the structure and morphology of the cocoons. These two architectural parameters appear to be far more important than the material properties of the silk fibres themselves. We consider tensile and compressive mechanical properties and gas permeation of the cocoon walls, and in each case identify mechanisms or models that relate these properties to cocoon structure, usually based upon non-woven fibre composites. These properties are of relevance also for synthetic non-woven composite materials and our studies will help formulate bio-inspired design principles for new materials.     

Biosynthesis and characterization of typical fibroin crystalline polypeptides of silkworm Bombyx mori

We aimed to investigate the self-organization/self-assembly mechanisms of silkworm fibroin-based material. In the present study, for the first time, we designed and multimerized four DNA “monomer” sequences from structurally simple fibroin crystalline peptides or analog, [GAGAGX] (X = A, S, Y and V) to encode polypeptides [GAGAGX]₁₆ (eGA, eGS, eGY and eGV) using a “head-to-tail” construction strategy. Multimers were cloned into pGEX-KG and fusion proteins GST-[GAGAGX]₁₆ (KGA, KGS, KGY and KGV) were efficiently expressed in Escherichia coli. These fusion proteins were isolated and purified by GST affinity chromatography and confirmed by SDS–PAGE and Western blot analysis using antibody reactive to GST. The polypeptides were cleavaged from GST fusion proteins by digesting with thrombin enzyme. The composition of the four polypeptides was confirmed by composition analysis of amino acids, and their abilities to form β-sheet structure were determined by ThT fluorescence spectral analysis. The content of β-sheet among the four polypeptides followed the order: eGS > eGV > eGY > eGA.     

Structure and properties of ultrafine silk fibers produced by <Emphasis Type="Italic">Theriodopteryx ephemeraeformis</Emphasis>

Theriodopteryx ephemeraeformis commonly known as bag worms produce ultrafine silk fibers that are remarkably different than the common domesticated (Bombyx mori) and wild (Saturniidae) silk fibers. Bag worms are considered as pests and commonly infect trees and shrubs. Although it has been known that the cocoons (bags) produced by bag worms are composed of silk, the structure and properties of the silk fibers in the bag worm cocoons have not been studied. In this research, the composition, morphology, physical structure, thermal stability, and tensile properties of silk fibers produced by bag worms were studied. Bag worm silk fibers have considerably different amino acid contents from those of the common silks. The physical structure of the bag worm silk fibers is also considerably different compared with B. mori and common wild silk fibers. Bag worm’s silk fibers have lower tensile strength (3.2 g/denier) and Young’s modulus (45 g/denier) but similar breaking elongation (15.3%) compared with B. mori silk. However, the tensile strength and Young’s modulus of bag worm fibers are similar to those of the common Saturniidae wild silk fibers. Bag worm silk fibers could be useful for some of the applications currently using the B. mori and wild silk fibers.     

Impact of different silkworm dietary supplements on its silk performance

This study aims to evaluate the effect of silkworm larva (Bombyx mori) diet supplementation with two amino acids (threonine and valine) on the cocoon production and on the structural and mechanical properties of the silk produced. Negligible morphological differences were observed in the silk fiber threads from silkworm larvae supplemented with the tested amino acids. Higher production (yield) of silk was obtained using threonine in the diet of the silkworm. The treatments with threonine have increased the limit of proportionality, tensile strength, toughness, and maximum deformation of the thread of silk fibers. No significant increment in these properties was observed due to the increase in the threonine content. The treatments with valine led to lower increase in tensile strength and toughness. The real density of the silk has decreased with the use of supplements. The present study contributes to engineering of advanced silk materials, which should be attractive candidates for multipurpose applications.     

Electrospun composites of PHBV,silk fibroin and nano-hydroxyapatite for bone tissue engineering

Electrospinning of fibrous scaffolds containing nano-hydroxyapatite (nHAp) embedded in a matrix of functional biomacromolecules offers an attractive route to mimicking the natural bone tissue architecture. Functional fibrous substrates will support cell attachment, proliferation and differentiation, while the role of HAp is to induce cells to secrete extracellular matrix (ECM) for mineralization to form bone. Electrospinning of biomaterials composed of polyhydroxybutyrate-co-(3-hydroxyvalerate) with 2% valerate fraction (PHBV), nano-hydroxyapatite (nHAp), and Bombyx mori silk fibroin essence (SF), Mw = 90KDa, has been achieved for nHAp and SF solution concentrations of 2 (w/vol) % each and 5 (w/vol) % each. The structure and properties of the nanocomposite fibrous membranes were investigated by means of Scanning Electron Microscopy in combination with Energy Dispersive X-Ray Analysis (SEM/EDX), Fourier Transformed Infrared Spectroscopy (FT-IR), uniaxial tensile and compressive mechanical testing, degradation tests and in vitro bioactivity tests. SEM images showed smooth, uniform and continuous fibre deposition with no bead formation, and fibre diameters of between 10 and 15 μm. EDX and FT-IR confirmed the presence of nHAp and SF. After one month in deionised water, tests showed less than 2% weight loss with the samples retaining their fibrous morphology, confirming that this material biodegrades slowly. After 28 days of immersion in Simulated Body Fluid (SBF) an apatite layer was visible on the surface of the fibres, proving their bioactivity. Preliminary in vitro biological assessment showed that after 1 and 3 days in culture, cells were attached to the fibres, retaining their morphology while presenting a flattened appearance and elongated shape on the surface of fibres. Young's modulus was found to increase from 0.7 kPa (± 0.33 kPa) for electrospun samples of PHBV only to 1.4 kPa (± 0.54 kPa) for samples with 2 (w/vol) % each of nHAp and SF. Samples prepared with 5 (w/vol) % each of nHAp and SF did not show a similar improvement.     

Structure and properties of cocoons and silk fibers produced by <Emphasis Type="Italic">Hyalophora cecropia</Emphasis>

This paper shows that silk fibers produced by cecropia (Hyalophora cecropia) have similar tensile properties but different amino acid composition than that of mulberry (Bombyx mori) silk. The cecropia fibers are also much finer and have better strength and modulus than tasar silk, the most common non-mulberry silk. Cecropia is one of the largest silk producing moths and has similar lifecycle to that of mulberry silk but is easier to grow and produces larger cocoons than mulberry silk. In this study, we have characterized the composition, morphology, physical and tensile properties, and thermal behavior of the cecropia silk. Cecropia cocoons have a three tier structure and are larger (750 mg) than the cocoons produced by B. mori (650 mg). Fibers in the three layers in cecropia cocoons have tensile properties similar to that of B. mori silk but are finer (1.7–2 denier) and have higher strength (3.8–4.3 g/denier) and modulus (68–92 g/denier) than tasar silk.     

In vitro study on silk fibroin textile structure for Anterior Cruciate Ligament regeneration

A novel hierarchical textile structure made of silk fibroin from Bombyx mori capable of matching the mechanical performance requirements of anterior cruciate ligament (ACL) and in vitro cell ingrowth is described. This sericin-free, Silk Fibroin Knitted Sheath with Braided Core (SF-KSBC) structure was fabricated using available textile technologies. Micro-CT analysis confirmed that the core was highly porous and had a higher degree of interconnectivity than that observed for the sheath. The in vivo cell colonization of the scaffolds is thus expected to penetrate even the internal parts of the structure. Tensile mechanical tests demonstrated a maximum load of 1212.4 ± 56.4 N (under hydrated conditions), confirming the scaffold's suitability for ACL reconstruction. The absence of cytotoxic substances in the extracts of the SF-KSBC structure in culture medium was verified by in vitro tests with L929 fibroblasts. In terms of extracellular matrix production, Human Periodontal Ligament Fibroblasts (HPdLFs) cultured in direct contact with SF-KSBC, compared to control samples, demonstrated an increased secretion of aggrecan (PG) and fibronectin (FBN) at 3 and 7 days of culture, and no change in IL-6 and TNF-α secretion. Altogether, the outcomes of this investigation confirm the significant utility of this novel scaffold for ACL tissue regeneration.     

Mechanical properties of silkworm cocoon pelades

The pelade, the innermost layer of silkworm cocoon next to the chrysalis, has special microstructures, mechanical properties and protective functions distinctly different from those of all the other layers. In the present paper, a series of static tensile tests and dynamic mechanical thermal analysis were performed for the first time to measure the mechanical properties of pelades, including Young’s modulus, tensile strength and thermomechanical parameters. The fracture process of precracked pelade specimens was observed by in-situ scanning electron microscopy under tension. It is found that the Young’s modulus, tensile strength, storage modulus and loss modulus of cocoon pelades are superior to the corresponding thickness-averaged values of a complete silkworm cocoon. The damage and fracture process of pelades involve delamination, silk breaking and damage localization band. The results indicate that silkworm caterpillars can be appreciated as sophisticated sewers to make anisotropic and optimized structures of cocoons with both protective functions and mechanical properties varying in their thickness direction. The present study might be helpful to guide biomimetic design of novel safe-guarding materials and structures from both the viewpoints of microstructures and spatial functional gradients.     

Development of a conductive composite based on polythiophene/Y-zeolite and its response towards sulfide ions

A new potentiometric sensor electrode for sulfide based on conducting polymer films is introduced. A composite of polythiophene (PTP) with Y-zeolite was prepared via chemical oxidative polymerization of thiophene (TP) in presence of a dispersion of Y-zeolite (powder) in CHCl3 solvent using anhydrous FeCl3 oxidant. Formation of polythiophene and its subsequent SEM and TEM analysis revealed formation of composite particles with average diameter in the range of 0.3–0.35 μm. DC conductivity value of the PTP-Y-zeolite composite was in the order of 10? 2 S/cm, which was indeed high compared to that of PTP, produced under identical conditions as above without the presence of Y-zeolite. This composite as active component with graphite powder and binding liquid was mixed and then used for preparation of solid state electrodes. The working temperature range for this electrode is between 20 and 40 °C. The linear dynamic range is 1 × 10? 7 – 1 × 10? 4 M and measures total sulfide concentration over a range of pH from 5 to 9. The composite electrode showed high selectivity for sulfide in the presence of many common interfering anions (? log KS, IO3?Pot = 6.3, ? log KS, BrO3?Pot = 5.5, ? log KS, S2O32 ?Pot = 4.8).     

Effect of stitch density on fatigue characteristics and damage mechanisms of stitched carbon/epoxy composites

The effect of stitch density (SD) on fatigue life, stiffness degradation and fatigue damage mechanisms in carbon/epoxy (T800SC/XNRH6813) stitched using Vectran thread is presented in this paper. Moderately stitched composite (SD = 0.028/mm²; ‘stitched 6 × 6’) and densely stitched composite (SD = 0.111/mm²; ‘stitched 3 × 3’) are tested and compared with composite without stitch thread (SD = 0.0; ‘unstitched’). The experiments show that the fatigue life of stitched 3 × 3 is moderately better than that of unstitched and stitched 6 × 6. Stitched 3 × 3 pattern is also able to postpone the stiffness degradation onset. The improvement of fatigue properties and postponement of stiffness degradation onset in stitched 3 × 3 is primarily due to an effective impediment of edge-delamination. Quantification of damage at various cycles and stress levels shows that stitch density primarily affects the growth rate of delamination.     

Variability in mechanical properties of Bombyx mori silk

In the present paper, the variability of mechanical properties of Bombyx mori silk is investigated at the intraspecific and intraindividual levels. We first reeled some complete silks of 700–1500 m in length from cocoons in a special procedure. A large number of tensile tests were performed to measure the variations of Young's modulus, ultimate tensile strength, yield stress, elongation and breaking energy of an entire silk along its length direction. Our experimental results and statistical analysis show that the diameter and mechanical properties of silk have significant variabilities at both the intraspecific and intraindividual levels. On one hand, this indicates that a single silkworm silk seems inappropriate to be applied as an engineering material to bear loading. On the other hand, however, it is interesting to note that the variability of silk does not exist as a disadvantage for a cocoon to achieve a superior ability against possible attacks from the outside. In addition, the dependence of such parameters as Young's modulus and tensile strength upon the diameter of silk was also analyzed, and distinct size effects were observed in these properties. This study not only provides a deeper understanding of the mechanical properties of silks and cocoons but is of interest for the design and tailoring of advanced biomimetic silk materials also.     

Low temperature sintering and microwave dielectric properties of Ba4Sm9.33Ti18O54 ceramics

The effect of BaCu(B₂O₅) (BCB) on the sinterability, microstructure and microwave dielectric properties of Ba₄Sm_9.33Ti₁₈O₅₄ (BST) has been investigated. Dilatometric measurements reveal that the sintering temperature of BST can be reduced by the addition of BCB. Microstructural analysis shows abnormal grain growth with large amount of BCB. A ceramic composite with Q × f = 4000 GHz, ?_r = 52 and τ_f = ?29 ppm/°C which can be sintered at 950 °C is obtained when 10 wt% BCB is added to BST. EDS analysis shows that the composite is chemically compatible with silver.     

Study on the genotoxicity of HA/ZrO2 composite particles in vitro

To evaluate the genotoxicity of the HA/ZrO₂ composite particles by using the micronucleus test (MNT) in vitro. HA/ZrO₂ composite particles prepared by sintering at high temperature and pressure, that used powder of HA and ZrO₂ of different proportions, were compared with pure HA particles and pure ZrO₂ particles. The effect of the composite particles on cell proliferation of rabbit mesenchymal stem cells, and its the genotoxicity to rabbit mesenchymal stem cells were detected by MNT method. The MTT test showed that both pure HA particles and composite particles which contained HA promoted cell proliferation of rabbit mesenchymal stem cells, while pure ZrO₂ particles did not, and there was a significant difference (P < 0.05). The MNT test showed no significant difference between the HA group and the negative control group (P > 0.05), but a significant difference between the HA group and the positive control group (P < 0.05). The difference between the ZrO₂ group and the negative control group was significant (P < 0.01), while the difference between the ZrO₂ group and the positive control group was insignificant (P > 0.05). The genotoxicity of the HA/ZrO₂ composite particle increased with a higher proportion of ZrO₂ and an increase in the concentration of the composite, and the 30 wt.% HA/70% ZrO₂ composite with 200 μg/mL concentration showed significant genotoxicity (P < 0.01).     

Poly(N,N-dimethylaminoethyl methacrylate) modification of activated carbon for copper ions removal

Poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) functionalization of rice husk-based activated carbon was prepared and its application in the removal of copper ions was investigated. The structural properties of the resulting composite material were characterized by means of N₂ adsorption/desorption, Fourier transform infrared (FT-IR) and thermogravimetric analysis (TGA). The obtained composite is observed to hold a relatively large pore diameter of 3.8 nm and high surface area of 789 m² g^?1 with 12 wt% of PDMAEMA coated, which is significant for its use as adsorbent. The ability of the composite material for removing Cu²⁺ from aqueous solution was studied by batch experiments. The adsorption data obeyed the Langmuir isotherms, which revealed that 1 g of the prepared material could adsorb 31.46 mg of Cu²⁺ from its aqueous solution. The PDMAEMA functionalized activated carbon is expected to be used as an efficient adsorbent for removing other heavy metal ions and dyes in water.     

Preparation and characterization of copper nanoparticles/zinc oxide composite modified electrode and its application to glucose sensing

We report a new method for selective detection of d(+)-glucose using a copper nanoparticles (Cu-NPs) attached zinc oxide (ZnO) film coated electrode. The ZnO and Cu-NPs were electrochemically deposited onto indium tin oxide (ITO) coated glass electrode and glassy carbon electrode (GCE) by layer-by-layer. In result, Cu-NPs/ZnO composite film topography was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. SEM and AFM confirmed the presence of nanometer sized Cu-NPs/ZnO composite particles on the electrode surface. In addition, X-ray diffraction pattern revealed that Cu-NPs and ZnO films were attached onto the electrode surface. Indeed, the Cu-NPs/ZnO composite modified electrode showed excellent electrocatalytic activity for glucose oxidation in alkaline (0.1 M NaOH) solution. Further, we utilized the Cu-NPs/ZnO composite modified electrode as an electrochemical sensor for detection of glucose. This glucose sensor showed a linear relationship in the range from 1 × 10^? 6 M to 1.53 × 10^? 3 M and the detection limit (S/N = 3) was found to be 2 × 10^? 7 M. The Cu-NPs/ZnO composite as a non-enzymatic glucose sensor presents a number of attractive features such as high sensitivity, stability, reproducibility, selectivity and fast response. The applicability of the proposed method to the determination of glucose in human urine samples was demonstrated with satisfactory results.