Mulberry non-engineered silk gland protein vis-à-vis silk cocoon protein engineered by silkworms as biomaterial matrices

Silk fibroin from silk gland of Bombyx mori 5th instar larvae was utilized to fabricate films, which may find possible applications as two-dimensional matrices for tissue engineering. Bombyx mori cocoon fibroin is well characterized as potential biomaterial by virtue of its good mechanical strength, water stability, thermal properties, surface roughness and biocompatibility. The present study aims to characterize the biophysical, thermal, mechanical, rheological, swelling properties along with spectroscopic analysis, surface morphology and biocompatibility of the silk gland fibroin films compared with cocoon fibroin. Fibroin solutions showed increased turbidity and shear thinning at higher concentration. The films after methanol treatment swelled moderately and were less hydrophilic compared to the untreated. The spectroscopic analysis of the films illustrated the presence of various amide peaks and conformational transition from random coil to beta sheet on methanol treatment. X-ray diffraction studies also confirmed the secondary structure. Thermogravimetric analysis showed distinct weight loss of the films. The films were mechanically stronger and AFM studies showed surfaces were rougher on methanol treatment. The matrices were biocompatible and supported L929 mouse fibroblast cell growth and proliferation. The results substantiate the silk gland fibroin films as potential biomaterial matrices.     

SILK.IT project: Silk Italian Technology for industrial biomanufacturing

Silk fibroin is a natural protein that constitutes the core fibre of the silkworm cocoon. Recently, a great deal of attention is being paid to the use of the regenerated silk fibroin (RSF) solution that can be obtained by water-processing of the cocoon fibre. Indeed, thanks to its chemical and physical properties it has been shown that substrates obtained by RSF can be used as biomaterials for several biomedical and technological applications. In this context, silk fibroin could be exploited as raw material that can become a platform for eco-sustainable manufacturing.The project SILK.IT, coordinated by the CNR-ISOF, within the framework of the Flagship Project “Factory of the Future”, a research programme approved by the Interministerial Committee for Economic Planning (CIPE), aims to support this assumption by developing methods and protocols to use this silk fibroin as new material for advanced biotechnologies and sustainable manufacturing.The main goal of the project is to establish and control the whole-chain underpinning the silk fibroin-based technology, and to promote its up-scaling from the laboratory to industrial scale, targeting bio-photonics for biomedical application.The recent results obtained by the project are herein reported as key building block towards proposed achievement to establish a sustainable and process-controlled silk-based bio-manufacturing of the future.     

Green synthesis of gold nanoparticles: Its effect on cocoon and silk traits of mulberry silkworm (Bombyx mori L.)

In the present century the small particles are unique phenomenon which can be developed by bottom-up and top-down processes. These small particles may be considered as nanoparticles which help to build up a technology called nanotechnology. Nanomaterials are those materials which possess the length scales below 100 nm and quite often they make a comparison with a human hair, which is about 80,000 nm wide. We have introduced this technology, specially the green synthesis of gold (Au) nanoparticles in silkworm (Bombyx mori L.). The gold nanoparticles clearly indicate that they have a tremendous effect on enhancement of silk proteins and thus the enhancement of the cocoon weight in silkworms. Gold nanoparticles were prepared from onion, Allium cepa L. The extracted green gold nanomaterials from A. cepa were confirmed by UV-Vis spectrophotometer, XRD, FTIR, SEM, TEM and AFM. The function of green gold nanomaterials extracted from A. cepa was tested on silkworm physiology. We have used UV for judgment of the nature of particles and spectrum peak wavelength showed an absorption peak at 535 nm and indicated the wavelength of the surface plasmon resonance in gold nanoparticles (Au NPs). In blank solutions no such absorption peak was observed at 535 nm. Moreover, the gold (Au) XRD spectrum is supposed to and does demonstrate (111), (200), (220), and (311) peaks in the assortment of superimpose on the background. The process includes the (002) trace graphite peaks, where the (111) peak appears to be exceptionally sharp and strong which helps to propose that it is gold in nature. The FTIR shows that the examined particles are gold in nature. In SEM where electrons interact with atoms in the sample, producing various signals that can be detected and that hold information about the sample's surface topography and composition. The electron beam in SEM is generally scanned in a raster scan pattern, and therefore the beam's position is combined and detects the signal to produce an image. SEM can attain a resolution better than 1 nanometer size. The transmission electron microscope helps to accelerate the electrons as a source of elucidation. The AFM measurement is made in three dimensions process and thus it may be measured as horizontal to X-Y plane. Therefore, decree (magnification) measured at Z–direction, which is normally higher than X-Y. The said repulsive force is major one in AFM. Thus the tip and sample may considered to be the specific force in AFM which may measured at Z–direction. The effect of green gold nanoparticles on mulberry silkworm (Bombyx mori L) can exaggerated the silkworm physiological function. Larvae at 50, 100, 200, and 300 ppm doses were studied right from 1st stage to 5th instar stage. Gold nano treatment resulted in significant alterations in the percentage of fibroin and sericin proteins in the 5th instar as compared to that of control. At a 300 ppm dose of green nano gold the percentage of fibroin was 78.07, while sericin decreased from 39.46 (control) to 21.92. It was observed that the green gold nanomaterials have the ability to not only alter the fibroin protein but also enhance the cocoon and silk traits. The aim of this study was to investigate the effect of extra foliation of mulberry leaves with G-GNPs extracted from A. cepa on larval duration, mature silk gland weight, pupal weight, cocoon weight, cocoon shell weight, fibroin and sericins contents, etc. Moreover, the enhanced production of fibroin will explore a new venture in bioengineering and also in biomedical field.     

Preparation of three-dimensional fibroin/collagen scaffolds in various pH conditions

Three dimensional (3-D) fibroin/collagen scaffolds are the novel fibroin based scaffolds derived from aqueous solution. In this article, we investigated the effect of pH on the formation of fibroin/collagen scaffolds. In the range of pH from 4 to 8.5, the fibroin/collagen scaffolds with good porous structures can be prepared using freeze-drying method, which would facilitate the adding of other biopolymers. The structures of different fibroin-based scaffolds were investigated with FTIR and DSC, which indicated that the interaction of fibroin and collagen affected the methanol-induced transformation of fibroin from random-coil to β-sheet conformation. The mechanical properties were also studied. The results mean that all the fibroin-based scaffolds prepared in various pH values had better mechanical properties than other reported fibroin scaffolds. Since the fibroin/collagen scaffolds can be prepared in the range of pH from 4 to 8.5, it is very easy to prepare different multifunctional scaffolds such as fibroin/collagen/chitosan scaffolds and fibroin/collagen/heparin scaffolds in acidic or neutral conditions. These new fibroin-based blend materials extend the range of biomaterial properties that can promote the use in biomedical applications such as drug release and tissue engineering.     

蚕丝在生物医用材料领域的应用研究

蚕丝既是优质的天然蛋白纤维,也是优质的高分子蛋白质材料,具有良好的力学性能、生物相容性和可控的生物降解性等。随着生物医用材料领域的不断发展和各学科的交叉融合,蚕丝作为生物医用材料已展示出很强的竞争力,其在该领域的应用潜力已逐渐展现。介绍了蚕丝的构成和特点,总结了蚕丝丝素及丝胶提取的方法,综述了近年来蚕丝及蚕丝蛋白在组织工程、载药、敷料等方面的应用,并客观分析了蚕丝及蚕丝蛋白在这些具体应用过程中所发挥的重要作用及各种蚕丝材料的优缺点,最后就蚕丝在生物医用材料领域的应用前景进行了展望。     

Blend films of silk fibroin and water-insoluble polyurethane prepared from an ionic liquid

In order to blend the water-insoluble and biocompatible polyurethane (PU) with silk fibroin (SF) to prepare water-insoluble films as a biomaterial, the ionic liquid, 1-butyl-3-methylimidazolium chloride (BMIMCl), was used to prepare a SF/BMIMCl solution, which was directly blended with PU solution in N, N-dimethylformamide (DMF). Infrared spectroscopy indicated that the solution blending led to chemical bonding between the two polymers and a β-sheet structure was the main conformation of SF in the films. The hydrophilicity of the films increased with the content of SF, based on contact angle measurements. The composite showed good blood compatibility in coagulation time tests and has potential biomaterial applications.     

Design,fabrication and characterization of silk fibroin-HPMC-PEG blended films as vehicle for transmucosal delivery

This study illustrates the fabrication of stable mucoadhesive films of silk protein fibroin as potential vehicle for transmucosal delivery by blending fibroin with hydroxy propyl methyl cellulose (HPMC) and poly ethylene glycol 400 (PEG). Investigations on mechanical properties, swelling ability in simulated saliva, bioadhesive strength by a specially designed instrument and study of in vitro stability in simulated saliva of goat buccal mucosa as model membrane was undertaken. Molecular interaction between blended materials was evaluated by FTIR spectroscopy. Increase in fibroin content of the blended films not only increased the mechanical properties and water stability but also the degree of swelling and stability of the films in simulated saliva. The FTIR spectrum shows an increase in water stability of the fibroin-HPMC blended films due to the formation of intermolecular hydrogen bonding between the HPMC and fibroin. The conformational transition of the silk fibroin molecule from the amorphous and random coil to β sheet structure has been observed. Fibroin-HPMC-PEG blended films can be used as a vehicle for transmucosal delivery by virtue of its good mechanical strength, water stability, ex vivo bioadhesive strength and ideal swellability as such characteristics are essential for rapid mucoadhesion.     

A versatile click-grafting approach to surface modification of silk fibroin films

Silk fibroin is a biocompatible, mechanically robust protein polymer that can be made optically transparent, and is widely used and studied as biomaterial for different applications. Its chemical modification is a fascinating way for tuning the properties and widening its application field. Herein, PEG grafting on the surface of regenerated silk fibroin films is obtained by direct linking based on a click reaction between the azido activated silk surface and an alkyne terminated PEG. The so obtained PEGylated films exhibit modified surface properties in comparison with the unmodified films. Through the same click approach, we also show that arrays of ordered fluorescent spots are steadily printed onto the film surface. This expands the versatility of our silk modification to different molecules and polymers, hence allowing for the realization of new functional hybrid materials.     

丝素蛋白对胶原膜性能改善的研究

天然高分子由于其良好的生物相容性而受到广泛的关注。本研究用酶法自制了具有一定水溶性的猪皮胶原。尝试利用丝素和胶原蛋白各自的优点。用简单的溶液浇铸法制备了胶原-丝素共混膜。并通过FTIR、TGA、SEM等手段对其结构进行了表征。结果表明。由于共混膜中俩组份间存在的分子间作用力，加入小于50％的丝素的胶原膜经乙醇处理后与纯胶原膜相比。其力学性能及热稳定性有所的改善。通过改变丝素比例可以调整共混膜的吸水性。由于两组分良好的生物相容性，预料该共混膜可用作生物材料。     

Surface modification and functionalization of silk fibroin fibers/fabric toward high performance applications

Silk from silkworms is composed of fibrous proteins with remarkable properties that have resulted in wide usage in the textile industry and as a biomaterial in the medical field. However, for some advanced applications, silk still has the disadvantages of wrinkling, photo-induced aging, deformation and even degradation caused by microorganism, biocompatibility problems. Based on sufficient supply of raw materials around the world and encouraged by the market demands for natural and smart materials, numerous attempts have been made in the surface modification and functionalization of silk fibroin fibers (SFFs) and silk fibroin fabric (SFF) using a wide range of functional materials and technology during last decade. Successful modification of silk may not only overcome its intrinsic shortcomings, but also enhance end-use performance. In this review, we summarize the recent progress of basic ideas, methodologies, and treatment techniques for surface modification and functionalization of SFF/SFFs with consideration of their enhanced properties and potential applications. Encouraged by achievements from experimental studies, the authors believe that such successes give silk products a promising future in both textile and biomaterial applications.     

Robust Biological Fibers Based on Widely Available Proteins: Facile Fabrication and Suturing Application

Lightweight and mechanically strong protein fibers are promising for many technical applications. Despite the widespread investigation of biological fibers based on spider silk and silkworm proteins, it remains a challenge to develop low‐cost proteins and convenient spinning technology for the fabrication of robust biological fibers. Since there are plenty of widely available proteins in nature, it is meaningful to investigate the preparation of fibers by the proteins and explore their biomedical applications. Here, a facile microfluidic strategy is developed for the scalable construction of biological fibers via a series of easily accessible spherical and linear proteins including chicken egg, quail egg, goose egg, bovine serum albumin, milk, and collagen. It is found that the crosslinking effect in microfluidic chips and double‐drawn treatment after spinning are crucial for the formation of fibers. Thus, high tensile strength and toughness are realized in the fibers, which are comparable or even higher than that of many recombinant spider silks or regenerated silkworm fibers. Moreover, the suturing applications in rat and minipig models are realized by employing the mechanically strong fibers. Therefore, this work opens a new direction for the production of biological fibers from natural sources.     

Flexible bio-composites based on silks and celluloses

Biomaterials have attracted worldwide attention due to the concerns regarding health and the environment. Silk, a natural protein produced by several species of insects, has been examined as a potential material for applications in many biotechnological and biomedical fields. However, regenerated silk fibroin has poor ductility and mechanical properties. Therefore, in this study, silk fibroin-cellulose composite films were prepared in an aqueous system to increase the ductility of regenerated silk fibroin. The morphology of the silk fibroin-cellulose composite film was observed by field emission scanning electron microscopy. The structure of the silk fibroin-cellulose composite films was examined by Fourier transform-infrared spectroscopy. The flexibility was analyzed using a bending test.     

Covalent binding of placental derived proteins to silk fibroin improves schwann cell adhesion and proliferation

Schwann cells play a key role in peripheral nerve regeneration. Failure in sufficient formation of Büngner bands due to impaired Schwann cell proliferation has significant effects on the functional outcome after regeneration. Therefore, the growth substrate for Schwann cells should be considered with highest priority in any peripheral nerve tissue engineering approach. Due to its excellent biocompatibility silk fibroin has most recently attracted considerable interest as a biomaterial for use as conduit material in peripheral nerve regeneration. In this study we established a protocol to covalently bind collagen and laminin, which have been isolated from human placenta, to silk fibroin utilizing carbodiimide chemistry. Altered adhesion, viability and proliferation of Schwann cells were evaluated. A cell adhesion assay revealed that the functionalization with both, laminin or collagen, significantly improved Schwann cell adhesion to silk fibroin. Moreover laminin drastically accelerated adhesion. Schwann cell proliferation and viability assessed with BrdU and MTT assay, respectively, were significantly increased in the laminin-functionalized groups. The results suggest beneficial effects of laminin on both, cell adhesion as well as proliferative behaviour of Schwann cells. To conclude, the covalent tailoring of silk fibroin drastically enhances its properties as a cell substratum for Schwann cells, which might help to overcome current hurdles bridging long distance gaps in peripheral nerve injuries with the use of silk-based nerve guidance conduits.     

Processing methods to control silk fibroin film biomaterial features

Control of silk structural and morphological features is reported for fibroin protein films via all aqueous processing, with and without polyethylene oxide (PEO). Silk films with thicknesses from 500 nm to 50 μm were generated with controllable surface morphologies by employing soft-lithography surface patterning or by adjusting PEO concentrations. FTIR analysis indicated that water-annealing, used to cure or set the films, resulted in increased β-sheet and α-helix content within the films. Steam sterilization provided an additional control point by increasing β-sheet content, while reducing random coil and turn structures, yet retaining film transparency and material integrity. Increased PEO concentration used during processing resulted in decreased sizes of surface globule structures, while simultaneously increasing uniformity of these features. The above results indicate that both surface and bulk morphologies and structures can be controlled by adjusting PEO concentration. The combined tool set for controlling silk film geometry and structure provides a foundation for further study of novel silk film biomaterial systems. These silk film biomaterials have potential applicability for a variety of optical and regenerative medicine applications due to their optical clarity, impressive mechanical properties, slow degradability, and biocompatibility.     

The interaction of the polyphenylacetylene surface with biological environments studied by XPS, RAIRS and biological tests

A π-conjugated polymer, polyphenylacetylene or PPA, has been tested for its possible applications as biosensor or biomaterial. Protein adsorption was investigated by incubating PPA films in solutions of bovine serum albumin (BSA) dissolved in phosphate buffer (PBS) having increasing protein concentration. Investigations on the PPA films were carried out by means of two surface analysis techniques, X-ray photoelectron spectroscopy (XPS) and reflection-absorption infrared spectroscopy (RAIRS). Desorption of BSA from the PPA surface was also investigated. Finally, the cytototoxicity of the PPA surface was checked by measuring viability and proliferation of lymphoma macrophages and SAOS osteoblasts grown in the presence of the polymer.     

Bovine pericardium coated with biopolymeric films as an alternative to prevent calcification: In vitro calcification and cytotoxicity results

Bovine pericardium, for cardiac valve fabrication, was coated with either chitosan or silk fibroin film. In vitro calcification tests of coated and non coated bovine pericardium were performed in simulated body fluid solution in order to investigate potential alternatives to minimize calcification on implanted heart valves. Complementary, morphology was assessed by scanning electron microscopy — SEM; X-ray diffraction (XRD) and infrared spectroscopy (FTIR-ATR) were performed for structural characterization of coatings and biocompatibility of chitosan. Silk fibroin films were assayed by in vitro cytotoxicity and endothelial cell growth tests. Bovine pericardium coated with silk fibroin or chitosan did not present calcification during in vitro calcification tests, indicating that these biopolymeric coatings do not induce bovine pericardium calcification. Chitosan and silk fibroin films were characterized as non cytotoxic and silk fibroin films presented high affinity to endothelial cells. The results indicate that bovine pericardium coated with silk fibroin is a potential candidate for cardiac valve fabrication, since the affinity of silk fibroin to endothelial cells can be explored to induce the tissue endothelization and therefore, increase valve durability by increasing their mechanical resistance and protecting them against calcification.     

Mechanical properties and structure of silkworm cocoons: A comparative study of Bombyx mori,Antheraea assamensis,Antheraea pernyi and Antheraea mylitta silkworm cocoons

As a protective shell against environmental damage and attack by natural predators, the silkworm cocoon has outstanding mechanical properties. In particular, this multilayer non-woven composite structure can be exceptionally tough to enhance the chance of survival for silkworms while supporting their metabolic activity. Peel, out-of-plane compression and nano-indentation tests and micro-structure analysis were performed on four types of silkworm cocoon walls (domesticated Bombyx mori, semi-domesticated Antheraea assamensis and wild Antheraea pernyi and Antheraea mylitta silkworm cocoons) to understand the structure and mechanical property relationships. The wild silkworm cocoons were shown to be uniquely tough composite structures. The maximum work-of-fracture for the wild cocoons (A. pernyi and A. mylitta) was approximately 1000 J/m², which was almost 10 times the value for the domesticated cocoon (Bombyx mori) and 3 ~ 4 times the value for the semi-domesticated cocoon (A. assamensis). Calcium oxalate crystals were found to deposit on the outer surfaces of the semi-domesticated and wild cocoons. They did not show influence in enhancing the interlaminar adhesion between cocoon layers but exhibited much higher hardness than the cocoon pelades.     

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.     

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.     

Characterization of proteins and fibroblasts on thin inorganic films

The ability of biomaterial surfaces to regulate cell behavior requires control over surface chemistry and material microstructure. One of the goals in the development of silicon-based biomedical devices such as biosensors or drug delivery systems is improved biocompatibility which may be achieved through the deposition or adsorption of thin films. In this study, films of single crystal silicon, stoichiometric and low stress silicon nitride, doped and undoped polysilicon, as well as Arg-Gly-Asp (RGD) peptide adsorbed surfaces characterized in terms of protein adsorption or cellular adhesion for a period of four days. Protein adsorption studies using fibrinogen and albumin, two proteins implicated in cellular adhesion and surface activity, reveal that low stress silicon nitride surfaces have a 223%±2.50% greater protein adsorption compared to undoped polysilicon surfaces, followed by silicon nitride, unmodified silicon, and doped polysilicon surfaces, respectively. The thickness of the adsorbed albumin and fibrinogen layer on various thin films was measured by ellipsometry and compared to contact angle measurements. The greatest cellular adhesion was observed on undoped polysilicon, followed by unmodified (control) silicon, low stress silicon nitride, silicon nitride, and doped polysilicon surfaces. Cellular binding supports the differential protein adsorption found on modified and unmodified silicon surfaces. Understanding the biological response to thin films will allow us to design more appropriate interfaces for implantable diagnostic and therapeutic silicon-based microdevices.