Bombyx Mori Silk Fibroin Research Articles

Tuning the structure and performance of silk biomaterials by combining mulberry and non-mulberry silk fibroin

Despite considerable efforts toward the biomedical applications of silk fibroin (SF), detailed control of structure and performance in silk biomaterials remains a major challenge. Silks are classified as mulberry and non-mulberry, which are produced by domesticated Bombyx mori and wild silkworm species, respectively. To achieve silk biomaterials with tunable performance, Bombyx mori silk fibroin (BSF) and non-mulberry Antheraea pernyi silk fibroin (ASF) were blended. These systems exploit the beneficial material and biological properties of both silk proteins. The sol-gel transition of ASF was much more rapid than that of BSF. Consequently, the gelation rate of SF solution could be controlled ranging from tens of minutes to days by changing the ASF ratio, providing useful options for the fabrication of silk hydrogels. Raising BSF ratio in the blend materials significantly increased mechanical performance, overcoming the inherent lack of mechanical performances of regenerated ASF materials. Conversely, increasing ASF ratio significantly enhanced degradability of the blend films, providing a wider range of regulation for the degradation rate of silk biomaterials. As a result, this simple blending strategy enables the process to be more controllable and flexible, controlling the performance of silk protein becomes feasible, and this would greatly expand the applications of silk as a biomedical material.

In vitro and in vivo evaluation of silk fibroin functionalized with GABA and allopregnanolone for Schwann cell and neuron survival.

This in vitro and in vivo study reports on silk fibroin (SF) scaffold, functionalized for in situ delivery of GABA and/or allopregnanolone (ALLO), as biomaterial for potential application in tissue engineering and nerve regeneration. We evaluated the feasibility to design 2D scaffolds (films) made of regenerated Bombyx mori SF, functionalized with GABA and/or ALLO to enhance in vitro biological functions, health, survival and growth of Schwann cells and sensitive neurons of the dorsal root ganglia. Our 2D-SF film showed an efficient loading and controllable release of drugs promoting nerve regeneration. SF functionalized film may be helpful for the development of bioengineered conduits and, in principle, have great potential for long-gap nerve injury repair.

Functionalized PVA-silk blended nanofibrous mats promote diabetic wound healing via regulation of extracellular matrix and tissue remodelling.

Chronic cutaneous ulcers, a complex pathophysiological diabetic condition, represent a critical clinical challenge in the current diabetes mellitus pandemic. Consequently, there is a compelling need for bioactive dressings that can trigger healing processes for complete wound repair. Silk fibroin (SF), a natural protein polymer from mulberry and non-mulberry silkworms, has properties that support accelerated wound healing rate. SF from non-mulberry variety possesses additional cell-binding motifs (arginine, glycine, and aspartate), offering cell-material interactions. This study is aimed to investigate wound healing efficacy of dressings made up of various SF varieties blended with poly(vinyl alcohol) biopolymer in alloxan-induced diabetic rabbit model. The nanofibrous mats have been developed using electrospinning and functionalized with growth factors and LL-37 antimicrobial peptide for sustained delivery. Following post 14-day treatment, non-mulberry SF (NMSF)-based dressings healed the wounds faster, in comparison with their mulberry Bombyx mori SF, poly(vinyl alcohol), and control counterparts (p<.01). NMSF-based dressings also supported faster granulation tissue development, angiogenesis, and reepithelialization of wounds. Gene expression study of matrix metalloproteinases and collagen proteins affirmed higher extent of tissue remodelling during the repair process. Furthermore, there was organized extracellular matrix deposition (collagen type I, collagen type III, elastin, and reticulin) and higher wound breaking strength in NMSF compared with other groups after 4weeks. These results validated the potential of NMSF-based bioactive dressings to regulate extracellular matrix deposition leading to faster and complete repair of chronic diabetic cutaneous wounds.

Bombyx mori Silk Fibroin Scaffolds with Antheraea pernyi Silk Fibroin Micro/Nano Fibers for Promoting EA. hy926 Cell Proliferation.

Achieving a high number of inter-pore channels and a nanofibrous structure similar to that of the extracellular matrix remains a challenge in the preparation of Bombyx mori silk fibroin (BSF) scaffolds for tissue engineering. In this study, Antheraea pernyi silk fibroin (ASF) micro/nano fibers with an average diameter of 324 nm were fabricated by electrospinning from an 8 wt % ASF solution in hexafluoroisopropanol. The electrospun fibers were cut into short fibers (~0.5 mm) and then dispersed in BSF solution. Next, BSF scaffolds with ASF micro/nano fibers were prepared by lyophilization. Scanning electron microscope images clearly showed connected channels between macropores after the addition of ASF micro/nano fibers; meanwhile, micro/nano fibers and micropores could be clearly observed on the pore walls. The results of in vitro cultures of human umbilical vein endothelial cells (EA. hy926) on BSF scaffolds showed that fibrous BSF scaffolds containing 150% ASF fibers significantly promoted cell proliferation during the initial stage.

Bioresorbable Scaffolds Based on Fibroin for Bone Tissue Regeneration

Using the tissue-engineered constructs based on scaffolds that imitate the extracellular matrix of living tissues unveils new opportunities in the treatment of various pathologies and injuries associated with tissue and organ damage. Silk fibroin of silkworm Bombyx mori is a biocompatible and bioresorbable polymer with high mechanical strength and elasticity that allows creating scaffolds on its basis for regeneration of various tissues, including bone. In the present work, fibroin scaffolds were obtained. They were designed in the form of porous sponges, films, and hybrid scaffolds of a bilayer structure in which the porous sponge threedimensional structure is limited on one side by a film. The structure of the scaffolds and their biocompatibility were studied: immortalized and primary fibroblasts, as well as the osteoblast-like cells, have been shown to successfully adhere and proliferate on the surface of the studied scaffolds. Numerous osteogenesis foci have been observed in the implant region 4 weeks after the fibroin porous scaffold implantation in the in vivo experiments in a rat femoral bone defect model indicating the osteoconduction of the scaffolds.

Methanol-Water-Dependent Structural Changes of Regenerated Silk Fibroin Probed Using Terahertz Spectroscopy.

The mechanism of β-sheet crystallization in silk fibroin remains unclear, due to the incomplete information of protein assembly and structural state. The emerging terahertz (THz) spectroscopy (<10 THz) has been taken as an important tool to detect new aspects of biomolecular structure and is used for the first time to analyze the methanol-water (MeOH) induced structural changes of Bombyx mori silk fibroin. Mid-infrared spectroscopy (IR) and X-ray diffraction (XRD) results show that silk fibroin initially exists in a typical silk I form and reassemble into a predominant silk II (antiparallel β-sheet crystal) structure after MeOH treatment. The samples treated with MeOH-H2O mixed solutions show a predominant silk I structure without any silk-II-related peaks. As the MeOH concentration approaches 40 vol%, the absorbance of the β-sheet-related IR bands and the XRD peaks gradually increase, indicating a formation of β-sheet crystal during this process. THz spectrum shows the absorption capacity below 3 THz as well as the absorbance at 5.1 THz and 7.9 THz is indeed affected by the MeOH-H2O treatment, implying a MeOH-H2O-dependent change of intermolecular H-bonds in silk fibroin. The THz spectrum for silk fibroin gives additional information to the existing studies on the MeOH-H2O induced β-sheet crystallization of silk fibroin, which may help us understanding the structural changes of natural silk.

Microwave assisted synthesis of luminescent carbonaceous nanoparticles from silk fibroin for bioimaging

Bombyx mori silk as a natural protein based biopolymer with high nitrogen content, is abundant and sustainable because of its mass product all over the world per year. In this study, we developed a facile and fast microwave-assisted synthesis of luminescent carbonaceous nanoparticles using Bombyx mori silk fibroin and silk solution as the precursors. As a result, the obtained carbonaceous nanoparticles exhibit a photoluminescence quantum yield of ~20%, high stability, low cytotoxicity, high biocompatibility. Most importantly, we successfully demonstrated bioimaging using these luminescent carbonaceous nanoparticles with excitation dependent luminescence. In addition, the microwave-assisted hydrothermal method can be extended to convert other biomass into functional nanomaterials.

Nucleation and Assembly of Silica into Protein-Based Nanocomposites as Effective Anticancer Drug Carriers Using Self-Assembled Silk Protein Nanostructures as Biotemplates.

Bombyx mori (B. mori) silk fibroin and sericin can act as a great candidate in delivering drugs or other bioactive substances. Silica also has a great application in the field of drug delivery. To the best of our knowledge, there has been no report on the design of a nanocomposite made of silk protein and silica for drug delivery. Here, for the first time, we used B. mori silk fibroin (SF) and sericin (SS), self-assembled into nanospheres and nanofibers in situ in the aqueous solution, respectively, as a biotemplate to regulate the nucleation and self-assembly of silica for designing anticancer drug delivery. SF and SS mediated the nucleation and assembly of silica into monodispersed nanospheres (termed Si/SF) and nanofibers (termed Si/SS), respectively. The size and topography of the silica assemblies were dependent on the concentration of SF or SS as well as reaction conditions. Both Si/SF nanospheres and Si/SS nanofibers showed a high loading capability and sustained release profile of an anticancer drug, doxorubicin (DOX), in vitro. Si/SF nanospheres were found to be efficiently internalized in human cervical carcinoma (HeLa) cells and accumulate around the cell nuclei. Si/SS nanofibers could only adhere to the surface of the cancer cells. This indicates that DOX-loaded Si/SF nanospheres and Si/SS nanofibers are more effective in cancer therapy than free DOX. Our results suggest that the self-assembled Si/SF spheres and Si/SS nanofibers are potential effective anticancer drug carriers.

Enhancement reactivity of Bombyx mori silk fibroins via genipin-mediated grafting of a tyrosine-rich polypeptide

Bombyx mori silk fibroin has potential applications in the biomedical field owing to its satisfactory biocompatibility. In the present work, a tyrosine-rich polypeptide (P) with the sequence of GKGYGGYGK was grafted onto fibroin molecules using a natural cross-linking agent of genipin (GP), aiming at improving the reactivity of the fibroin-based materials. Incubation of the polypeptide-grafted fibroin membrane was subsequently carried out, with a mushroom tyrosinase (MT) and a model functional macromolecule of ε-poly-l-lysine (ε-PL). The changes in the structure and composition of the silk fibroin before and after polypeptide grafting were examined by means of size exclusion chromatography and amino acid analysis. The results indicated that the polypeptide was covalently bonded to fibroin chains and evidently promoted the succeeding enzymatic coupling of ε-PL with fibroins, resulting in a higher content of ε-PL compared to the control. The obtained antibacterial activities against S. aureus and E. coli for the fibroin membrane treated with GP/P and MT/ε-PL was more satisfactory than others. Cytotoxicity testing reveals that polypeptide grafting did less impact on the biocompatibility of silk fibroins. The present work provides a novel method to improve the reactivity of silk fibroins and it can be utilized for bio-functionalization of silk fibroin.

Magnetic silk fibroin e-gel scaffolds for bone tissue engineering applications

Recently, the incorporation of magnetic nanoparticles into standard scaffolds has emerged as a promising approach for tissue engineering applications. This strategy can promote not only tissue regeneration but also reloading of scaffolds through an external supervising center that adsorbs growth factors, preserving their stability and biological activity. In this study, novel magnetic silk fibroin e-gel scaffolds were prepared by the electrogelation process of concentrated Bombyx mori silk fibroin (8 wt%) aqueous solution. In addition, basic fibroblast growth factor was conjugated physically to human serum albumin = Fe3O4 nanoparticles (71.52 ± 2.3 nm in size) with 97.5% binding yield. Scanning electron microscopy images of the prepared human serum albumin = Fe3O4-basic fibroblast growth factor-loaded silk fibroin e-gel scaffolds showed a three-dimensional porous morphology. In terms of water uptake, basic fibroblast growth factor-conjugated scaffolds had the highest water absorbability among all groups. In vitro cell culture studies showed that both the human serum albumin coating of Fe3O4 nanoparticle surface and basic fibroblast growth factor conjugation had an inductive effect on cell viability. One of the most used markers of bone formation and osteoblast differentiation is alkaline phosphatase activity; human serum albumin = Fe3O4-basic fibroblast growth factor-loaded silk fibroin e-gels showed significantly enhanced alkaline phosphatase activity (p &lt; 0.05). SaOS-2 cells cultured on human serum albumin = Fe3O4-basic fibroblast growth factor-loaded silk fibroin e-gels deposited more calcium compared with those cultured on bare silk fibroin e-gels. These results indicated that the proposed e-gel scaffolds are valuable candidates for magnetic guiding in bone tissue regeneration, and they will present new perspectives for magnetic field application in regenerative medicine.

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.

Packing arrangement of 13C selectively labeled sequence model peptides of Samia cynthia ricini silk fibroin fibers studied by solid-state NMR.

Samia cynthia ricini (S. c. ricini) is one of the wild silkworms. Their silk fibroins have been paid attention as potentially valuable biomedical materials as well as Bombyx mori silk fibroins, but detailed information on the packing arrangement of the fibers is still not currently well understood at a molecular level. In this study, 34 mer model peptides, GGAGGGYGGDGG(A)12GGAGDGYGAG with different 13C labeled positions have been synthesized as a typical sequence of the primary structure of S. c. ricini silk fibroins made up of tandemly repeated sequences of polyalanine as the crystalline region and glycin-rich sequences as the non-crystalline region. The heterogeneous structure was obtained from the determination of the fraction of several conformations depending on the position of the Ala residue by 13C cross polarization/magic angle spinning NMR. The packing arrangement was studied by 13C dipolar assisted rotational resonance NMR and packing in a staggered arrangement rather than a rectangular arrangement of this peptide with an anti-parallel β-sheet structure was clarified, which is in good agreement with our previous report on the packing arrangement of (Ala)7 with an anti-parallel β-sheet structure.

Hydration of Bombyx mori silk cocoon, silk sericin and silk fibroin and their interactions with water as studied by 13C NMR and 2H NMR relaxation.

The mechanical properties of Bombyx mori silk fibers, such as elasticity and tensile strength, change remarkably upon hydration. However, changes in the local conformation and dynamics of individual amino acid residues and change in the dynamics of water molecules due to hydration are not currently well understood on the molecular level. In this work, the conformations and dynamics of the hydrated Bombyx mori silk fibers, including silk cocoon (SC), silk sericin (SS) and silk fibroin (SF), were determined after sustained immersion in water by using 13C refocused insensitive nuclei enhanced by polarization transfer (INEPT) NMR, 13C cross-polarization/magic angle spinning (CP/MAS) NMR and 13C dipolar decoupled-magic angle spinning (DD/MAS) NMR. The 13C INEPT NMR spectrum reflects their mobile domain, the 13C CP/MAS NMR spectrum their rigid domain, and the 13C DD/MAS NMR spectrum both domains. The mobile domain of the hydrated SC fiber originates mainly from the hydrated SS part and the rigid domain of the hydrated SC fiber from the hydrated SF part. Moreover, the dynamics of mobile water molecules interacting with the silk fiber was studied by 2H solution NMR relaxation measurements in the silk fiber-2H2O system. Using an inverse Laplace transform algorithm, we were able to identify distinct mobile components in the relaxation times for 2H2O. Our measurements provide new insight relating to the characteristics of the hydrated structure of SC, SS and SF fibers, and the water molecules that interact with them in water. The information is relevant in light of current interest in the design of novel silk-based biomaterials which are usually in contact with blood and other body fluids.

Ultrathin nanoporous membranes derived from protein-based nanospheres for high-performance smart molecular filtration

Flexible ultrathin nanoporous membranes composed of closely packed nanospheres with diameters of ∼11 nm are fabricated from natural Bombyx mori silk fibroin via a vacuum filtration process.

Electrospinning of silk fibroin from all aqueous solution at low concentration

Non-woven mats of Bombyx mori silk fibroin were fabricated using electrospinning with an all aqueous solution at <10wt% without any co-existing water soluble polymer such as PEO. The fibroin aqueous solution electrospinnability was affected by the fibroin molecular weight and the spinning solution pH. Hot-water treatment without any alkaline reagent or soap produced higher molecular weight fibroin than the typical degumming process did. The higher molecular weight fibroin provided good electrospinnability. Results show that the basic solution (pH10–11) is important for electrospinning at low concentrations of 5wt%. Evaluation of structural and mechanical properties of the non-woven mat fabricated with water solvent revealed that it is safe for use in the human body. It is anticipated for wider use in medical materials such as cellular scaffolds for tissue engineering.

Reloadable Silk-Hydrogel Hybrid Scaffolds for Sustained and Targeted Delivery of Molecules.

Tunable repeated drug administration is often inevitable in a number of pathological cases. Reloadable 3D matrices for sustained drug delivery are predicted as a prospective avenue to realize this objective. This study was directed toward sonication-induced fabrication of novel reloadable Bombyx mori silk fibroin (SF) (4, 6, and 8 wt %) hydrogel, injected within 3D porous (8 wt %) scaffolds. The focus was to develop a dual-barrier reloadable depot system for sustained molecular cargo release. Both the varying SF concentration (4, 6, and 8 wt %) and the sonication time (30, 45, and 60 s) dictated the extent of cross-linking, β-sheet content, and porosity (1-10 μm) influencing the release behavior of model molecules. Release studies of model molecules (trypan blue, TB, 961 Da and bovine serum albumin, BSA, 66 kDa) for 28 days attested that the variations in their molecular weight, the matrix cross-linking density, and the scaffold-hydrogel interactions dictated the release behavior. The Ritger and Peppas equation was further fitted into the release behavior of model molecules from various SF matrices. The hybrid constructs exhibited high compressive strength along with in vitro compatibility using primary porcine chondrocytes and tunable enzymatic degradation as assessed for 28 days. The aptness of the constructs was evinced as a reloadable model molecule (BSA and fluorescein isothiocyanate-inulin, 3.9 kDa) depot system through UV-visible and fluorescence spectroscopic analyses. The novel affordable platform developed using silk scaffold-hydrogel hybrid constructs could serve as a sustained and reloadable drug depot system for administration of multiple and repeated drugs.

Characterization of Bombyx mori and Antheraea pernyi silk fibroins and their blends as potential biomaterials.

Fibroin proteins isolated from the cocoons of certain silk-producing insects have been widely investigated as biomaterials for tissue engineering applications. In this study, fibroins were isolated from cocoons of domesticated Bombyx mori (BM) and wild Antheraea pernyi (AP) silkworms following a degumming process. The object of this study was to obtain an assessment on certain properties of these fibroins in order that a concept might be had regarding the feasibility of using their blends as biomaterials. Membranes, 10–20 μm thick, which are water-insoluble, flexible and transparent, were prepared from pure fibroins and from their blends, and subjected to water vapor annealing in vacuum, with the aim of providing materials sufficiently strong for manipulation. The resulting materials were characterized by electrophoretic analysis and infrared spectrometry. The tensile properties of the membranes were measured and correlated with the results of infrared analysis. At low concentrations of any of the two fibroins, the mechanical characteristics of the membranes appeared to be adequate for surgical manipulation, as the modulus and strength surpassed those of BM silk fibroin alone. It was noticed that high concentrations of AP silk fibroin led to a significant reduction in the elasticity of membranes.

Role of non-mulberry silk fibroin in deposition and regulation of extracellular matrix towards accelerated wound healing

A facile fabrication of a ready-to-use bioactive wound dressing capable of concomitantly accelerating the healing process as well as deposition of the extracellular matrix (ECM) to circumvent further scarring complicacies has become a focal point of research. In this backdrop, our present work is based on non-mulberry silk fibroin (NMSF) electrospun antibiotic loaded semi-occlusive mats, mimicking the ECM of skin in terms of morphology, topology, microporous structure and mechanical stiffness. Regulation of ECM deposition and isotropic orientation evinced the potential of the mat as an instructive platform for skin regeneration. The unique peptide motifs of NMSF assisted the augmented recruitment of fibroblast, keratinocytes and endothelial cells leading to accelerated wound healing. Early progression of mature granulation, faster re-epithelialization and angiogenesis in the wounds in in vivo rabbit model forwarded the blended nanofibrous mats of NMSF and PVA ferrying EGF, apt for scarless healing.

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.

Synthesis of Silver Nanoparticles Using Bombyxmori Silk Fibroin and Their Antibacterial Activity

Present work describes the synthesis of colloidal silver nanoparticles using Bombyx mori silk fibroin under white light environment at room temperature. The bio reduction of silver ions showed the unique surface plasmon resonance (SPR) band at 420 nm which was confirmed by UV-visible spectroscopy. Transmission electron microscopy (TEM) showed the synthesized AgNPs are spherical in shape with the average particle size of 35-40 nm. X-ray diffraction (XRD) pattren evidenced the crystalline nature of the AgNPs with FCC structure. The biosynthesized AgNPs showed effective antibacterial activity against bacterial stains Bacillus subtilis, and Salmonella typhi.