HRP-mediated graft polymerization of acrylic acid onto silk fibroins and in situ biomimetic mineralization.
Zhou Buguang,Zhou Qian,Wang Ping,Yuan Jiugang,Yu Yuanyuan,Deng Chao,Wang Qiang,Fan Xuerong
Journal of materials science. Materials in medicine
Silk fibroin (SF) can be extensively utilized in biomedical areas owing to its appreciable bioactivity. In this study, biocompatible composites of SF and hydroxyapatite (HAp) were fabricated through in situ biomimetic mineralization process. Graft copolymerization of acrylic acid (AA) onto SF was conducted by using the catalytic system of acetylacetone (ACAC), hydrogen peroxide (HO) and horseradish peroxidase (HRP), for enhancing the deposition of apatite onto the fibroin chains. Subsequently, biomimetic mineralization of the prepared fibroin-based membrane was performed in Ca/P solutions to synthesize the organized SF/HAp composites. The efficacies of graft copolymerization and biomimetic mineralization were evaluated by means of ATR-FTIR, GPC, EDS-Mapping, XRD and others. The results denoted that AA was successfully graft-copolymerized with fibroin and formed the copolymer of silk fibroin-graft-polyacrylic acid (SF-g-PAA), and the grafting percentage (GP) and grafting efficiency (GE) under the optimal condition reached to 23.2% and 29.4%, respectively. More mineral phases were detected on the surface of SF-g-PAA membrane after mineralization process when compared to that of the untreated fibroin membrane, companying with an improved mechanical property. According to MG-63 cell viability and fluorescent adhesion assays, the mineralized SF-g-PAA composite showed satisfactory biocompatibility and exceptional adhesive effects as well. The synthetized composite of SF-g-PAA/HAp can be potentially applied in the fields of bone tissue engineering.
3D Architectures of Co P Using Silk Fibroin Scaffolds: An Active and Stable Electrocatalyst for Hydrogen Generation in Acidic and Alkaline Media.
Kim Taek-Seung,Song Hee Jo,Kim Jae-Chan,Ju Bobae,Kim Dong-Wan
Small (Weinheim an der Bergstrasse, Germany)
Developing nonprecious, highly active, and stable catalysts is essential for efficient electrocatalytic hydrogen evolution reaction in water splitting. In this study, the facile synthesis of a 3D flower-like Co P/carbon architecture is proposed composed of an assembly of nanosheets interconnected by silk fibroin that acts as 3D scaffolds and a carbon source. This unique 3D architecture coupled with a carbon matrix enhances catalytic activity by exposing more active sites and increasing charge transport. The flower-like Co P/carbon can facilitate a lower overpotential, Tafel slope, charge transfer resistance, and a higher electrochemically active surface than carbon-free and silk-free Co P. The nanostructured architecture exhibits excellent catalytic performance with low overpotentials of 109 and 121 mV at 10 mA cm and Tafel slopes of 55 and 62 mV dec in acidic and alkaline media, respectively. Furthermore, it minimally degrades the overpotential and current density after long-term stability tests 10 000 cyclic voltammetry cycles and a chronoamperometric test over 40 h, respectively, in acidic media, which confirms the high durability and stability of the flower-like Co P/carbon.
Dual-layer aligned-random nanofibrous scaffolds for improving gradient microstructure of tendon-to-bone healing in a rabbit extra-articular model.
Cai Jiangyu,Wang Juan,Ye Kaiqiang,Li Dandan,Ai Chengchong,Sheng Dandan,Jin Wenhe,Liu Xingwang,Zhi Yunlong,Jiang Jia,Chen Jun,Mo Xiumei,Chen Shiyi
International journal of nanomedicine
BACKGROUND:Tendon/ligament injuries are common sports injuries. Clinically, the repair of a ruptured tendon or ligament to its bony insertion is needed, but the enthesis structure is not well reestablished following surgical repair. Herein, we fabricated dual-layer aligned-random scaffold (ARS) by electrospinning and aimed to investigate the effect of the scaffold on tendon-to-bone healing in vivo. MATERIALS AND METHODS:The random and dual-layer aligned-random silk fbroin poly(L-lactic acid-co-e-caprolactone) (P(LLA-CL)) nanofibrous scaffolds were successfully fabricated by electrospinning methods. Ninety New Zealand white rabbits were randomly divided into three groups (random scaffold [RS], ARS, and control groups), and they were subjected to surgery to establish an extra-articular tendon-to-bone healing model with autologous Achilles tendon. RESULTS:Histological assessment showed that the ARS significantly increased the area of metachromasia, decreased the interface width, and improved collagen maturation and organization at the tendon-bone interface compared with the RS and control groups. Microcomputed tomography analysis showed that the bone tunnel area of RS and ARS groups was significantly smaller than those of the control group. Real-time polymerase chain reaction showed that BMP-2 and osteopontin expression levels of the tissue at the interface between the bone and graft in the RS and ARS groups were higher than those of the control group at 6 weeks. Collagen I expression level of the ARS group was significantly higher than those of the RS and control groups at 6 and 12 weeks. Moreover, the ARS groups had a better ultimate load-to-failure and stiffness than the RS and control groups. CONCLUSION:ARS could effectively augment the tendon-to-bone integration and improve gradient microstructure in a rabbit extra-articular model by inducing the new bone formation, increasing the area of fibrocartilage, and improving collagen organization and maturation. The dual-layer aligned-random silk fibroin/P(LLA-CL) nanofibrous scaffold is proved to be a promising biomaterial for tendon-to-bone healing.
Electrospun silk fibroin fibers for storage and controlled release of human platelet lysate.
Pignatelli Cataldo,Perotto Giovanni,Nardini Marta,Cancedda Ranieri,Mastrogiacomo Maddalena,Athanassiou Athanassia
Human platelet lysate (hPL) is a pool of growth factors and cytokines able to induce regeneration of different tissues. Despite its good potentiality as therapeutic tool for regenerative medicine applications, hPL has been only moderately exploited in this field. A more widespread adoption has been limited because of its rapid degradation at room temperature that decreases its functionality. Another limiting factor for its extensive use is the difficulty of handling the hPL gels. In this work, silk fibroin-based patches were developed to address several points: improving the handling of hPL, enabling their delivery in a controlled manner and facilitating their storage by creating a device ready to use with expanded shelf life. Patches of fibroin loaded with hPL were synthesized by electrospinning to take advantage of the fibrous morphology. The release kinetics of the material was characterized and tuned through the control of fibroin crystallinity. Cell viability assays, performed with primary human dermal fibroblasts, demonstrated that fibroin is able to preserve the hPL biological activity and prolong its shelf-life. The strategy of storing and preserving small active molecules within a naturally-derived, protein-based fibrous scaffold was successfully implemented, leading to the design of a biocompatible device, which can potentially simplify the storage and the application of the hPL on a human patient, undergoing medical procedures such as surgery and wound care. STATEMENT OF SIGNIFICANCE:Human platelets lysate (hPL) is a mixture of growth factors and cytokines able to induce the regeneration of damaged tissues. This study aims at enclosing hPL in a silk fibroin electrospun matrix to expand its utilization. Silk fibroin showed the ability to preserve the hPL activity at temperature up to 60 °C and the manipulation of fibroin's crystallinity provided a tool to modulate the hPL release kinetic. This entails the possibility to fabricate the hPL silk fibroin patches in advance and store them, resulting in an easy and fast accessibility and an expanded use of hPL for wound healing.
Protein-Based Electronic Skin Akin to Biological Tissues.
Jo Minsik,Min Kyungtaek,Roy Biswajit,Kim Sookyoung,Lee Sangmin,Park Ji-Yong,Kim Sunghwan
Human skin provides an interface that transduces external stimuli into electrical signals for communication with the brain. There has been considerable effort to produce soft, flexible, and stretchable electronic skin (E-skin) devices. However, common polymers cannot imitate human skin perfectly due to their poor biocompatibility, biofunctionality, and permeability to many chemicals and biomolecules. Herein, we report on highly flexible, stretchable, conformal, molecule-permeable, and skin-adhering E-skins that combine a metallic nanowire (NW) network and silk protein hydrogel. The silk protein hydrogels offer high stretchability and stability under hydration through the addition of Ca ions and glycerol. The NW electrodes exhibit stable operation when subjected to large deformations and hydration. Meanwhile, the hydrogel window provides water and biomolecules to the electrodes (communication between the environment and the electrode). These favorable characteristics allow the E-skin to be capable of sensing strain, electrochemical, and electrophysiological signals.
Silk nanofibril self-assembly versus electrospinning.
Humenik Martin,Lang Gregor,Scheibel Thomas
Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology
Natural silk fibers represent one of the most advanced blueprints for (bio)polymer scientists, displaying highly optimized mechanical properties due to their hierarchical structures. Biotechnological production of silk proteins and implementation of advanced processing methods enabled harnessing the potential of these biopolymer not just based on the mechanical properties. In addition to fibers, diverse morphologies can be produced, such as nonwoven meshes, films, hydrogels, foams, capsules and particles. Among them, nanoscale fibrils and fibers are particularly interesting concerning medical and technical applications due to their biocompatibility, environmental and mechanical robustness as well as high surface-to-volume ratio. Therefore, we introduce here self-assembly of silk proteins into hierarchically organized structures such as supramolecular nanofibrils and fabricated materials based thereon. As an alternative to self-assembly, we also present electrospinning a technique to produce nanofibers and nanofibrous mats. Accordingly, we introduce a broad range of silk-based dopes, used in self-assembly and electrospinning: natural silk proteins originating from natural spinning glands, natural silk protein solutions reconstituted from fibers, engineered recombinant silk proteins designed from natural blueprints, genetic fusions of recombinant silk proteins with other structural or functional peptides and moieties, as well as hybrids of recombinant silk proteins chemically conjugated with nonproteinaceous biotic or abiotic molecules. We highlight the advantages but also point out drawbacks of each particular production route. The scope includes studies of the natural self-assembly mechanism during natural silk spinning, production of silk fibrils as new nanostructured non-native scaffolds allowing dynamic morphological switches, as well as studying potential applications. This article is categorized under: Biology-Inspired Nanomaterials > Peptide-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.
Nanofibrous silk fibroin/reduced graphene oxide scaffolds for tissue engineering and cell culture applications.
Nalvuran Hande,Elçin Ayşe Eser,Elçin Yaşar Murat
International journal of biological macromolecules
Graphene and silk fibroin (SF) have been extensively investigated in the literature. Hybrid scaffolds of SF and graphene combine the properties of both of the materials and provide promising applications for tissue engineering purposes. In this study, reduced graphene oxide (RGO) (0.5%, 1.0% and 2.0% (w/v)) was incorporated into SF and fabricated into composite nanofibers through electrospinning. The fibers were characterized and analyzed by SEM, XRD, FTIR, TGA, circular dichroism analysis, contact angle measurements and tensile tests. Here, we document that the presence of RGO increases intermolecular forces between RGO and SF molecular chains in the SF matrix, which results in an increased silk II content. Upon the incorporation of RGO, thermal stability and mechanical properties of the fibers significantly improved. Furthermore, in-vitro findings showed that composite nanofibers supported cell viability and were hemocompatible. Finally, bone marrow mesenchymal stem cells were induced osteogenically on electrospun SF/RGO mats for 30days, which showed that the substrate supported osteogenic differentiation. In this study, a feasible method is proposed to generate biocompatible and versatile SF/RGO-composite nanofibers that can influence biomedical applications.
Iontophoresis-stimulated silk fibroin films as a peptide delivery system for wound healing.
Lemos Camila Nunes,Cubayachi Camila,Dias Karina,Mendonça Jacqueline Nakau,Lopes Norberto Peporine,Furtado Niege Araçari Jacometti Cardoso,Lopez Renata F V
European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V
Silk fibroin (SF) films containing a peptide, neurotensin (NT), stimulated by iontophoresis were developed aiming to modulate the inflammatory process and prevent the growth of microorganisms typical of wounds. NT-loaded SF films composition shows predominance of β-sheet structures that conferred adequate mechanical properties, transparency, moderate roughness and low swelling index to fibroin films. Infrared spectroscopy and thermal analysis suggested the presence of non-covalent interactions between NT and fibroin. Using the MALDI imaging technique, it was possible to visualize the homogeneous NT distribution throughout the film surface, in addition to its prolonged release for up to 72 h. In vitro studies in E. coli liposaccharide-stimulated macrophages showed a significant reduction of interleukins production after NT-loaded film application, whereas NT solution did not reduce them. Bi-laminated NT-loaded fibroin films containing silver electrodes provided a burst release of NT when anodic iontophoresis was applied, enabling a rapid onset of drug action. In addition, anodic iontophoresis presented a bacteriostatic effect against gram-positive microorganisms. Different iontophoresis densities, from 0.2 to 0.6 mA/cm, did not significantly reduce fibroblast viability after 30 min of application. In conclusion, iontophoretic-stimulated peptide-loaded fibroin films could be a promising platform for the treatment of wounds.
Silk protein nanowires patterned using electron beam lithography.
Pal Ramendra K,Yadavalli Vamsi K
Nanofabrication approaches to pattern proteins at the nanoscale are useful in applications ranging from organic bioelectronics to cellular engineering. Specifically, functional materials based on natural polymers offer sustainable and environment-friendly substitutes to synthetic polymers. Silk proteins (fibroin and sericin) have emerged as an important class of biomaterials for next generation applications owing to excellent optical and mechanical properties, inherent biocompatibility, and biodegradability. However, the ability to precisely control their spatial positioning at the nanoscale via high throughput tools continues to remain a challenge. In this study electron beam lithography (EBL) is used to provide nanoscale patterning using methacrylate conjugated silk proteins that are photoreactive 'photoresists' materials. Very low energy electron beam radiation can be used to pattern silk proteins at the nanoscale and over large areas, whereby such nanostructure fabrication can be performed without specialized EBL tools. Significantly, using conducting polymers in conjunction with these silk proteins, the formation of protein nanowires down to 100 nm is shown. These wires can be easily degraded using enzymatic degradation. Thus, proteins can be precisely and scalably patterned and doped with conducting polymers and enzymes to form degradable, organic bioelectronic devices.
Overview of Silk Fibroin Use in Wound Dressings.
Farokhi Mehdi,Mottaghitalab Fatemeh,Fatahi Yousef,Khademhosseini Ali,Kaplan David L
Trends in biotechnology
Recently, biomimetic wound dressings were introduced as potential replacements for treating skin injuries. Although there are some clinically available skin replacements, the range of wound types and locations necessitates a broader range of options for the clinic. Natural polymeric-based dressings are of central interest in this area due to their outstanding biocompatibility, biodegradability, low toxicity, and non-allergenic nature. Among them, silk fibroin (SF) has exceptional characteristics as a wound dressing. SF-based dressings can also be used as carriers for delivering drugs, growth factors, and bioactive agents to the wound area, while providing appropriate support for complete healing. In this review, we describe recent advances in the development of SF-based wound dressings for skin regeneration.
In vitro and in vivo evaluation of etoposide - silk wafers for neuroblastoma treatment.
Yavuz Burcin,Zeki Jasmine,Coburn Jeannine M,Ikegaki Naohiko,Levitin Daniel,Kaplan David L,Chiu Bill
Journal of controlled release : official journal of the Controlled Release Society
High-risk neuroblastoma requires surgical resection and multi-drug chemotherapy. This study aimed to develop an extended release, implantable and degradable delivery system for etoposide, commonly used for neuroblastoma treatment. Different concentrations of silk, a biodegradable, non-toxic, non-immunogenic material were employed to prepare etoposide-loaded wafer formulations. Secondary structure of silk in the formulations was characterized using Fourier Transform Infrared (FTIR) spectroscopy and optimized based on the crystalline structure. Accelerated in vitro degradation studies under different conditions such as acidic, alkaline, oxidizing mediums and high temperature, were performed. The integrity of the silk wafer structure was maintained unless exposed to 0.1 N NaOH for 24 h. In vitro release of etoposide was performed in PBS (phosphate buffered saline) at 37 °C. Silk coated 6% wafers released the drug up to 45 days, while uncoated wafers released the drug for 30 days. Cytotoxicity study was performed on KELLY cells to evaluate the etoposide cytotoxicity (LC50) and the long-term efficacy of the etoposide wafer formulations. The results showed that etoposide killed 50% of the cells at 1 μg/mL concentration and the wafer formulations demonstrated significant cytotoxicity up to 22 days when compared to untreated cells. Using an orthotopic neuroblastoma mouse model, intra-tumoral implantation of the coated 6%, uncoated 6%, or uncoated 3% silk wafers were all effective at decreasing tumor growth. Histological examination revealed tumor cell necrosis adjacent to the drug-loaded silk wafer.
In vitro studies on space-conforming self-assembling silk hydrogels as a mesenchymal stem cell-support matrix suitable for minimally invasive brain application.
Osama I,Gorenkova N,McKittrick C M,Wongpinyochit T,Goudie A,Seib F P,Carswell H V O
Advanced cell therapies require robust delivery materials and silk is a promising contender with a long clinical track record. Our aim was to optimise self-assembling silk hydrogels as a mesenchymal stem cell (MSC)-support matrix that would allow future minimally invasive brain application. We used sonication energy to programme the transition of silk (1-5% w/v) secondary structure from a random coil to a stable β-sheet configuration. This allowed fine tuning of self-assembling silk hydrogels to achieve space conformity in the absence of any silk hydrogel swelling and to support uniform cell distribution as well as cell viability. Embedded cells underwent significant proliferation over 14 days in vitro, with the best proliferation achieved with 2% w/v hydrogels. Embedded MSCs showed significantly better viability in vitro after injection through a 30G needle when the gels were in the pre-gelled versus post-gelled state. Silk hydrogels (4% w/v) with physical characteristics matching brain tissue were visualised in preliminary in vivo experiments to exhibit good space conformity in an ischemic cavity (intraluminal thread middle cerebral artery occlusion model) in adult male Sprague-Dawley rats (n = 3). This study informs on optimal MSC-hydrogel matrix conditions for minimally invasive application as a platform for future experiments targeting brain repair.
Soft freezing-induced self-assembly of silk fibroin for tunable gelation.
Li Xiufang,Yan Shuqin,Qu Jing,Li Mingzhong,Ye Dezhan,You Renchuan,Zhang Qiang,Wang Dong
International journal of biological macromolecules
Silk fibroin (SF) hydrogel is a promising candidate in biomaterial field; however its application is quite limited by long-gelation time. In the present study, we developed a novel strategy named soft freezing to accelerate the process and control the sol-gel transition of SF protein. SF protein was induced to self-assembly by soft freezing process for achieving the reconstructed SF solution with metastable structure. It was found that the soft freezing process triggers the structural transition from random structure to ordered structure-rich conformation. Gelation kinetics showed that the gelation time of SF protein could be regulated by changing freezing time and initial concentration. The reconstructed SF solution allowed enhanced sol-gel transition within 6 hours, even at extremely low concentration. The attractive features of the method described here include the accelerated gelation, free of chemical agents, and reducing processing complexity. The SF solution with short gelation time will be applicable as cell encapsulation and injectable applications for tissue engineering and regenerative medicine, which greatly expand the applications of SF hydrogels.
Endowing polyetheretherketone with synergistic bactericidal effects and improved osteogenic ability.
Yan Jianglong,Zhou Wenhao,Jia Zhaojun,Xiong Pan,Li Yangyang,Wang Pei,Li Qiyao,Cheng Yan,Zheng Yufeng
Biomedical associated infections (BAI) are difficult to treat and may even lead to amputation and death, especially after the emergence of drug-resistant bacteria. The aim of this study was to harness the potential synergistic effects of multiple bactericidal agents to endow polyetheretherketone (PEEK) with the ability of achieving full eradication of planktonic and adherent bacteria while maintaining acceptable biocompatibility. In this work, a mussel inspired, silver nanoparticles (AgNPs) incorporated silk fibroin (SF)/gentamicin sulfate (GS) coating was constructed upon porous PEEK surface. The obtained coating greatly enhanced the bactericidal efficiency to Gram-positive bacteria and Gram-negative bacteria. The number of bacteria survived in the culture medium after treated with this coating was 10 fold lower than that survived after treated with PEEK sample, while the number of viable bacteria adhered to this coating was 10 lower than that adhered to PEEK sample. Furthermore, release of Ag and GS increased with decreasing pH, indicating great potential of this coating to be a "smart" bacteria-triggered self-defensive coating. Meanwhile, this functional coating shows favorable cytocompatibility and osteogenic ability. The mechanism behind this dual function is also partially revealed. Expectedly, this "smart" dual function coating can give a promise for PEEK to become a solution to increasingly deteriorated BAI. STATEMENT OF SIGNIFICANCE:In this study, a mussel inspired, silver nanoparticles (AgNPs) incorporated silk fibroin (SF)/gentamicin sulfate (GS) coating was constructed upon porous polyetheretherketone (PEEK) surface. This design was aimed to provide a solution to the increasingly deteriorated biomedical associated infections (BAI). Actually, this design endowed PEEK with dual function: bacteria-triggered synergistic bactericidal effect and improved osteogenic ability. The combination of silver and GS exhibited synergistic bacteria killing effect on both Gram-positive and Gram-negative bacteria, which showed 10 times higher in releasing-killing and 10 times higher in anti-adhesion than that of untreated PEEK. Furthermore, release of bactericidal agents increased with decreasing pH, indicating great potential of this coating to be a bacteria-triggered self-defensive coating. More interestingly, this study revealed the mechanism of synergistic effect between silver and GS.
Sericin nanomicelles with enhanced cellular uptake and pH-triggered release of doxorubicin reverse cancer drug resistance.
Guo Weihong,Deng Lizhi,Yu Jiang,Chen Zhaoyu,Woo Yanghee,Liu Hao,Li Tuanjie,Lin Tian,Chen Hao,Zhao Mingli,Zhang Liming,Li Guoxin,Hu Yanfeng
Drug resistance is the major challenge facing cancer chemotherapy and nanoscale delivery systems based on natural materials, such as sericin, are a promising means of overcoming drug resistance. Yet, no attempt of introducing synthetic poly(γ-benzyl-L-glutamate) (PBLG) onto sericin polypeptide to fabricate a facile biocompatible and biodegradable micelle has been tried. Here, we prepared a polypeptide-based amphiphilic polymer containing hydrophilic sericin polypeptide backbone and PBLG side chains via ring-opening polymerization (ROP) strategy. The introduction of PBLG side chains remarkably enhances the stability of sericin micelles in water. Meanwhile, the micelles exhibited a high loading capacity and pH-responsive release ability for antitumor drug doxorubicin (DOX), called sericin-PBLG-DOX. Owing to the excellent cell membrane penetration of sericin-PBLG, the cellular uptake of DOX when loaded into micelles was improved. Subsequently, sericin-PBLG-DOX was transferred into perinuclear lysosomes, where the release rate of DOX was accelerated. Compared to the same dose of DOX, sericin-PBLG-DOX could induce a more efficient anti-tumor effect both in vitro and in vivo, and these micelles have promise for future clinical applications in overcoming cancer drug resistance with good biosafety, enhanced cellular uptake, pH-triggered drug release, efficient anti-tumor effects, and minimized systemic toxicity.
Functionalization of silk fibroin through anionic fibroin derived polypeptides.
Griffanti Gabriele,James-Bhasin Mark,Donelli Ilaria,Freddi Giuliano,Nazhat Showan N
Biomedical materials (Bristol, England)
While silk fibroin (SF)-based fibrous matrices are often considered as templates to mimic the native biomineralization process, their limited ability to induce apatite deposition hinders their potential applications in bone tissue engineering. In this study, it was hypothesized that the incorporation of anionic fibroin derived polypeptides (Cs), generated through the α-chymotrypsin digestion of SF, into SF would induce apatite deposition. The effect of Cs incorporation and content on the mineralization of fibrous, electrospun (ES) SF matrices, was assessed in simulated body fluid (SBF). Moreover, the potential role of Cs in mediating the proliferation and osteoblastic differentiation of seeded mesenchymal stem cells (MSCs), in vitro, was also investigated. Methylene blue staining indicated that the ES SF matrices became increasingly negatively charged with an increase in Cs content. Furthermore, the mechanical properties of the ES SF matrices were modulated through variations in Cs content. Their subsequent immersion in SBF demonstrated rapid mineralization, attributable to the carboxyl groups provided by the negatively charged Cs polypeptides, which served as nucleation sites for apatite deposition. Seeded MSCs attached on all scaffold types with differences observed in metabolic activities when cultured in osteogenic medium. Relative to basal medium, there was an up-regulation of alkaline phosphatase, runt related transcription factor 2 and osteocalcin in osteogenic medium (at days 14 and 21). Cell-induced mineralized matrix deposition appeared to be accelerated on Cs incorporated ES SF suggesting an osteoinductive potential of these polypeptides. In sum, the ability to incorporate Cs into SF scaffolds offers promise in bone tissue engineering applications.
Reinforcement of calcium phosphate cement using alkaline-treated silk fibroin.
Hu Muli,He Zhiwei,Han Fengxuan,Shi Chen,Zhou Pinghui,Ling Feng,Zhu Xuesong,Yang Huilin,Li Bin
International journal of nanomedicine
BACKGROUND:Bone cement plays an important role in the treatment of osteoporotic vertebral compression fractures. Calcium phosphate cement (CPC) is a potential alternative to poly(methyl methacrylate), currently the gold standard of bone cements. However, the poor mechanical properties of CPCs limit their clinical applications. The objective of this study was to develop reinforced CPCs for minimally invasive orthopedic surgeries by compositing silk fibroin (SF) with α-tricalcium phosphate. METHODS:SF solution was treated with calcium hydroxide and characterized by Zeta potential analyzer and Fourier transform infrared spectroscopy. The alkaline-treated SF (tSF) was com-posited with α-tricalcium phosphate to obtain tSF/CPC composite, which was characterized using mechanical tests, scanning electron microscopy, handling property and biocompatibility tests, and sheep vertebral augmentation tests. RESULTS:Upon treatment with calcium hydroxide, larger SF particles and more abundant negative charge appeared in tSF solution. The tSF/CPCs exhibited a compact structure, which consisted of numerous SF -CPC clusters and needle-like hydroxyapatite (HAp) crystals. In addition, high transition rate of HAp in tSF/CPCs was achieved. As a result, the mechanical property of tSF/ CPC composite cements was enhanced remarkably, with the compressive strength reaching as high as 56.3±1.1 MPa. Moreover, the tSF/CPC cements showed good injectability, anti-washout property, and decent biocompatibility. The tSF/CPCs could be used to augment defected sheep vertebrae to restore their mechanical strength. CONCLUSION:tSF/CPC may be a promising composite bone cement for minimally invasive orthopedic surgeries.
Enhancing neural differentiation of induced pluripotent stem cells by conductive graphene/silk fibroin films.
Niu Yimeng,Chen Xiaofang,Yao Danyu,Peng Ge,Liu Haifeng,Fan Yubo
Journal of biomedical materials research. Part A
Nerve regeneration and function recovery remain challenges for tissue engineering. The application of suitable scaffold in tissue engineering has been demonstrated to be able to enhance nerve regeneration and differentiation. However, a desired scaffold must meet the requirements of good cytocompatibility and high electrical conductivity simultaneously. In this study, a conductive film composed of SF and graphene was successfully fabricated, which was applied to evaluate its effect on the neural differentiation of iPSCs. The conductive film was found to enhance the differentiation of iPSCs toward neurons. In addition, the differentiation was enhanced with graphene contents and highest value was obtained at graphene content of 4%. Thus, the results in this study suggested that 4% G/SF film might be a suitable biomaterial scaffold for application in neural regeneration. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2973-2983, 2018.
Fabrication of Air-Stable and Conductive Silk Fibroin Gels.
Yao Meng,Su Dihan,Wang Wenqi,Chen Xin,Shao Zhengzhong
ACS applied materials & interfaces
Owing to their promising applications in flexible electronics, researchers have extensively explored flexible and conductive gels. However, these gels have unsatisfactory strength and flexibility as well as easily dry in air. Herein, a rationally designed robust regenerated silk fibroin (RSF)-based gel with significant flexibility and strength, favorable conductivity, and excellent air stability is fabricated by inducing the conformation transition of RSF from random coil to β-sheet in ionic liquid (IL)/water mixtures. We found that such RSF-based gels have a unique homogeneous network structure of RSF nanofibers, which is likely formed because of evenly distributed cross-links dominated by small-sized β-sheet domains created during the conformation transition of RSF. Although the unique homogeneous nanostructure/network contributes toward improving the mechanical properties of these gels, it also provides pathways for ionic transport to help the gels preserve high conductivity of ILs. The prepared RSF-based gels display a remarkable air stability and reversible loss/absorption water capability in a wide humidity range environment primarily because of the distinguished combination of the IL and water. Therefore, the novel RSF-based gels hold a great potential in various applications as multifunctional, flexible, conductive materials, which are dispensed with encapsulation.
Composite Silk-Extracellular Matrix Scaffolds for Enhanced Chondrogenesis of Mesenchymal Stem Cells.
Zhang Xiao,Zhai Chenjun,Fei Hao,Liu Yang,Wang Zhen,Luo Chunyang,Zhang Jiyong,Ding Yanzi,Xu Tao,Fan Weimin
Tissue engineering. Part C, Methods
IMPACT STATEMENT:This study presented a new method to fabricate SF-ECM scaffolds that potentially promote chondrogenesis of BMSCs, and open up new possibilities for using SF-ECM scaffolds as an off-the-shelf strategy for joint cartilage regeneration. It is worthy of further investigation in knee joints of animals, and beyond knee cartilage, this scaffold may also serve as an ideal biomaterial for the regeneration of other joint cartilages.
Conductive Silk-Polypyrrole Composite Scaffolds with Bioinspired Nanotopographic Cues for Cardiac Tissue Engineering.
Tsui Jonathan H,Ostrovsky-Snider Nicholas A,Yama David M P,Donohue Jordan D,Choi Jong Seob,Chavanachat Rakchanok,Larson Jesse D,Murphy Amanda R,Kim Deok-Ho
Journal of materials chemistry. B
We report on the development of bioinspired cardiac scaffolds made from electroconductive acid-modified silk fibroin-poly(pyrrole) (AMSF+PPy) substrates patterned with nanoscale ridges and grooves reminiscent of native myocardial extracellular matrix (ECM) topography to enhance the structural and functional properties of cultured human pluripotent stem cells (hPSC)-derived cardiomyocytes. Nanopattern fidelity was maintained throughout the fabrication and functionalization processes, and no loss in conductive behavior occurred due to the presence of the nanotopographical features. AMSF+PPy substrates were biocompatible and stable, maintaining high cell viability over a 21-day culture period while displaying no signs of PPy delamination. The presence of anisotropic topographical cues led to increased cellular organization and sarcomere development, and electroconductive cues promoted a significant improvement in the expression and polarization of connexin 43 (Cx43), a critical regulator of cell-cell electrical coupling. The combination of biomimetic topography and electroconductivity also increased the expression of genes that encode key proteins involved in regulating the contractile and electrophysiological function of mature human cardiac tissue.
Fabrication and characterization of silk microfiber-reinforced methacrylated gelatin hydrogel with turnable properties.
Xiao Wenqian,Tan Yunfei,Li Jiale,Gu ChengFu,Li Hang,Li Bo,Liao Xiaoling
Journal of biomaterials science. Polymer edition
Despite considerable research effort, the natural hydrogels presently available for tissue engineering suffer from several major drawbacks, one of the significant issue is their poor mechanical strength which are unable to satisfy some mechanical requirements for successful outcomes. Herein, to mimic the composition and structure of the natural extracellular matrix, the micron-sized silk fibers obtained by alkaline hydrolysis were used as a reinforcement phase in a GelMA hydrogel, resulting in a material with significantly greater stiffness than pure GelMA hydrogel alone. In addition, the hydrogel demonstrated tunable compressive strength, swelling capacity, and degradation properties based on the silk fiber length. Experiments with cells indicated that MC3T3-E1 pre-osteoblasts quickly adhered to and proliferated on the surface of the composite hydrogels, as revealed by FDA/PI staining and CCK-8 assays. In addition, various cellular responses, including cell adhesion, changes in cellular morphology and cell proliferation behavior, occurred on the composite hydrogel and varied with fiber length. Overall, this study introduces a series of fiber-reinforced, tunable composite hydrogels that could be useful for various tissue engineering applications.
Enhanced cellular response elicited by addition of amniotic fluid to alginate hydrogel-electrospun silk fibroin fibers for potential wound dressing application.
Ghalei Sama,Nourmohammadi Jhamak,Solouk Atefeh,Mirzadeh Hamid
Colloids and surfaces. B, Biointerfaces
This study aimed to evaluate a novel bioactive wound dressing from alginate hydrogel-electrospun silk fibroin (SF) fibers with the ability to deliver amniotic fluid (AF) to the wound site. AF is highly enriched with multiple therapeutic agents that can promote cellular response and wound healing. In this study, electrospun SF fibers were first fabricated by electrospinning method and then combined with the alginate hydrogel (ALG) containing AF. Different dressings were made by changing the alginate to AF ratio. The scanning electron microscopy images revealed that SF fibers were fully covered with alginate hydrogel. In addition, the modulus of the dressing decreased by lowering ALG/AF ratios, while a reverse trend was observed for the elongation-at-break. In vitro AF release profiles indicated that an increase in the concentration of ALG leads to a reduction in the AF amount. Fibroblast culturing on the fabricated dressings demonstrated that cellular proliferation, spreading, and secretion of collagen enhanced with increasing AF. Taken together, our results provide a novel bioactive dressing with great potentials for speeding up the healing process in severe wounds.
Cross-linking of dialdehyde carboxymethyl cellulose with silk sericin to reinforce sericin film for potential biomedical application.
Wang Peng,He Huawei,Cai Rui,Tao Gang,Yang Meirong,Zuo Hua,Umar Ahmad,Wang Yejing
Developing biomaterials based on the natural biomacromolecule silk sericin from Bombyx mori cocoon is of great interest for biomedical application. Dialdehyde carboxymethyl cellulose (DCMC) is derived from periodate oxidation of carboxy- methyl cellulose. Here, we developed a novel strategy of cross-linking of sericin with DCMC via the Schiff's base reaction. Fourier transform infrared spectroscopy and scanning electron microscopy indicated the formation of Schiff's base via the blending of sericin and DCMC. The mechanical properties tests suggested the covalent cross-linking effectively enhanced the tensile strength of sericin. The swelling test and water contact angle indicated the DCMC/SS film had excellent hydrophilicity, swellability. Additionally, we demonstrated the DCMC/SS film had excellent blood compatibility, cytocompatibility and promoting cell proliferation activity by the hemolysis ratio analysis, cell adhesion, cells viability and proliferation assays. The prepared DCMC/SS film has shown great promise in biomedical applications such as wound dressing, artificial skin and tissue engineering.
Silk scaffolds connected with different naturally occurring biomaterials for prostate cancer cell cultivation in 3D.
Bäcker Anne,Erhardt Olga,Wietbrock Lukas,Schel Natalia,Göppert Bettina,Dirschka Marian,Abaffy Paul,Sollich Thomas,Cecilia Angelica,Gruhl Friederike J
In the present work, different biopolymer blend scaffolds based on the silk protein fibroin from Bombyx mori (BM) were prepared via freeze-drying method. The chemical, structural, and mechanical properties of the three dimensional (3D) porous silk fibroin (SF) composite scaffolds of gelatin, collagen, and chitosan as well as SF from Antheraea pernyi (AP) and the recombinant spider silk protein spidroin (SSP1) have been systematically investigated, followed by cell culture experiments with epithelial prostate cancer cells (LNCaP) up to 14 days. Compared to the pure SF scaffold of BM, the blend scaffolds differ in porous morphology, elasticity, swelling behavior, and biochemical composition. The new composite scaffold with SSP1 showed an increased swelling degree and soft tissue like elastic properties. Whereas, in vitro cultivation of LNCaP cells demonstrated an increased growth behavior and spheroid formation within chitosan blended scaffolds based on its remarkable porosity, which supports nutrient supply matrix. Results of this study suggest that silk fibroin matrices are sufficient and certain SF composite scaffolds even improve 3D cell cultivation for prostate cancer research compared to matrices based on pure biomaterials or synthetic polymers.
Silkworms as a factory of functional wearable energy storage fabrics.
Ali Basant A,Allam Nageh K
Feeding Bombyx mori larvae with chemically-modified diets affects the structure and properties of the resulted silk. Herein, we provide a road map for the use of silkworms as a factory to produce semiconducting/metallic natural silk that can be used in many technological applications such as supercapacitor electrodes. The silkworms were fed with four different types of chemicals; carbon material (graphite), sulfide (MoS), oxide (TiO nanotubes), and a mixture of reactive chemicals (KMnO/MnCl). All the fed materials were successfully integrated into the resulted silk. The capacitive performance of the resulted silk was evaluated as self-standing fabric electrodes as well as on glassy carbon substrates. The self-standing silk and the silk@glassy carbon substrate showed a great enhancement in the capacitive performance over that of the unmodified counterparts. The specific capacitance of the self-standing blank silk negative and positive electrodes was enhanced 4 and 5 folds at 10 mV/s, respectively upon the modification with KMnO/MnCl compared to that of the plain silk electrodes.
Mimicking Hierarchical Complexity of the Osteochondral Interface Using Electrospun Silk-Bioactive Glass Composites.
M Joseph Christakiran,Reardon Philip J T,Konwarh Rocktotpal,Knowles Jonathan C,Mandal Biman B
ACS applied materials & interfaces
The anatomical complexity and slow regeneration capacity of hyaline cartilage at the osteochondral interface pose a great challenge in the repair of osteochondral defects (OCD). In this study, we utilized the processing feasibility offered by the sol derived 70S bioactive glass and silk fibroin (mulberry Bombyx mori and endemic Indian non-mulberry Antheraea assama), in fabricating a well-integrated, biomimetic scaffolding matrix with a coherent interface. Differences in surface properties such as wettability and amorphousness between the two silk groups resulted in profound variations in cell attachment and extracellular matrix protein deposition. Mechanical assessment showed that the biphasic composites exhibited both an elastic region pertinent for cartilage tissue and a stiff compression resistant region simulating the bone phase. In vitro biological studies revealed that the biphasic mats presented spatial confinement for the growth and maturation of both osteoblasts and chondrocytes, marked by increased alkaline phosphatase (ALP) activity, osteopontin (OPN), sulfated glycosaminoglycan (sGAG) and collagen secretion in the cocultured mats. The non-mulberry silk based biphasic composite mats performed better than their mulberry counterpart, as evidenced by enhanced expression levels of key cartilage and bone specific marker genes. Therefore, the developed biphasic scaffold show great promise for improving the current clinical strategies for osteochondral tissue repair.
Polymorphic regenerated silk fibers assembled through bioinspired spinning.
Ling Shengjie,Qin Zhao,Li Chunmei,Huang Wenwen,Kaplan David L,Buehler Markus J
A variety of artificial spinning methods have been applied to produce regenerated silk fibers; however, how to spin regenerated silk fibers that retain the advantages of natural silks in terms of structural hierarchy and mechanical properties remains challenging. Here, we show a bioinspired approach to spin regenerated silk fibers. First, we develop a nematic silk microfibril solution, highly viscous and stable, by partially dissolving silk fibers into microfibrils. This solution maintains the hierarchical structures in natural silks and serves as spinning dope. It is then spun into regenerated silk fibers by direct extrusion in the air, offering a useful route to generate polymorphic and hierarchical regenerated silk fibers with physical properties beyond natural fiber construction. The materials maintain the structural hierarchy and mechanical properties of natural silks, including a modulus of 11 ± 4 GPa, even higher than natural spider silk. It can further be functionalized with a conductive silk/carbon nanotube coating, responsive to changes in humidity and temperature.
Water mediated dielectric polarizability and electron charge transport properties of high resistance natural fibers.
Kumar Ankit,Jash Amit,Dubey Amarish,Bajpai Alok,Philip Deepu,Bhargava Kalpana,Singh Sushil K,Das Mainak,Banerjee S S
Recent studies showed that silk and human hair fibers develop thermoelectric properties at optimal water, temperature and light conditions. The nature of charge carriers and the role of water in mediating charge conduction in these fibers is an unexplored issue. By studying four different classes of natural fibers, viz., silk cocoon, human hair, jute and corn silk, we uncover their common electrical transport properties and its dependence on water concentration and temperature. All these fibers uniformly exhibit nonlinear, hysteretic current - voltage characteristics, which scale with water concentration. The optimal electrical conductivity shows thermally activated hopping transport mechanism. Scanning tunneling microscope (STM) and dielectric measurements of silk cocoon fibers showed the electronic density of states and dielectric properties of the hydrated medium enhances with water concentration. Electron paramagnetic resonance (EPR) study reveals that the charge carriers in these membranes are electronic in nature. Our results are explained through the mechanism of hopping of a Polaron, which is an electron surrounded by positive charge fluctuations created by water molecules. The mechanism unravels the peculiar role water plays in mediating electrical activity in these membranes and also opens the possibility for exploring such charge transport mechanism in other biological membranes.
Silk based scaffolds with immunomodulatory capacity: anti-inflammatory effects of nicotinic acid.
Zakeri Siavashani Abdollah,Mohammadi Javad,Maniura-Weber Katharina,Senturk Berna,Nourmohammadi Jhamak,Sadeghi Behnam,Huber Lukas,Rottmar Markus
Implantation of temporary and permanent biomaterials in the body leads to a foreign body reaction (FBR), which may adversely affect tissue repair processes and functional integration of the biomaterial. However, modulation of the inflammatory response towards biomaterials can potentially enable a favorable healing response associated with functional tissue formation and tissue regeneration. In this work, incorporation of nicotinic acid in 3D silk scaffolds is explored as an immunomodulatory strategy for implantable biomaterials. Silk scaffolds were fabricated from dissolved Bombyx mori silk fibers by freeze-drying, resulting in silk scaffolds with high porosity (>94%), well-connected macropores, a high swelling degree (>550%) and resistance to in vitro degradation. Furthermore, drug-loaded scaffolds displayed a sustained drug release and excellent cytocompatibility could be observed with osteoblast-like MG63 cells. Cultivating M1-like macrophages on the scaffolds revealed that scaffolds loaded with nicotinic acid suppress gene expression of pro-inflammatory markers TNF-α, CXCL10 and CD197 as well as secretion of TNF-α in a concentration dependant manner. Hence, this study provides insights into the possible application of nicotinic acid in tissue engineering to control inflammatory responses towards biomaterials and potentially help minimizing FBR.
Modifications of silk film for dual delivery.
Dhyani Vartika,Singh Neetu
Biomedical materials (Bristol, England)
Silk biomaterials can be designed to provide an architectural framework comparable to connate extracellular matrix in order to boost cell growth and eventual tissue regeneration. Silk (Bombyx mori) fibroins self-assemble into hydrophobic crystalline β sheets, which provide mechanical strength and tunable degradability. The next generation of tissue engineering scaffolds aim to provide spatially controlled modulation of cell adhesion and differentiation, which can be achieved by spatially controlled surface functionalization of the scaffolds. In this respect, it is even more important to be able to release molecules at timescales ranging from hours to days, as many biological processes require signals early on to initiate processes, and over prolonged periods to sustain them. Unfortunately, achieving spatio-temporal control over multiple release profiles from silk based substrates is challenging due to their intrinsic slow release behaviour. Here, we report a simple strategy that provides spatio-temporal control over the release of drugs from silk films (SFs). We have developed a UV based strategy to modify the SFs with nanogels, which can provide a fast as well as slow release profile from a single platform. We demonstrate that the release profile of encapsulated molecules on the SF substrate can be tuned from fast (within hours) to slow (within days), thus resulting in a dual release system, which can be eventually utilized to deliver bioactive molecules at specific regions with different rates to achieve the desired multiple biological effects.
The Wonders of Silk Fibroin Biomaterials in the Treatment of Breast Cancer.
Tulay Pinar,Galam Nanyak,Adali Terin
Critical reviews in eukaryotic gene expression
Breast cancer has continued to be a cause of increasing morbidity and mortality in women, being the most common cause of cancer-related deaths among them. Its management using chemotherapy is continually plagued with problems of systemic toxicity, lack of compliance by patients, and inadequate targeting of cancer cells. The future of breast cancer chemotherapy will likely involve the use of biocompatible high cell-targeting capacity drug-delivery vehicles like silk fibroin to ameliorate these problems. The utilization of silk fibroin nanoparticles to deliver cytotoxic drugs provides specificity, optimal entrapment, improved therapeutic index, and maximal breast cancer cell toxicity with minimal or no collateral damage to surrounding normal cells. The silk fibroin obtained from the cocoon of the Bombyx morii worm is processed and degummed to remove the sericin component; it is then made into nanoparticles utilizing the desolvation, ionic gelation, or electrospray method. It is then loaded with an appropriate chemotherapeutic drug (e.g., carboplatin), and characterization is performed using physico-chemical methods such as fourier-transform infrared spectroscopy, dynamic light scattering, or transmission electron microscopy. The nanoparticles are then tested for cytoxicity, and the induction of apoptosis on breast cancer cell lines MC-7 (Her2-) and MDA-MB-453 (Her2+). Toxicity and apoptosis are assessed using the MTT assay and ELISA methods, respectively. Silk fibroin has been demonstrated in various studies to be a very useful tool in specific active or passive drug delivery to target cancer cells, thus ensuring a maximum destruction and minimum damage to normal surrounding cells, which decreases systemic toxicity and enhances drug efficacy.
Visible light biophotosensors using biliverdin from Antheraea yamamai.
Leem Jung Woo,Allcca Andres E Llacsahuanga,Chen Junjie,Kim Seong-Wan,Kim Kee-Young,Choi Kwang-Ho,Chen Yong P,Kim Seong-Ryul,Kim Young L
We report an endogenous photoelectric biomolecule and demonstrate that such a biomolecule can be used to detect visible light. We identify the green pigment abundantly present in natural silk cocoons of Antheraea yamamai (Japanese oak silkmoth) as biliverdin, using mass spectroscopy and optical spectroscopy. Biliverdin extracted from the green silk cocoons generates photocurrent upon light illumination with distinct colors. We further characterize the basic performance, responsiveness, and stability of the biliverdin-based biophotosensors at a photovoltaic device level using blue, green, orange, and red light illumination. Biliverdin could potentially serve as an optoelectric biomolecule toward the development of next-generation implantable photosensors and artificial photoreceptors.
Comparison of the knitted silk vascular grafts coated with fibroin sponges prepared using glycerin, poly(ethylene glycol diglycidyl ether) and poly(ethylene glycol) as porogens.
Tanaka Takashi,Uemura Akiko,Tanaka Ryo,Tasei Yugo,Asakura Tetsuo
Journal of biomaterials applications
Development of a small-diameter artificial vascular graft is urgent because existing materials often occlude within a short time. We have shown that small-diameter vascular graft using Bombyx mori silk fibroin is a potential candidate. Silk fibroin grafts are fabricated by coating silk fibroin on the knit tube prepared from silk fibroin fibers. However, there is a serious problem that the coated silk fibroin portion hardens when alcohol is used for insolubilization of the coated silk fibroin. This hardening prevents the desired biodegradation of the coated silk fibroin. In this study, we improved the silk fibroin coating method of the knit silk fibroin tube. Namely, the silk fibroin sponge coating was performed using glycerin, poly(ethylene glycol diglycidyl ether) or poly(ethylene glycol). In addition, silk fibroin grafts were prepared avoiding dryness during the coating process and were kept in the hydrated state until implantation into the abdominal aorta was complete. After implantation of the hydrated silk fibroin grafts, grafts were taken out at two weeks or three months, and histopathological examination was performed. The grafts coated with three types of silk fibroin sponges had a higher tissue infiltration rate than alcohol-treated grafts and were superior in the formation of smooth muscle cell and vascular endothelial cell remodeling. Biodegradations of the silk fibroin grafts prepared using the three types of silk fibroin sponge coatings and alcohol-treated silk fibroin grafts were also examined with protease XIV in vitro, and the grafts were observed by scanning electron microscopy before and 24 h after biodegradation. Faster biodegradations were observed for grafts coated with the three types of silk fibroin sponges. C solid-state nuclear magnetic resonance studies showed that the conformation of the silk fibroin sponge prepared using porogen was a random coil with high mobility in the hydrated state. We believe that small-diameter silk fibroin vascular grafts coated with quick biodegradable silk fibroin sponges can be developed based on these findings.
Cooperative Assembly of a Peptide Gelator and Silk Fibroin Afford an Injectable Hydrogel for Tissue Engineering.
Cheng Baochang,Yan Yufei,Qi Jingjing,Deng Lianfu,Shao Zeng-Wu,Zhang Ke-Qin,Li Bin,Sun Ziling,Li Xinming
ACS applied materials & interfaces
Silk fibroin (SF) from Bombyx mori has received increasing interest in biomedical fields, because of its slow biodegradability, good biocompatibility, and low immunogenicity. Although SF-based hydrogels have been studied intensively as a potential matrix for tissue engineering, weak gelation performance and low mechanical strength are major limitations that hamper their widespread applicability. Therefore, searching for new strategies to improve the SF gelation property is highly desirable in tissue engineering research. Herein, we report a facile approach to induce rapid gelation of SF by a small peptide gelator (e.g., NapFF). Following the simple mixing of SF and NapFF in water, a stable hydrogel of SF was obtained in a short time period at physiological pH, and the minimum gelation concentration of SF can reach as low as 0.1%. In this process of gelation, NapFF not only can behave itself as a gelator for supramolecular self-assembly, but also can trigger the conformational transition of the SF molecule from random coil to β-sheet structure via hydrophobic and hydrogen-bonding interactions. More importantly, for the generation of a scaffold with favorable cell-surface interactions, a new peptide gelator (NapFFRGD) with Arg-Gly-Asp (RGD) domain was applied to functionalize SF hydrogel with improved bioactivity for cell adhesion and growth. Following encapsulating the vascular endothelial growth factor (VEGF), the SF gel was subcutaneously injected in mice, and served as an effective matrix to trigger the generation of new blood capillaries in vivo.
Silk fibroin as a platform for dual sensing of vitamin B using photoluminescence and electrical techniques.
Chakravarty Sudesna,Gogoi Bedanta,Mandal Biman B,Bhardwaj Nandana,Sarma Neelotpal Sen
Biosensors & bioelectronics
The amalgamation of natural origin materials and technologies for label-free and real time detection is pertinent in analytical field. In this study, Bombyx mori silk fibroin protein (BMSF) has been utilized for the dual sensing of vitamin B via fluorescence and electrical impedance techniques. The processing of BMSF is done to generate an aqueous solution of BMSF along with the development of micro patterned thin films via soft lithography technique. The BMSF aqueous solution exhibit auto fluorescence property and thereby utilized for label free detection of vitamin B with an estimated limit of detection (LOD) of about 0.003 × 10 g/uL. This is followed by impedimetric detection of vitamin B using the micro patterned BMSF thin films. A LOD of 17.8 ppm and 0.25 ppm are achieved in aqueous solution and human blood serum, respectively. Taken together, this work demonstrates a potential label free dual sensing mode for sensitive detection of micronutrient vitamin B.
Silk fibroin-bioactive glass based advanced biomaterials: towards patient-specific bone grafts.
Midha Swati,Kumar Sumit,Sharma Aarushi,Kaur Kulwinder,Shi Xiaomeng,Naruphontjirakul Parichart,Jones Julian R,Ghosh Sourabh
Biomedical materials (Bristol, England)
A major challenge in bone tissue engineering is to develop patient-specific, defect-site specific grafts capable of triggering specific cell signaling pathways. We could programmably fabricate the 3D printed bone constructs via direct ink writing of silk-gelatin-bioactive glass (SF-G-BG) hybrids using two different compositions of melt-derived bioactive glasses (with and without strontium) and compared against commercial 45S5 Bioglass. Physico-chemical characterization revealed that released ions from bioactive glasses inhibited the conformational change of Bombyx mori silk fibroin protein (from random coil to β-sheet conformation), affecting printability of the SF-G-BG ink. In-depth molecular investigations showed that strontium containing SF-G-BG constructs demonstrated superior osteogenic differentiation of mesenchymal stem cells (TVA-BMSCs) over 21 days towards osteoblastic (marked by upregulated expression of runt related transcription factor, alkaline phosphatase, osteopontin, osteonectin, integrin bone sialoprotein, osteocalcin) and osteocytic (marked by podoplanin, dentin matrix acidic phosphoprotein, sclerostin) phenotype compared to other BG compositions and silk-gelatin alone. Moreover, ionic release from bioactive glasses in the silk-gelatin ink triggered the activation of signaling pathways (BMP-2, BMP-4 and IHH), which are critical in regulating bone formation in vivo. Overall, the presence of strontium containing bioactive glass in silk-gelatin matrices provided appropriate cues in regulating the development of custom-made 3D in vitro human bone constructs.
Evaluating the nutritive profile of three insect meals and their effects to replace soya bean in broiler diet.
Khan S,Khan R U,Alam W,Sultan A
Journal of animal physiology and animal nutrition
This study was conducted to evaluate the comparative effect of maggot meal, silkworm meal and mealworm as dietary protein source on the production performance and some aspects of meat quality in broilers. In this regard, maggot meal was reared on chicken offal and poultry waste. Silkworm meal was obtained from silk industry, while mealworm was developed through beetles rearing. A total of 120-day-old broiler chicks were randomly divided into four groups where soya bean meal (M0) was replaced with maggot meal (M1), silkworm meal (M2) and mealworm (M3) respectively. Each group was further divided into three replicates. The study was carried out for a period of 5 weeks. Diets containing mealworm significantly reduced overall feed consumption and resulted into higher weight gain (p < .05). Lowest feed conversion ratio (FCR) was recorded for birds fed with mealworm diet (p < .05). Tenderness and juiciness of meat were higher (p < .05) in M3 compared to the control and other treatments. Mortality did not vary between the control and the treated groups. Therefore, it is concluded that insect meal is rich in essential nutrients and could be successfully used in broiler ration without compromising acceptability. In the light of this study, mealworm is the best choice in broiler ration, in comparison with maggot and silkworm.
Metabolic profiling of natural and cultured Cordyceps by NMR spectroscopy.
Lu Yi,Zhi Yuee,Miyakawa Takuya,Tanokura Masaru
Cordyceps, a type of Chinese herbal medicine that exhibits anti-angiogenesis and tumor growth suppression effects, has recently gained increasing popularity. However, high-quality, natural Cordyceps, such as Ophiocordyceps sinensis, is very rare and difficult to obtain in large amounts. Cordyceps is cultured instead of harvested from natural sources, but the quality with respect to the ingredients has not been fully studied. In this study, we performed an NMR metabolic profiling of aqueous extracts of Cordyceps without any sample treatment to evaluate the proper species and medium and influence of two different disinfection methods. It was discovered that Cordyceps militaris fungus and silkworm chrysalis medium were suitable for cultivation of Cordyceps. Furthermore, cordycepin, a Cordyceps-specific functional compound, was produced at different growth stages during different cultivation processes, even at the mycelial stage, and was found at three times higher concentrations in cultured C. militaris compared to that in naturally occurring C. militaris.
Polydopamine-Coated ( ) Silk Fibroin Films Promote Cell Adhesion and Wound Healing in Skin Tissue Repair.
Wang Jie,Chen Yuping,Zhou Guanshan,Chen Yuyin,Mao Chuanbin,Yang Mingying
ACS applied materials & interfaces
Wound dressings are important materials for the successful recovery of skin trauma. Traditional wound dressings such as gauzes are not efficient in wound healing. Here we show that silk fibroin, spun from a wild silkworm () and rich in Arg-Gly-Asp (RGD) sequences, can be developed into a wound dressing after proper modification for improving the cell adhesion to accelerate the skin repair. Specifically, polydopamine (PDA) was coated on an silk fibroin (AF) film to form the PAF film to achieve enhanced cell adhesion and would healing. The PDA coating significantly increased the roughness and hydrophilicity of the AF film and thus its protein absorption capability. Furthermore, the PAF films promoted the adhesion and migration of mesenchymal stem cells (MSCs) in the in vitro wound healing assay. In vivo testing confirmed that wound covered with the PAF film was completely healed with the formation of the new skin and hair within 14 days post trauma. Histological examination indicated that, compared to the AF film and gauze control, the PAF film did not cause significant inflammation in the wound but promoted the epithelialization and well-organized collagen deposition in the dermis. This work indicates that AF films coated with PDA are promising wound dressings for skin tissue repair.
Preparation of new natural silk non-woven fabrics by using adhesion characteristics of sericin and their characterization.
Lee Ji Hye,Bae Yeon Su,Kim Su Jin,Song Dae Woong,Park Young Hwan,Bae Do Gyu,Choi Jin Hyun,Um In Chul
International journal of biological macromolecules
Electro-spun regenerated silk webs have been extensively studied for biomedical applications because of the simplicity of their fabrication methods However, the productivity of the electro-spinning process is low for web fabrication and the mechanical properties of the electro-spun silk web are not satisfactory, which restricts its commercialization. In this study, a new silk non-woven fabric was successfully fabricated by wetting and hot press treatments using the excellent binding characteristic of sericin. The effects of the press temperature and residual sericin content on the preparation, structure, and properties of the silk non-woven fabric were examined. A press temperature of 200°C was optimum for obtaining non-woven fabrics with best mechanical properties, without yellowing. The silk non-woven fabric could not be fabricated without sericin, and a minimum of 8% sericin was required to fabricate it. As the sericin content was increased, the strength and Young's modulus of the silk non-woven fabric increased, while the tensile elongation remained constant. Regardless of the press temperature and sericin content, all the silk non-woven fabrics showed good cell viability, comparable to that of the tissue culture plate (TCP) used as a control until 4days, which however decreased compared to that of TCP after 7days.
Swellable silk fibroin microneedles for transdermal drug delivery.
Yin Zhuping,Kuang Dajiang,Wang Shiyi,Zheng Zhaozhu,Yadavalli Vamsi K,Lu Shenzhou
International journal of biological macromolecules
In this paper, a swelling-modified silk fibroin (SF) microneedle for transdermal drug delivery is presented. The microneedles undergo a phase transition from a dried and rigid state to a semi-solid, acerose hydrogel state with a controlled 3-dimensional (3D) porous network structure. Different micromolecular reagents have been studied for mixing with aqueous silk fibroin to endow a swellable and insoluble capacity to the SF. The aqueous SF composite is poured on a polydimethylsiloxane (PDMS) mold with arranged micropores on its surface to fabricate SF microneedles with high fidelity and mechanical robustness. The results demonstrate that 2-ethoxyethanol (ECS) modified SF microneedles can easily pierce porcine skin with a depth of ∼200μm in vitro, and transform into semi-solid hydrogels with 50-700nm porous network inside. These swelling-modified microneedles can accomplish a significantly enhanced transdermal drug release capacity in proportion to their swelling characteristics. The better swelling capacity of the microneedles produces larger pores, resulting in higher transdermal drug release kinetics. There is also a relationship between swollen pore dimensions and the molecular weights of encapsulated therapeutics. The controllable properties of these SF microneedles coupled with their high biocompatibility, render swell-to-release ECS/SF composites as viable transdermal delivery devices.
Silk-sericin degummed wastewater solution-derived and nitrogen enriched porous carbon nanosheets for robust biological imaging of stem cells.
Chuang Chuan-Chung,Prasannan Adhimoorthy,Hong Po-Da,Chiang Ming-Yu
International journal of biological macromolecules
Appreciated raw materials like silk-sericin can be recovered from silk-textile industrial waste for the production of novel functional nanomaterials. In this study, highly fluorescent sericin based carbon nanosheets (SCN) were produced from industrial wastewater containing silk-sericin as a precursor, and was applied as bio-imaging application for oral fat stem cells. A simple one-pot, hydrothermal carbonization method was used to produce SCN at a 180°C. The obtained hydrothermal carbons exhibited strong fluorescence properties due to the presence of strong polar groups, such as carboxyl, amino and amide groups in the surface. Heteroatom functionalization of the SCN leads to the property of fluorescence due to enriched nitrogen and was confirmed by X-ray photoelectron and Fourier transform infrared spectroscopy. The plate-like morphology of SCN about 35nm in size was evaluated by transmission electron microscopy. The carbon 13 nuclear magnetic resonance results revealed that nano-sized fluorescent SCN formed during carbonization and functionalization occurred through dehydration of the sericin protein. Moreover, the prepared SCNs demonstrated low toxicity and their suitability for bio-imaging applications was demonstrated to the oral fat stem cells. Overall, sericin degumming wastewater from the silk textile industry can be utilized for the production of SCNs for stem cells bio-imaging applications.
In vitro cartilage construct generation from silk fibroin- chitosan porous scaffold and umbilical cord blood derived human mesenchymal stem cells in dynamic culture condition.
Agrawal Parinita,Pramanik Krishna,Biswas Amit,Ku Patra Ranjan
Journal of biomedical materials research. Part A
Cartilage construct generation includes a scaffold with appropriate composition to mimic matrix of the damaged tissue on which the stem cells grow and differentiate. In this study, umbilical cord blood (UCB) derived human mesenchymal stem cells (hMSCs) were seeded on freeze dried porous silk-fibroin (SF)/chitosan (CS) scaffolds. Influence of static and dynamic (spinner flask bioreactor) culture conditions on the developing cartilage construct were studied by in-vitro characterization for viability, proliferation, distribution, and chondrogenic differentiation of hMSCs over the scaffold. Constructs developed in spinner flask consisted of 62% live cells, and exhibited 543% more cell density at the core than constructs cultured in static system. Quantification of DNA and glycosaminoglycans accumulation after 21 days showed the progression of chondrogenic differentiation of hMSCs was higher in dynamic culture compared to static one. In constructs generated under dynamic condition, histology staining for proteoglycan matrix, and fluorescence staining for collagen-II and aggrecan showed positive correlation between early and late stage chondrogenic markers, which was further confirmed by quantitative PCR analysis, showing low collagen-I expression and highly expressed Sox9, collagen-II and aggrecan. The present study demonstrated that construct generated by combining 3D SF/CS scaffold with UCB-hMSCs under dynamic condition using spinner flask bioreactor can be used for cartilage tissue regeneration for future medical treatments. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 397-407, 2018.
Single step fabrication of antimicrobial fibre mats from a bioengineered protein-based polymer.
da Costa A,Pereira A M,Gomes A C,Rodriguez-Cabello J C,Sencadas V,Casal M,Machado R
Biomedical materials (Bristol, England)
Genetically engineered protein polymers functionalized with bioactive domains have potential as multifunctional versatile materials for biomedical use. The present work describes the fabrication and characterisation of antimicrobial fibre mats comprising the antimicrobial elastin-like recombinamer (ELR) CM4-A200. The CM4-A200 protein polymer derives from the genetic fusion of the ABP-CM4 antimicrobial peptide from Bombyx mori with 200 repetitions of the pentamer VPAVG. This is the first report on non-crosslinked fibre mats fabricated with an antimicrobial ELR stable in solution. Thermal gravimetric analysis of CM4-A200 fibre mats shows one single degradation step at temperatures above 300 °C, with fibres displaying a higher thermal degradation activation. The electrospun CM4-A200 fibres display high antimicrobial activity against Gram-positive and Gram-negative bacteria with no detectable cytotoxic effects against normal human skin fibroblasts and keratinocytes, revealing the great potential of these polymers for the fabrication of biomedical materials.
Antioxidant potential of mulberry and non-mulberry silk sericin and its implications in biomedicine.
Kumar Jadi Praveen,Mandal Biman B
Free radical biology & medicine
Sericin, a principal constituent of silk, is widely used in various biomedical applications. In addition, conferring protection against free radicals and oxidative damage add more value to its therapeutic potential. However, the antioxidant (AO) properties of silk sericin (SS) remains contingent on extraction procedures. In the present study, we have evaluated the effect of different extraction methods (conventional, autoclaving, urea, alkali and acid-degradation) on AO properties of SS from three Indian silk varieties [Antheraea assamensis (AA), Philosamia ricini (PR) and Bombyx mori (BM)]. The physico-chemical characterization studies revealed that the molecular weight of SS isolates of each method ranged from 10 to 220kDa along with varied protein structural biochemistry. SS extracts using urea-degradation (BM, PR and AA), conventional method and alkali-degradation (BM) displayed high percentage of β-sheets, random coils and turns. Acid-degraded SS (PR, followed by AA and BM) showed the highest total flavonoid content while conventional method (PR), autoclaving (AA) and alkali-degradation (BM) displayed lowest flavonoid levels. Interestingly, SS extracted by autoclaving (BM and AA), acid-degradation (PR), conventional and alkali-degradation (BM, AA and PR) methods exhibited 50% reduction of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical. Moreover, the efficacy of antioxidant potential of SS extracted by different methods was found to be in the order of "alkali>autoclaving>conventional" as demonstrated in L929 cells. Correspondingly, the anti-lipid peroxidation activity of SS extracted by alkali method (AA, BM and PR) further confirmed better AO properties amid others. Thus, the present study demonstrates that the extraction methods may significantly affect AO activity of SS which might be of importance for potential cosmetic applications.
Biological effects of silk fibroin 3D scaffolds on stem cells from human exfoliated deciduous teeth (SHEDs).
Collado-González M,Pecci-Lloret M P,García-Bernal D,Aznar-Cervantes S,Oñate-Sánchez R E,Moraleda J M,Cenis J L,Rodríguez-Lozano F J
The aim is to investigate in vitro biological effects of silk fibroin 3D scaffolds on stem cells from human exfoliated deciduous teeth (SHEDs) in terms of proliferation, morphological appearance, cell viability, and expression of mesenchymal stem cell markers. Silk fibroin 3D scaffolding materials may represent promising suitable scaffolds for their application in regenerative endodontic therapy approaches. SHEDs were cultured in silk fibroin 3D scaffolds. Then, cell numbers were counted and the Alamar blue colorimetric assay was used to analyse cell proliferation after 24, 48, 72, and 168 h of culture. The morphological features of SHEDs cultured on silk fibroin scaffolds were evaluated by scanning electron microscopy (SEM). Finally, cell viability and the expression of mesenchymal stem cell markers were analysed by flow cytometry. One-way analysis of variance (ANOVA) followed by a Bonferroni post-test was performed (P < 0.05). At 24 and 48 h of culture, SHED proliferation on scaffolds was modest compared to the control although still significant (p < 0.05). However, cell proliferation progressively increased from 72 to 168 h compared with the control (p < 0.001; p < 0.01). In addition, flow cytometry analysis showed that the culture of SHEDs on silk fibroin scaffolds did not significantly alter the level of expression of the mesenchymal markers CD73, CD90, or CD105 up to 168 h; in addition, cell viability in silk fibroin was similar to than obtained in plastic. Moreover, SEM studies revealed a suitable degree of proliferation, cell spreading, and attachment, especially after 168 h of culture. The findings from the current study suggest that silk fibroin 3D scaffolds had a favourable effect on the biological responses of SHEDs. Further in vivo investigations are required to confirm these results.
Development of biomimetic thermoplastic polyurethane/fibroin small-diameter vascular grafts via a novel electrospinning approach.
Yu Emily,Mi Hao-Yang,Zhang Jue,Thomson James A,Turng Lih-Sheng
Journal of biomedical materials research. Part A
A new electrospinning approach for fabricating vascular grafts with a layered, circumferentially aligned, and micro-wavy fibrous structure similar to natural elastic tissues has been developed. The customized electrospinning collector was able to generate wavy fibers using the dynamic "jump rope" collecting process, which also solved the sample removal problem for mandrel-type collectors. In this study, natural silk fibroin and synthetic thermoplastic polyurethane (TPU) were combined at different weight ratios to produce hybrid small-diameter vascular grafts. The purpose of combining these two materials was to leverage the bioactivity and tunable mechanical properties of these natural and synthetic materials. Results showed that the electrospun fiber morphology was highly influenced by the material compositions and solvents employed. All of the TPU/fibroin hybrid grafts had mechanical properties comparable to natural blood vessels. The circumferentially aligned and wavy biomimetic configuration provided the grafts with a sufficient toe region and the capacity for long-term usage under repeated dilatation and contraction. Cell culture tests with human endothelial cells (EC) also revealed high cell viability and good biocompatibility for these grafts. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 985-996, 2018.
Functionalized silk fibroin film scaffold using β-Carotene for cornea endothelial cell regeneration.
Kim Do Kyung,Sim Bo Ra,Kim Jeong In,Khang Gilson
Colloids and surfaces. B, Biointerfaces
Design of corneal endothelium substitute is important for replacement of cadaveric cornea tissue. Our previous study has shown the suitability of silk fibroin (SF) as a biomaterial for cornea scaffold. In this study, we used β-Carotene (β-C) to enhance the regeneration of corneal endothelial cells (CEnCs) and maintain CEnC specific function. The fabricated film scaffolds showed desired transparency and hydrophilic properties which are crucial factors for vision recovery. The cell viability, phenotype and gene expression was examined using MTT assay, immunofluorescence and reverse transcription polymerase chain reactions. Compared with pristine SF scaffold, proper amount of β-C incorporated with SF scaffolds showed higher initial cell attachment, cell viability and mRNA expression. The results indicate that the fabricated SF film scaffold incorporated with small amount of β-C might be the suitable alternative corneal endothelium substitute for transplantation.
Protective effect of Thai silk extracts on drug-induced phototoxicity in human epidermal A431 cells and a reconstructed human epidermis model.
Rosena Apiwan,Koobkokkruad Thongchai,Eaknai Waleewan,Bunwatcharaphansakun Phichaporn,Maniratanachote Rawiwan,Aueviriyavit Sasitorn
Journal of photochemistry and photobiology. B, Biology
Bombyx mori silk extracts, derived from the cocoon degumming process of draw and dye silk in the textile industry, are mainly composed of sericin protein. To add value to the Thai silk extracts, and hence the silk industry, a simple enrichment process was recently developed and the enriched silk extracts were then applied in nano-cosmeceutical products and nano-delivery systems. In this study, the protective effect of Thai silk extracts from three different strains of Bombyx mori on the drug-induced phototoxicity was evaluated in vitro using chlorpromazine (CPZ), a commonly used antipsychotic drug, as a representative phototoxic drug. The human epidermal A431 cell line and reconstructed human epidermis (RhE) model were used as the in vitro skin model. The silk extracts significantly improved the viability of A431 cells after CPZ exposure and ultraviolet A (UVA) irradiation, as shown by the significantly increased CPZ and UVA IC values and the decreased proportion of apoptotic cells. The protective effect of these silk extracts against the CPZ-induced UVA-phototoxicity in A431 cells was associated with the attenuation of intracellular oxidative stress via an increased intracellular glutathione level. Likewise, the silk extracts exhibited a protective effect on the CPZ-induced UVA-phototoxicity in the RhE model, in terms of an improved tissue viability and attenuation of the released inflammatory cytokine, interleukin-1α. These findings support the potential usefulness of silk extracts in novel applications, especially in the protection of drug-induced phototoxicity.
Treatment with solubilized Silk-Derived Protein (SDP) enhances rabbit corneal epithelial wound healing.
Abdel-Naby Waleed,Cole Brigette,Liu Aihong,Liu Jingbo,Wan Pengxia,Schreiner Ryan,Infanger David W,Paulson Nicholas B,Lawrence Brian D,Rosenblatt Mark I
There is a significant clinical need to improve current therapeutic approaches to treat ocular surface injuries and disease, which affect hundreds of millions of people annually worldwide. The work presented here demonstrates that the presence of Silk-Derived Protein (SDP) on the healing rabbit corneal surface, administered in an eye drop formulation, corresponds with an enhanced epithelial wound healing profile. Rabbit corneas were denuded of their epithelial surface, and then treated for 72-hours with either PBS or PBS containing 5 or 20 mg/mL SDP in solution four times per day. Post-injury treatment with SDP formulations was found to accelerate the acute healing phase of the injured rabbit corneal epithelium. In addition, the use of SDP corresponded with an enhanced tissue healing profile through the formation of a multi-layered epithelial surface with increased tight junction formation. Additional biological effects were also revealed that included increased epithelial proliferation, and increased focal adhesion formation with a corresponding reduction in the presence of MMP-9 enzyme. These in vivo findings demonstrate for the first time that the presence of SDP on the injured ocular surface may aid to improve various steps of rabbit corneal wound healing, and provides evidence that SDP may have applicability as an ingredient in therapeutic ophthalmic formulations.
Silk Composite Electronic Textile Sensor for High Space Precision 2D Combo Temperature-Pressure Sensing.
Wu Ronghui,Ma Liyun,Hou Chen,Meng Zhaohui,Guo Wenxi,Yu Weidong,Yu Rui,Hu Fan,Liu Xiang Yang
Small (Weinheim an der Bergstrasse, Germany)
Wearable electronic textiles based on natural biocompatible/biodegradable materials have attracted great attention due to applications in health care and smart clothes. Silkworm fibers are durable, good heat conductors, insulating, and biocompatible, and are therefore regarded as excellent mediating materials for flexible electronics. In this paper, a strategy on the design and fabrication of highly flexible multimode electronic textiles (E-textile) based on functionalized silkworm fiber coiled yarns and weaving technology is presented. To achieve enhanced temperature sensing performance, a mixture of carbon nanotubes and an ionic liquid ([EMIM]Tf N) is embedded, which displays top sensitivity of 1.23% °C and stability compared with others. Furthermore, fibrous pressure sensing based on the capacitance change of each cross-point of two yarns gives rise to highly position dependent and sensitivity sensing of 0.136 kPa . Based on weaving technologies, a unique combo textile sensor, which can sense temperature and pressure independently with a position precision of 1 mm , is obtained. The application to intelligent gloves endows the position dependent sensing of the weight, and temperature distribution sensing of the temperature.
Silk fibroin-poly(lactic acid) biocomposites: Effect of protein-synthetic polymer interactions and miscibility on material properties and biological responses.
Wang Fang,Wu Hao,Venkataraman Venkat,Hu Xiao
Materials science & engineering. C, Materials for biological applications
A protein-polymer blend system based on silkworm silk fibroin (SF) and polylactic acid (PLA) was systematically investigated to understand the interaction and miscibility of proteins and synthetic biocompatible polymers in the macro- and micro-meter scales, which can dramatically control the cell responses and enzyme biodegradation on the biomaterial interface. Silk fibroin, a semicrystalline protein with beta-sheet crystals, provides controllable crystal content and biodegradability; while noncrystallizable PDLLA provides hydrophobicity and thermal stability in the system. Differential scanning calorimetry (DSC) combined with scanning electron microscope (SEM) showed that the morphology of the blend films was uniform on a macroscopic scale, yet with tunable micro-phase patterns at different mixing ratios. Fourier transform infrared analysis (FTIR) revealed that structures of the blend system, such as beta-sheet crystal content, gradually changed with the mixing ratios. All blended samples have better stability than pure SF and PLA samples as evidenced by thermogravimetric analysis. Protease XIV enzymatic study showed that the biodegradability of the blend samples varied with their blending ratios and microscale morphologies. Significantly, the topology of the micro-phase patterns on the blends can promote cell attachment and manipulate the cell growth and proliferation. This study provided a useful platform for understanding the fabrication strategies of protein-synthetic polymer composites that have direct biomedical and green chemistry applications.
Molecular Interactions and Toughening Mechanisms in Silk Fibroin-Epoxy Resin Blend Films.
Yang Kang,Yazawa Kenjiro,Tsuchiya Kousuke,Numata Keiji,Guan Juan
Natural silkworm silks have been applied to reinforce epoxy resin to achieve sub-ambient and impact toughness in the composite. However, the molecular interactions at the silk fiber-matrix interface of the composite are poorly understood. In this work, silk fibroin extracted from Bombyx mori silk is blended with an epoxy resin polymer system to study the molecular interactions between silk fibroin, epoxy compounds, and hardeners. The effects of chemical crosslinks between epoxy groups and hardeners or silk fibroin, as well as physical crosslinks in the β-sheet structure of silk fibroin, were discussed on the thermal stability, glass transition behavior, and mechanical properties of the blend films. A relationship between the crosslinking structure and mechanical properties for the films is proposed to enlighten on the toughening mechanisms. The findings would provide insights into forming strong and tough silk fibroin material as well as understanding the interface interactions of silk-epoxy composites.
Silk fibres grafted with 2-hydroxyethyl methacrylate (HEMA) and 4-hydroxybutyl acrylate (HBA) for biomedical applications.
Taddei Paola,Di Foggia Michele,Martinotti Simona,Ranzato Elia,Carmagnola Irene,Chiono Valeria,Tsukada Masuhiro
International journal of biological macromolecules
Silk fibroin may be chemically modified by grafting, with the purpose of improving its properties according to the desired function. In this study, silk fabrics from Bombyx mori silk fibres were grafted with 2-hydroxyethyl methacrylate (HEMA), as well as a binary mixture of HEMA and 4-hydroxybutyl acrylate (HBA). The samples were then electrospun from trifluoroacetic acid and treated with aqueous methanol. The% weight gains ascribable to HEMA and HBA were successfully determined through Raman spectroscopy. PolyHEMA made the fibres more hydrophilic and hindered crystallization into β-sheet only upon electrospinning and treatment with aqueous methanol; the presence of the HBA component in the grafting mixture did not further decrease the ability of silk fibroin to rearrange into β-sheet, due to its low contents (below 5%) under the used experimental conditions. Fibrillation partially occurred in the grafted fabrics; the electrospun samples maintained their nanostructured morphology. The surface of the substrates under investigation was compatible with cell attachment and growth, which were higher for the nanofibres. Cell adhesion and proliferation may be modulated by varying the surface chemistry and topography of the fabrics; grafting improved the surface properties of silk fibroin for enhanced functional performance in view of biomedical applications.
Functional hepatocyte clusters on bioactive blend silk matrices towards generating bioartificial liver constructs.
Janani G,Nandi Samit K,Mandal Biman B
The creation of in vitro functional hepatic tissue simulating micro-environmental niche of native liver is a keen area of research due to its demand in bioartificial liver (BAL) and cell-based tissue engineering. Here, we investigated the potential of novel blend (BA) silk scaffold fabricated by blending mulberry (Bombyx mori, BM) silk fibroin with cell adhesion motif (RGD) rich non-mulberry (Antheraea assamensis, AA) silk fibroin, in generating a functional liver construct. Three-dimensional (3D) porous silk scaffolds (BM, AA and BA) were physico-chemically characterized and functionally evaluated using human hepatocarcinoma cells (HepG2) and primary neonatal rat hepatocytes. The growth and distribution of hepatocytes within the scaffolds were tracked by FESEM, alamar blue proliferation assay and live/dead staining. Hemocompatible BA scaffolds supported the formation of high density hepatocyte clusters, facilitating cell-matrix and cell-cell interactions. Blend scaffolds evinced enhanced liver-specific functions of cultured hepatocytes in terms of albumin synthesis, urea synthesis and cytochrome P450 enzyme activity over 21 days. Subcutaneous implantation of scaffolds demonstrated minimal macrophage infiltration in blend scaffolds. These findings substantiate that the integral property of blend (BA) scaffold offers a befitting environment by influencing spheroidal growth of hepatocytes with enhanced biological activity. Collectively, the present study provides a new 3D bio-matrix niche for growing functional liver cells that would have future prospects in BAL as well as regenerative medicine. STATEMENT OF SIGNIFICANCE:An end stage liver disease called cirrhosis perturbs the self-healing ability and physiological functions of liver. Due to the scarcity of healthy donors, a functional in vitro hepatic construct retaining the liver-specific functions is in great demand for its prospects in bioartificial liver (BAL) and cell-based tissue engineering. Physicochemical attributes of a matrix influence the behavior of cultured hepatocytes in terms of attachment, morphology and functionality. Mulberry and non-mulberry silk fibroin presents unique amino acid sequence with difference in hydrophobicity and crystallinity. Considering this, the present study focuses on the development of a suitable three-dimensional (3D) bioactive matrix incorporating both mulberry silk fibroin and cell adhesion motif (RGD) rich non-mulberry silk fibroin. Porous silk blend scaffolds facilitated the formation of hepatocyte clusters with enhanced liver-specific functions emphasizing both cell-cell and cell-matrix interactions. Hemocompatibility and integral property of blend scaffolds offers a biological niche for seeding functional liver cells that would have future prospects in biohybrid devices.
In vitro and in vivo evaluation of silk fibroin functionalized with GABA and allopregnanolone for Schwann cell and neuron survival.
Gennari Chiara Gm,Cilurzo Francesco,Mitro Nico,Caruso Donatella,Minghetti Paola,Magnaghi Valerio
AIM: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. MATERIALS & METHODS: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. RESULTS & CONCLUSION: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.
Impact of ionic liquid type on the structure, morphology and properties of silk-cellulose biocomposite materials.
Stanton John,Xue Ye,Pandher Prabhdeep,Malek Laura,Brown Tyler,Hu Xiao,Salas-de la Cruz David
International journal of biological macromolecules
Microcrystalline cellulose and Bombyx mori silk blended biocomposite films were regenerated using various imidazolium-based ionic liquids. The films were characterized to understand the effect of the inter- and intra-molecular interactions upon the morphology and thermal properties. Various techniques were implemented to investigate structural, morphological and thermal properties of the biocomposite films, including Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC) and X-ray scattering. Results show that the type of ionic liquid has strong influence on the structure of silk-cellulose composites that can form either amorphous or semicrystalline structures. While the thermal properties are independent of the type of cation in ionic liquids, the levels of β-sheet configuration are dependent on the type of anion, which further causes changes on the biocomposite thermal properties. The topological image provided information to support morphological effects on the varying ionic liquids and X-ray scattering allowed for insight on the role of ionic liquids on the crystallinity and the spacing differences in biocomposite films. The results have demonstrated that there is a direct relationship between the intermolecular interactions in films and the anion structure of the ionic liquids.
Functionalized PVA-silk blended nanofibrous mats promote diabetic wound healing via regulation of extracellular matrix and tissue remodelling.
Chouhan Dimple,Janani G,Chakraborty Bijayashree,Nandi Samit K,Mandal Biman B
Journal of tissue engineering and regenerative medicine
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 4 weeks. 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.
Silkworm Silk Matrices Coated with Functionalized Spider Silk Accelerate Healing of Diabetic Wounds.
Chouhan Dimple,Das Piyali,Thatikonda Naresh,Nandi Samit K,Hedhammar My,Mandal Biman B
ACS biomaterials science & engineering
Complex cutaneous wounds like diabetic foot ulcers represent a critical clinical challenge and demand a large-scale and low-cost strategy for effective treatment. Herein, we use a rabbit animal model to investigate efficacy of bioactive wound dressings made up of silk biomaterials. Nanofibrous mats of silkworm silk fibroin (AaSF) are coated with various recombinant spider silk fusion proteins through silk-silk interactions to fabricate multifunctional wound dressings. Two different types of spider silk coatings are used to compare their healing efficiency: FN-4RepCT (contains a cell binding motif derived from fibronectin) and Lac-4RepCT (contains a cationic antimicrobial peptide from lactoferricin). AaSF mats coated with spider silk show accelerated wound healing properties in comparison to the uncoated mats. Among the spider silk coated variants, dual coating of FN-4RepCT and Lac-4RepCT on top of AaSF mat demonstrated better wound healing efficiency, followed by FN-4RepCT and Lac-4RepCT single coated counterparts. The study also reveals excellent skin regeneration by the functionalized silk dressings in comparison to commercially used Duoderm dressing and untreated wounds. The spider silk coatings demonstrate early granulation tissue development, re-epithelialization, and efficient matrix remodelling of wounds. The results thus validate potential of bioactive silk matrices in faster repair of diabetic wounds.
Dataset on the synthesis and physicochemical characterization of blank and curcumin encapsulated sericin nanoparticles obtained from silkworm cocoons.
Bhuyan Devangana,Greene George Warren,Das Ratul Kumar
Data in brief
The dataset presents the synthesis and physicochemical characterization of blank and curcumin encapsulated sericin protein nanoparticles obtained from (also known as Ahimsa silk or Peace silk or Eri). Reports on application of sericin protein obtained from are scanty at best. Sericin was extracted from the cocoons by high temperature and high pressure method. Synthesis of sericin nanoparticles was carried out by desolvation method using acetone as the desolvating agent. Curcumin was used as a hydrophobic model drug and was encapsulated into the sericin nanoparticles. Physicochemical characterization of the blank and curcumin encapsulated sericin nanoparticles were carried out by different instrumental analyses. The size and surface charges of sericin nanoparticles changed with the variation of applied sericin concentration. Encapsulation efficiency and loading capacity of the encapsulated sericin nanoparticles showed variation with curcumin concentration. The obtained data indicated the applicative potentials of sericin protein extracted from silkworm cocoons.
Silk micrococoons for protein stabilisation and molecular encapsulation.
Shimanovich Ulyana,Ruggeri Francesco S,De Genst Erwin,Adamcik Jozef,Barros Teresa P,Porter David,Müller Thomas,Mezzenga Raffaele,Dobson Christopher M,Vollrath Fritz,Holland Chris,Knowles Tuomas P J
Naturally spun silks generate fibres with unique properties, including strength, elasticity and biocompatibility. Here we describe a microfluidics-based strategy to spin liquid native silk, obtained directly from the silk gland of Bombyx mori silkworms, into micron-scale capsules with controllable geometry and variable levels of intermolecular β-sheet content in their protein shells. We demonstrate that such micrococoons can store internally the otherwise highly unstable liquid native silk for several months and without apparent effect on its functionality. We further demonstrate that these native silk micrococoons enable the effective encapsulation, storage and release of other aggregation-prone proteins, such as functional antibodies. These results show that native silk micrococoons are capable of preserving the full activity of sensitive cargo proteins that can aggregate and lose function under conditions of bulk storage, and thus represent an attractive class of materials for the storage and release of active biomolecules.
Influence of hyperproteinemia on reproductive development in an invertebrate model.
Wang Yong-Feng,Chen Xue-Dong,Wang Guang,Li Qiu-Ying,Liang Xin-Yin,Sima Yang-Hu,Xu Shi-Qing
International journal of biological sciences
Hyperproteinemia is a severe metabolic disease characterized by abnormally elevated plasma protein concentrations (PPC). However, there is currently no reliable animal model for PPC, and the pathological mechanism of hyperproteinemia thus remains unclear. In this study, we evaluated the effects of hyperproteinemia on reproductive development in an invertebrate silkworm model with a controllable PPC and no primary disease effects. High PPC inhibited the synthesis of vitellogenin and 30K protein essential for female ovarian development in the fat body of metabolic tissues, and inhibited their transport through the hemolymph to the ovary. High PPC also induced programmed cell death in testis and ovary cells, slowed the development of germ cells, and significantly reduced the reproductive coefficient. Furthermore, the intensities and mechanisms of high-PPC-induced reproductive toxicity differed between sexes in this silkworm model.
One-pot facile synthesis of silk sericin-capped gold nanoparticles by UVC radiation: Investigation of stability, biocompatibility, and antibacterial activity.
Akturk Omer,Gun Gok Zehra,Erdemli Ozge,Yigitoglu Mustafa
Journal of biomedical materials research. Part A
Herein, an easy one-pot synthesis method for gold nanoparticles (AuNPs), involving only gold salt and sericin extracted from silkworm cocoon in the presence of ultraviolet C (UVC) radiation, was developed. Nanoparticle formation was confirmed by characteristic surface plasmon resonance peaks at 520-540 nm wavelengths, and the influence of silk sericin on enhancing the colloidal stability of AuNPs was confirmed. Transmission electron microscopy examination showed the average size (<10 nm) and size distribution decreased significantly with higher sericin concentration. No antibacterial activity was observed on Gram-positive Bacillus subtilis or Gram-negative Escherichia coli for sole AuNPs (0.065-0.26 mg/ml), but the conjugation of AuNPs with streptomycin antibiotic decreased significantly the required minimum inhibitory concentration doses, as also confirmed with agar plating, Scanning Electron Microscopy and Atomic Force Microscopy analyses. Furthermore, sericin-capped AuNPs showed high cell viabilities (>100%) and no sign of any detectable apoptosis or necrosis in 1-day incubation. Also, high real-time cell proliferation results of AuNPs competitive with positive control groups implied excellent in vitro biocompatibility. These results evidenced that sericin enhanced the colloidal stability of AuNPs and the biological activities of sericin-capped AuNPs reported here could render them suitable nanoscale vehicles for biomedical applications.
Development and validation of scFv-conjugated affinity silk protein for specific detection of carcinoembryonic antigen.
Sato Mitsuru,Kitani Hiroshi,Kojima Katsura
The production costs for monoclonal antibodies (MAbs) utilized in medical diagnostic kits are inevitably high because the MAbs are mostly obtained from hybridoma cell culture. Here, we report the development and validation of a novel affinity silk protein produced by transgenic silkworm technology as a possible alternative diagnostic tool for cancers. We generated a transgenic silkworm expressing a cDNA construct containing fibroin L-chain fused to a single-chain variable fragment (scFv) derived from a MAb against carcinoembryonic antigen (CEA). The transgenic cocoons were dissolved in aqueous lithium bromide solution, applied to 96-well plates, and analysed by enzyme-linked immunosorbent assay. The scFv-conjugated affinity silk protein specifically recognized CEA as well as the parental MAb. The binding activity was retained after several months of storage in coated plates or concentrated solution. Thus, the scFv-conjugated affinity silk protein provides a potentially useful alternative to conventional MAbs in medical diagnostic kits.
Fabrication and Characterization of Reconstituted Silk Microgels for the Storage and Release of Small Molecules.
Liu Xizhou,Toprakcioglu Zenon,Dear Alexander J,Levin Aviad,Ruggeri Francesco Simone,Taylor Christopher G,Hu Mengsha,Kumita Janet R,Andreasen Maria,Dobson Christopher M,Shimanovich Ulyana,Knowles Tuomas P J
Macromolecular rapid communications
Silk fibroin is a natural protein obtained from the Bombyx mori silkworm. In addition to being the key structural component in silkworm cocoons, it also has the propensity to self-assemble in vitro into hierarchical structures with desirable properties such as high levels of mechanical strength and robustness. Furthermore, it is an appealing biopolymer due to its biocompatability, low immunogenicity, and lack of toxicity, making it a prime candidate for biomedical material applications. Here, it is demonstrated that nanofibrils formed by reconstituted silk fibroin can be engineered into supramolecular microgels using a soft lithography-based microfluidic approach. Building on these results, a potential application for these protein microgels to encapsulate and release small molecules in a controlled manner is illustrated. Taken together, these results suggest that the tailored self-assembly of biocompatible and biodegradable silk nanofibrils can be used to generate functional micromaterials for a range of potential applications in the biomedical and pharmaceutical fields.
Fabrication and Mechanical Properties of Engineered Protein-Based Adhesives and Fibers.
Sun Jing,Su Juanjuan,Ma Chao,Göstl Robert,Herrmann Andreas,Liu Kai,Zhang Hongjie
Advanced materials (Deerfield Beach, Fla.)
Protein-based structural biomaterials are of great interest for various applications because the sequence flexibility within the proteins may result in their improved mechanical and structural integrity and tunability. As the two representative examples, protein-based adhesives and fibers have attracted tremendous attention. The typical protein adhesives, which are secreted by mussels, sandcastle worms, barnacles, and caddisfly larvae, exhibit robust underwater adhesion performance. In order to mimic the adhesion performance of these marine organisms, two main biological adhesives are presented, including genetically engineered protein-based adhesives and biomimetic chemically synthetized adhesives. Moreover, various protein-based fibers inspired by spider and silkworm proteins, collagen, elastin, and resilin are studied extensively. The achievements in synthesis and fabrication of structural biomaterials by DNA recombinant technology and chemical regeneration certainly will accelerate the explorations and applications of protein-based adhesives and fibers in wound healing, tissue regeneration, drug delivery, biosensors, and other high-tech applications. However, the mechanical properties of the biological structural materials still do not match those of natural systems. More efforts need to be devoted to the study of the interplay of the protein structure, cohesion and adhesion effects, fiber processing, and mechanical performance.
Silk-Derived Protein Enhances Corneal Epithelial Migration, Adhesion, and Proliferation.
Abdel-Naby Waleed,Cole Brigette,Liu Aihong,Liu Jingbo,Wan Pengxia,Guaiquil Victor H,Schreiner Ryan,Infanger David,Lawrence Brian D,Rosenblatt Mark I
Investigative ophthalmology & visual science
Purpose:The corneal surface is vulnerable to a myriad of traumatic insults including mechanical, chemical, and thermal injuries. The resulting trauma may render the naturally occurring regenerative properties of the cornea incapable of restoring a healthy epithelial surface, and may result in the loss of corneal transparency and vision. Healing of the corneal epithelium requires a complex cascade of biological processes that work to restore the tissue after injury. New therapeutic agents that act on the multiple steps of the corneal wound-healing process would offer a potential for improving patient outcomes. Here, a novel silk fibroin-derived protein (SDP) was studied for potential impacts on wound healing through studying an in vitro model. Methods:Solubilized SDP, produced from the Bombyx mori silkworm cocoon, was added to human corneal limbal-epithelial (hCLE) cultures to evaluate the material's effects on epithelial cell migration, proliferation, and adhesion through the use of various scratch wound assays and flow chamber studies. Results:Results indicated that the addition of SDP to culture increased hCLE migration rate by over 50%, and produced an approximate 60% increase in cell proliferation. This resulted in a nearly 30% enhancement of in vitro scratch wound closure time. In addition, cultures treated with SDP experienced increased cell-matrix focal adhesion formation by over 95% when compared to controls. Conclusions:The addition of SDP to culture media significantly enhanced hCLE cell sheet migration, proliferation, and attachment when compared to untreated controls, and indicates SDP's potential utility as an ophthalmic therapeutic agent.
[Experience of Professor Fan Yongsheng in treating arthralgia with insect drugs].
Wu Guo-Lin,Li Tian-Yi,Fan Yong-Sheng
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica
National-level famous traditional Chinese medicine( TCM) doctor Professor Fan Yong-sheng has high-level accomplishments in the treatment of arthralgia. According to clinical diagnosis and dialectic,TCM with appropriate medicinal properties were flexibly applied,with a remarkable efficacy. Especially in the application of insect drugs,he has accumulated abundant clinical experience.According to the main syndromes of arthralgia and pathogenic site,patient' s constitution,Scorpio,Scolopendra,Pheretima,Vespae Nidus,Bombyx Batryticatus,Manis Squama,Bungarus Parvus,Zaocys,Agkistrodon,Cicadae Periostracum,Aspongopus,Eupolyphaga Steleophaga,Silkworm Sand were properly selected. Attention was also paid to the compatibility between insect drugs and antirheumatic drugs,blood-activating drugs and tonifying drugs. In the premise of safe and effective application in the treatment of arthralgia,insect drugs show such efficacies as antipyretic,activating collaterals and relieving pain.
Bombyx mori Silk Fibroin Scaffolds with Antheraea pernyi Silk Fibroin Micro/Nano Fibers for Promoting EA. hy926 Cell Proliferation.
Chen Yongchun,Yang Weichao,Wang Weiwei,Zhang Min,Li Mingzhong
Materials (Basel, Switzerland)
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.
Silk fibroin-Thelebolan matrix: A promising chemopreventive scaffold for soft tissue cancer.
Mukhopadhyay Sourav K,Naskar Deboki,Bhattacharjee Promita,Mishra Abheepsa,Kundu Subhas C,Dey Satyahari
Colloids and surfaces. B, Biointerfaces
Research of improved functional bio-mimetic matrix for regenerative medicine is currently one of the rapidly growing fields in tissue engineering and medical sciences. This study reports a novel bio-polymeric matrix, which is fabricated using silk protein fibroin from Bombyx mori silkworm and fungal exopolysaccharide Thelebolan from Antarctic fungus Thelebolus sp. IITKGP-BT12 by solvent evaporation and freeze drying method. Natural cross linker genipin is used to imprison the Thelebolan within the fibroin network. Different cross-linked and non-cross-linked fibroin/Thelebolan matrices are fabricated and biophysically characterized. Cross-linked thin films show robustness, good mechanical strength and high temperature stability in comparison to non-cross-linked and pure matrices. The 3D sponge matrices demonstrate good cytocompatibility. Interestingly, sustained release of the Thelebolan from the cross-linked matrices induce apoptosis in colon cancer cell line (HT-29) in time dependent manner while it is nontoxic to the normal fibroblast cells (L929).The findings indicate that the cross-linked fibroin/Thelebolan matrices can be used as potential topical chemopreventive scaffold for preclusion of soft tissue carcinoma.
Growth factor-functionalized silk membranes support wound healing in vitro.
Bienert Michaela,Hoss M,Bartneck M,Weinandy S,Böbel M,Jockenhövel S,Knüchel R,Pottbacker K,Wöltje M,Jahnen-Dechent W,Neuss S
Biomedical materials (Bristol, England)
Chronic wounds represent a serious problem in daily medical routine requiring improved wound care. Silk of the domesticated silkworm (Bombyx mori) has been used to form a variety of biomaterials for medical applications. We genetically engineered B. mori to produce silk functionalized with growth factors to promote wound healing in vitro. In this study FGF-, EGF-, KGF-, PDGF- or VEGF-functionalized silk membranes were compared to native B. mori silk membranes without growth factors for their ability to support wound healing in vitro. All silk membranes were cytocompatible and supported macrophage secretion of neutrophil recruiting factor CXCL1 and monocyte chemoattractant protein 1 (MCP-1). VEGF-functionalized silk significantly outperformed other growth factor-functionalized silk membranes, but not native silk in angiogenesis assays. In addition, EGF- and VEGF-functionalized silk membranes slightly enhanced macrophage adhesion compared to silk without growth factors. In wound healing assays in vitro (reduction of wound lesion), dermal equivalents showed a higher wound healing capacity when covered with EGF-, FGF- or VEGF-functionalized silk membranes compared to native, KGF- or PDGF-functionalized silk membranes. Keratinocyte migration and growth is overstimulated by KGF- and VEGF-functionalized silk membranes. In conclusion, growth factor-functionalized silk membranes prepared from genetically engineered silk worm glands are promising wound dressings for future wound healing therapies.
The Usefulness of Silkworms as a Model Animal for Evaluating the Effectiveness of Medicine and Food.
Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan
Development of novel medicines is an important responsibility of researchers in the field of pharmaceutical sciences. However, the discovery of new therapeutically effective without side effects is not an easy job. I think the limiting step of drug discovery is the process of evaluating the therapeutic effects of candidate drugs. To overcome this problem, I would like to propose a novel approach, "drug discovery with silkworms".
Silk peptide treatment potentiates natural killer cell activity and induces natural killer cell maturation and activation in mouse splenocytes.
Jang Sun-Hee,Oh Mi-Sun,Baek Hyang-Im,Ha Ki-Chan,Lee Jeong-Yong,Jang Yong-Suk
Silk peptide from cocoons of silkworm ( L., Bombycidae) has been employed as a biomedical material and exhibits various bioactivities, including immune-modulating activity. We analyzed whether silk peptide exerts direct modulating effects on NK cells using an NK cell line and splenocytes. We also attempted to delineate the mechanism underlying the modulation. activity of silk peptide on NK cells was determined by measurement of cytolytic activity against K562 cells at an effector-to-target ratio of 5:1 after incubation of NK-92MI cells with silk peptide (0-2000 μg/mL) for 48 and 72 h. activity of silk peptide on mouse splenic NK cells was determined similarly by using YAC-1 cells. Treatment of NK-92MI NK cells with silk peptide (500-2000 μg/mL) significantly increased cytolytic activity on target cells by 2- to 4-fold. The same concentrations (500-2000 μg/mL) of silk peptide treatment also significantly enhanced the cytolytic activity of splenic NK cells against YAC-1 cells. Silk peptide treatment of IL-2-stimulated splenocytes induced enhanced expression of Th1, 2 and 17 cytokines including TNF-α, IFN-γ, IL-6, IL-4 and IL-17. Finally, treatment with silk peptide on mouse splenocytes significantly enhanced the degree of NK cell maturation in a dose-dependent manner from 3.49 to 23.79%. These findings suggest that silk peptide stimulates NK cells, thereby influencing systemic immune functions and improving natural immunity. Thus, silk peptide could be useful as a complementary therapy in cancer patients.
Silk Fibroin-Based Hydrogels and Scaffolds for Osteochondral Repair and Regeneration.
Ribeiro Viviana P,Pina Sandra,Oliveira J Miguel,Reis Rui L
Advances in experimental medicine and biology
Osteochondral lesions treatment and regeneration demands biomimetic strategies aiming physicochemical and biological properties of both bone and cartilage tissues, with long-term clinical outcomes. Hydrogels and scaffolds appeared as assertive approaches to guide the development and structure of the new osteochondral engineered tissue. Moreover, these structures alone or in combination with cells and bioactive molecules bring the mechanical support after in vitro and in vivo implantation. Moreover, multilayered structures designed with continuous interfaces furnish appropriate features of the cartilage and subchondral regions, namely microstructure, composition, and mechanical properties. Owing the potential as scaffolding materials, natural and synthetic polymers, bioceramics, and composites have been employed. Particularly, significance is attributed to the natural-based biopolymer silk fibroin from the Bombyx mori silkworm, considering its unique mechanical and biological properties. The significant studies on silk fibroin-based structures, namely hydrogels and scaffolds, towards bone, cartilage, and osteochondral tissue repair and regeneration are overviewed herein. The developed biomimetic strategies, processing methodologies, and final properties of the structures are summarized and discussed in depth.
Synthetic Engineering of Spider Silk Fiber as Implantable Optical Waveguides for Low-Loss Light Guiding.
Qiao Xin,Qian Zhigang,Li Junjie,Sun Hongji,Han Yao,Xia Xiaoxia,Zhou Jin,Wang Chunlan,Wang Yan,Wang Changyong
ACS applied materials & interfaces
A variety of devices used for biomedical engineering have been fabricated using protein polymer because of their excellent properties, such as strength, toughness, biocompatibility, and biodegradability. In this study, we fabricated an optical waveguide using genetically engineered spider silk protein. This method has two significant advantages: (1) recombinant spider silk optical waveguide exhibits excellent optical and biological properties and (2) biosynthesis of spider silk protein can overcome the limitation to the research on spider silk optical waveguide due to the low yield of natural spider silk. In detail, two kinds of protein-based optical waveguides made from recombinant spider silk protein and regenerative silkworm silk protein were successfully prepared. Results suggested that the recombinant spider silk optical waveguide showed a smoother surface and a higher refractive index when compared with regenerative silkworm silk protein. The optical loss of recombinant spider silk optical waveguide was 0.8 ± 0.1 dB/cm in air and 1.9 ± 0.3 dB/cm in mouse muscles, which were significantly lower than those of regenerative silkworm silk optical waveguide. Moreover, recombinant spider silk optical waveguide can meet the demand to guide and efficiently deliver light through biological tissue. In addition, recombinant spider silk optical waveguide showed low toxicity to cells in vitro and low-level inflammatory reaction with surrounding tissue in vivo. Therefore, recombinant spider silk optical waveguide is a promising implantable device to guide and deliver light with low loss.
Purification and characterization of a metalloprotease produced by the C8 isolate of Serratia marcescens using silkworm pupae or casein as a protein source.
Vélez-Gómez Jenny Marcela,Melchor-Moncada Jhon Jairo,Veloza Luz Angela,Sepúlveda-Arias Juan Carlos
International journal of biological macromolecules
Serratiopeptidase, a metalloprotease produced by Serratia marcescens, is produced through a fermentation process using carbohydrates and proteins as carbon and nitrogen sources. However, some byproducts of the silk industry could be an alternative source for serratiopeptidase production. Therefore, the present work is focused on the purification and characterization of a serratiopeptidase produced from the C8 isolate of Serratia marcescens and obtained from a Colombian silkworm hybrid using casein or silkworm pupae. The protease was purified using ultrafiltration, anion-exchange, and size-exclusion chromatography. The purified enzyme showed a molecular weight of ~50 kDa with a purity above 96%, an isoelectric point of ~4.6, optimum pH and temperature of 6 and 50 °C, and stability at 4 °C for one month. The kinetic constants using azocasein as substrate were 0.63 mM (K), 2,016 μM/min (V), 41.41 s (K), and 6.56 × 10 M s (K/K). Inhibition by 5 mM EDTA or 1,10-phenanthroline was recovered by adding Zn at the same concentration. Mass spectrometry analysis indicated 94% homology with the sequence of serratiopeptidase produced by the E-15 strain. We purified and characterized a serratiopeptidase produced by the C8 isolate of S. marcescens in a culture medium based on a renewable source from the silk industry.
Silkworm Silk Scaffolds Functionalized with Recombinant Spider Silk Containing a Fibronectin Motif Promotes Healing of Full-Thickness Burn Wounds.
Chouhan Dimple,Lohe Tshewuzo-U,Thatikonda Naresh,Naidu Vgm,Hedhammar My,Mandal Biman B
ACS biomaterials science & engineering
Full-thickness cutaneous wounds, such as deep burns, are complex wounds that often require surgical interventions. Herein, we show the efficacy of acellular grafts that can be made available off-the-shelf at an affordable cost using silk biomaterials. Silkworm silk fibroin (SF), being a cost-effective and natural biopolymer, provides essential features required for the fabrication of three-dimensional constructs for wound-healing applications. We report the treatment of third-degree burn wounds using a freeze-dried microporous scaffold of SF (AaSF) functionalized with a recombinant spider silk fusion protein FN-4RepCT (FN-4RC) that holds the fibronectin cell binding motif. In order to examine the healing efficiency of functionalized silk scaffolds, an in vivo burn rat model was used, and the scaffolds were implanted by a one-step grafting procedure. The aim of our work is to investigate the efficacy of the developed acellular silk grafts for treating full-thickness wounds as well as to examine the effect of recombinant spider silk coatings on the healing outcomes. Following 14-day treatment, AaSF scaffolds coated with FN-4RC demonstrated accelerated wound healing when compared to the uncoated counterpart, commercially used DuoDERM dressing patch, and untreated wounds. Histological assessments of wounds over time further confirmed that functionalized silk scaffolds promoted wound healing, showing vascularization and re-epithelialization in the initial phase. In addition, higher extent of tissue remodeling was affirmed by the gene expression study of collagen type I and type III, indicating advanced stage of healing by the silk treatments. Thus, the present study validates the potential of scaffolds of combined silkworm silk and FN-4RC for skin regeneration.
Rational design of a high-strength bone scaffold platform based on in situ hybridization of bacterial cellulose/nano-hydroxyapatite framework and silk fibroin reinforcing phase.
Jiang Pei,Ran Jiabing,Yan Pan,Zheng Lingyue,Shen Xinyu,Tong Hua
Journal of biomaterials science. Polymer edition
Bacterial cellulose/hydroxyapatite (BC/HAp) composite had favourable bioaffinity but its poor mechanical strength limited its widespread applications in bone tissue engineering (BTE). Silk fibroin, which possesses special crystalline structure, has been widely used as organic reinforcing material, and different SFs have different amino acid sequences, which exhibit different bioaffinity and mechanical properties. In this regard, bacterial cellulose-Antheraea yamamai silk fibroin/hydroxyapatite (BC-AYSF/HAp), bacterial cellulose-Bombyx mori silk fibroin/hydroxyapatite (BC-BMSF/HAp), and BC/HAp nano-composites were synthesized via a novel in situ hybridization method. Compared with BC/HAp and BC-BMSF/HAp, the BC-AYSF/HAp exhibited better interpenetration, which may benefit for the transportation of nutrients and wastes, the adhesion of cells as well. Additionally, the BC-AYSF/HAp also presented superior thermal stability than the other two composites revealed by differential thermal analysis (DTA) and thermogravimetric analysis (TGA). Compression testing indicated that the mechanical strength of BC-BMSF/HAp was greatly reinforced compared with BC/HAp and was even a little higher than that of BC-AYSF/HAp. Tensile testing showed that BC-AYSF/HAp possesses extraordinary mechanical properties with a higher elastic modulus at low strain and higher fracture strength simultaneously than the other two composites. In vitro cell culture exhibited that MC3T3-E1 cells on the BC-AYSF/HAp membrane took on higher proliferative potential than those on the BC-BMSF/HAp membrane. These results suggested that compared with BC-BMSF/HAp, the BC-AYSF/HAp composite was more appropriate as an ideal bone scaffold platform or biomedical membrane to be used in BTE.
Green Fabrication of Silkworm Cocoon-like Silicon-Based Composite for High-Performance Li-Ion Batteries.
Du Fei-Hu,Ni Yizhou,Wang Ye,Wang Dong,Ge Qi,Chen Shuo,Yang Hui Ying
Designing yolk-shell nanostructures is an effective way of addressing the huge volume expansion issue for large-capacity anode and cathode materials in Li-ion batteries (LIBs). Previous studies mainly focused on adopting a SiO template through HF etching to create yolk-shell nanostructures. However, HF etching is highly corrosive and may result in a significant reduction of Si content in the composite. Herein, a silkworm cocoon-like silicon-based composite is prepared through a green approach in which AlO was selected as a sacrificial template. The void space between the outer nitrogen-doped carbon (NC) shell formed by chemical vapor deposition using a pyridine precursor and the inside porous silicon nanorods (p-Si NRs) synthesized by magnesiothermic reduction of ordered mesoporous silica nanorods can be generated by etching AlO with diluted HCl. The obtained p-Si NRs@void@NC composite is utilized as an anode material for LIBs, which exhibits a large initial discharge capacity of 3161 mAh g at 0.5 A g, excellent cycling behavior up to 300 cycles, and super rate performance. Furthermore, a deep understanding of the mechanism for the yolk-shell nanostructure during the Li-alloying process is revealed by in situ transmission electron microscopy and finite element simulation.
Multi-channel silk sponge mimicking bone marrow vascular niche for platelet production.
Tozzi Lorenzo,Laurent Pierre-Alexandre,Di Buduo Christian A,Mu Xuan,Massaro Angelo,Bretherton Ross,Stoppel Whitney,Kaplan David L,Balduini Alessandra
In the bone marrow, the interaction of progenitor cells with the vasculature is fundamental for the release of blood cells into circulation. Silk fibroin, derived from Bombyx mori silkworm cocoons, is a promising protein biomaterial for bone marrow tissue engineering, because of its tunable architecture and mechanical properties, the capacity to incorporate labile compounds without loss of bioactivity and the demonstrated ability to support blood cell formation without premature activation. In this study, we fabricated a custom perfusion chamber to contain a multi-channel lyophilized silk sponge mimicking the vascular network in the bone marrow niche. The perfusion system consisted in an inlet and an outlet and 2 splitters that allowed funneling flow in each single channel of the silk sponge. Computational Fluid Dynamic analysis demonstrated that this design permitted confined flow inside the vascular channels. The silk channeled sponge supported efficient platelet release from megakaryocytes (Mks). After seeding, the Mks localized along SDF-1α functionalized vascular channels in the sponge. Perfusion of the channels allowed the recovery of functional platelets as demonstrated by increased PAC-1 binding upon thrombin stimulation. Further, increasing the number of channels in the silk sponge resulted in a proportional increase in the numbers of platelets recovered, suggesting applicability to scale-up for platelet production. In conclusion, we have developed a scalable system consisting of a multi-channeled silk sponge incorporated in a perfusion chamber that can provide useful technology for functional platelet production ex vivo.
[Development of Enzyme Drugs Derived from Transgenic Silkworms to Treat Lysosomal Diseases].
Itoh Kohji,Nishioka So-Ichiro,Hidaka Tomo,Tsuji Daisuke,Maita Nobuo
Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan
Lysosomal storage diseases (LSDs) are inborn errors caused by genetic defects of lysosomal enzymes associated with the excessive accumulation of natural substrates and neurovisceral manifestations. Until now, enzyme replacement therapy (ERT) with human lysosomal enzymes produced by genetically engineered mammalian cell lines has been applied clinically to treat several LSDs. ERT is based on the incorporation of N-glycosylated lysosomal enzymes through binding to glycan receptors on the surface of target cells and delivery to lysosomes. However, ERT has several disadvantages, including difficulty in mass producing human enzymes, dangers of pathogen contamination, and high cost. Recently, we have succeeded in producing transgenic silkworms which overexpress human lysosomal enzymes in silk glands, and have purified active and functional enzymes from middle silk glands and cocoons. Silk gland- and cocoon-derived human enzymes carrying high-mannose and pauci-mannose N-glycans are endocytosed by monocytes via the mannose receptor pathway; these were then delivered to lysosomes. Human cathepsin A (Ctsa) precursor proteins purified from the cocoons have been found to suppress microglial activation in the brains of Ctsa-deficient mice; this deficiency is caused by a splicing defect, and serves as a galactosialidosis model associated with the combination of a deficiency of lysosomal neuraminidase 1 (NEU1) and the accumulation of sialyloligosaccharides. Transgenic silkworms overexpressing human lysosomal enzymes in silk glands could serve as a future bioresource to provide safe therapeutic enzymes for the treatment of LSDs. The combination of recent developments in transglycosylation technology with microbial endoglycosidases will aid in the development of therapeutic glycoproteins as bio-medicines.
Porous, Aligned, and Biomimetic Fibers of Regenerated Silk Fibroin Produced by Solution Blow Spinning.
Magaz Adrián,Roberts Aled D,Faraji Sheida,Nascimento Tatiana R L,Medeiros Eliton S,Zhang Wenzhao,Greenhalgh Ryan D,Mautner Andreas,Li Xu,Blaker Jonny J
Solution blow spinning (SBS) has emerged as a rapid and scalable technique for the production of polymeric and ceramic materials into micro-/nanofibers. Here, SBS was employed to produce submicrometer fibers of regenerated silk fibroin (RSF) from Bombyx mori (silkworm) cocoons based on formic acid or aqueous systems. Spinning in the presence of vapor permitted the production of fibers from aqueous solutions, and high alignment could be obtained by modifying the SBS setup to give a concentrated channeled airflow. The combination of SBS and a thermally induced phase separation technique (TIPS) resulted in the production of macro-/microporous fibers with 3D interconnected pores. Furthermore, a coaxial SBS system enabled a pH gradient and kosmotropic salts to be applied at the point of fiber formation, mimicking some of the aspects of the natural spinning process, fostering fiber formation by self-assembly of the spinning dope. This scalable and fast production of various types of silk-based fibrous scaffolds could be suitable for a myriad of biomedical applications.
Expression and characterization of recombinant human VEGF165 in the middle silk gland of transgenic silkworms.
Zhang Tianyang,Liu Rongpeng,Luo Qin,Qu Dawei,Chen Tao,Yao Ou,Xu Hanfu
Recombinant human vascular endothelial growth factor (rhVEGF) has important applications in therapeutic angiogenesis and inhibition of VEGF-mediated pathological angiogenesis. Previous studies have shown that rhVEGF can be produced in several expression systems, including Escherichia coli, yeasts, insect cells and mammalian cells. However, little is known regarding the effective production of this protein in organs of live organisms. Here, we report for the first time the expression and characterization of rhVEGF165 in the middle silk gland (MSG) of the transgenic silkworm line S1-V165. Our results confirmed that (1) rhVEGF165 was highly expressed in MSG cells and was secreted into the cocoon of S1-V165; (2) the dimeric form of rhVEGF165 could be easily dissolved from S1-V165 cocoons using an alkaline solution; (3) rhVEGF165 extracted from S1-V165 cocoons exhibited slightly better cell proliferative activity than the hVEGF165 standard in cultured human umbilical vein endothelial cells. This study provides an alternative strategy for the production of bioactive rhVEGF165 using the MSG of transgenic silkworms.
CRISPR/Cas9 Initiated Transgenic Silkworms as a Natural Spinner of Spider Silk.
Zhang Xiaoli,Xia Lijin,Day Breton A,Harris Thomas I,Oliveira Paula,Knittel Chelsea,Licon Ana Laura,Gong Chengliang,Dion Geneviève,Lewis Randolph V,Jones Justin A
Using transgenic silkworms with their natural spinning apparatus has proven to be a promising way to spin spider silk-like fibers. The challenges are incorporating native-size spider silk proteins and achieving an inheritable transgenic silkworm strain. In this study, a CRISPR/Cas9 initiated fixed-point strategy was used to successfully incorporate spider silk protein genes into the Bombyx mori genome. Native-size spider silk genes (up to 10 kb) were inserted into an intron of the fibroin heavy or light chain (FibH or FibL) ensuring that any sequence changes induced by the CRISPR/Cas9 would not impact protein production. The resulting fibers are as strong as native spider silks (1.2 GPa tensile strength). The transgenic silkworms have been tracked for several generations with normal inheritance of the transgenes. This strategy demonstrates the feasibility of using silkworms as a natural spider silk spinner for industrial production of high-performance fibers.
Analysis of the pressure requirements for silk spinning reveals a pultrusion dominated process.
Sparkes James,Holland Chris
Silks are remarkable materials with desirable mechanical properties, yet the fine details of natural production remain elusive and subsequently inaccessible to biomimetic strategies. Improved knowledge of the natural processes could therefore unlock development of a host of bio inspired fibre spinning systems. Here, we use the Chinese silkworm Bombyx mori to review the pressure requirements for natural spinning and discuss the limits of a biological extrusion domain. This provides a target for finite element analysis of the flow of silk proteins, with the aim of bringing the simulated and natural domains into closer alignment. Supported by two parallel routes of experimental validation, our results indicate that natural spinning is achieved, not by extruding the feedstock, but by the pulling of nascent silk fibres. This helps unravel the oft-debated question of whether silk is pushed or pulled from the animal, and provides impetus to the development of pultrusion-based biomimetic spinning devices.The natural production of silks remains elusive and subsequently inaccessible to biomimetic strategies. Here the authors show that silks cannot be spun by pushing alone, and that natural spinning is dominated by pultrusion, which provides design guidelines for future biomimetic spinning systems.
Modular flow chamber for engineering bone marrow architecture and function.
Di Buduo Christian A,Soprano Paolo M,Tozzi Lorenzo,Marconi Stefania,Auricchio Ferdinando,Kaplan David L,Balduini Alessandra
The bone marrow is a soft, spongy, gelatinous tissue found in the hollow cavities of flat and long bones that support hematopoiesis in order to maintain the physiologic turnover of all blood cells. Silk fibroin, derived from Bombyx mori silkworm cocoons, is a promising biomaterial for bone marrow engineering, because of its tunable architecture and mechanical properties, the capacity of incorporating labile compounds without loss of bioactivity and demonstrated ability to support blood cell formation. In this study, we developed a bone marrow scaffold consisting of a modular flow chamber made of polydimethylsiloxane, holding a silk sponge, prepared with salt leaching methods and functionalized with extracellular matrix components. The silk sponge was able to support efficient platelet formation when megakaryocytes were seeded in the system. Perfusion of the chamber allowed the recovery of functional platelets based on multiple activation tests. Further, inhibition of AKT signaling molecule, which has been shown to be crucial in regulating physiologic platelet formation, significantly reduced the number of collected platelets, suggesting the applicability of this tissue model for evaluation of the effects of bone marrow exposure to compounds that may affect platelet formation. In conclusion, we have bioengineered a novel modular system that, along with multi-porous silk sponges, can provide a useful technology for reproducing a simplified bone marrow scaffold for blood cell production ex vivo.
Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms.
Kumano-Kuramochi Miyuki,Tatematsu Ken-Ichiro,Ohnishi-Kameyama Mayumi,Maeda-Yamamoto Mari,Kobori Toshiro,Sezutsu Hideki,Machida Sachiko
Here, we demonstrated the expression of the N-glycosylated extracellular ligand binding domain of receptor for advanced glycation end products (sRAGE) in middle silk glands (MSGs) of transgenic silkworms using the GAL4/UAS system. Over 1 mg of sRAGE was obtained from one transgenic silkworm. sRAGE purified from the silkworm exhibited good stability and maintained specific ligand-binding ability. In addition, N-glycan analysis of sRAGE revealed that N-glucan completely lacked potentially allergenic fucose. Moreover, co-expression of biotin ligase (BirA) with C-terminal BioEase-tagged sRAGE in MSGs resulted in efficient biotinylation of sRAGE after addition of biotin bait. C-terminal biotinylated sRAGE could be immobilized onto a solid surface in one direction through binding to streptavidin without any loss of ability. The dissociation constant of sRAGE with fructose-BSA, a typical RAGE ligand, was 7.25 × 10 M, consistent with that on the mammalian cell surface. Thus, we developed a novel, innovative silkworm expression system for efficient expression of recombinant sRAGE, which could serve as a basis for the elucidation of RAGE-ligand interactions and facilitate the search for new ligands and inhibitors.
Neospora caninum antigens displaying virus-like particles as a bivalent vaccine candidate against neosporosis.
Xu Jian,Hiramatsu Rikito,Suhaimi Hamizah,Kato Tatsuya,Fujimoto Akari,Tokiwa Toshihiro,Ike Kazunori,Park Enoch Y
Neospora caninum is a causative and transmissible agent of dog and bovine neosporosis. The resulting reproductive failures in infected cattle lead to significant economic losses worldwide. However, there is no satisfactory treatment or vaccine currently available to combat this pathogen. Thus, the development of appropriate vaccines to manage its infection and transmission is urgently needed. In this study, we expressed Rous sarcoma virus-like particles (RSV-LP) that displayed dual N. caninum antigens in silkworms. The antigen candidates are modified by adding a transmembrane domain of GP64 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) to the C-terminus of surface antigen 1 (NcSAG1) and SAG1-related sequence 2 (NcSRS2). The NcSRS2 alone or the NcSAG1/NcSRS2 bivalent form displaying RSV-LPs were purified using sucrose density gradient centrifugation. These purified VLPs were then used for immunizations in gerbils, Meriones unguiculatus, to evaluate the anti-N. caninum effects in vivo. The results demonstrated that antigens displaying RSV-LPs in immunized gerbils produced the antigen-specific antibody, leading to a relatively lower parasite load after infections of N. caninum. To the best of our knowledge, this is the first study to present an RSV-LP vaccine displaying bivalent antigens from neosporosis. Taken together, our strategy suggests that silkworm-expressed virus-like particles (VLPs) are promising bivalent vaccine candidates against N. caninum infections.
Molecular mechanisms of Bombyx batryticatus ethanol extract inducing gastric cancer SGC-7901 cells apoptosis.
Wu Jin-Yi,Sheikho Almutamad,Ma He,Li Tian-Ci,Zhao Ya-Qi,Zhang Ya-Lin,Wang Dun
Bombyx batryticatus is a traditional Chinese medicine. To understand apoptotic effect of B. batryticatus ethanol extract (BBE), we investigated the role of BBE in inducing apoptosis of human gastric cancer cells SGC-7901. Cells treated with BBE and apoptosis was assessed by methyl thiazolyl tetrazolium (MTT) assay, morphological changes, DNA fragmentation and flow cytometry assays. The expression of Bcl-2, Bax and P21 were evaluated by western blot analysis and real time polymerase chain reaction. MTT assay showed that the cytotoxicity of BBE extract on SGC-7901 cells was correlated with treatment time and concentration. After treatment with 6 mg/mL of BBE the microscopy showed that, the majority of SGC-7901 cells were obviously reduced, distorted and grew slowly. Annexin-V/propidium iodide double-staining assay emerge the early apoptosis and the late apoptosis after treatment with different times by laser confocal fluorescence microscopy and flow cytometer. Cell cycle analysis of SGC 79 cells showed that BBE induced cell cycle arrest in the G1 and G2 phases. DNA fragmentation indicated the trend of BBE inducing apoptosis on SGC-7901 cells. The qRT-PCR and western blot analysis indicated that the mRNA and protein expressions of Bax and P21 were significantly up-regulated whereas that of Bc1-2 was down-regulated after treatment with BBE for 24 h. Our results revealed a correlation between gene regulation and BBE-induced apoptosis, which might indicate the potential of BBE in cancer therapy.
Porous Fe@C Composites Derived from Silkworm Excrement for Effective Separation of Anisole Compounds.
Wu Yuxiang,Huang Yan,Huang Hong,Muhammad Yaseen,Huang Zuqiang,Winarta Joseph,Zhang Yanjuan,Nie Shuangxi,Zhao Zhongxing,Mu Bin
Silkworm excrement is a very useful biomass waste, composed of layer-structured fats and proteins, which are great precursors for carbon composite materials. In this work, new porous composites derived from silkworm excrement were prepared for selective separation of flavor 4-methylanisole from the binary 4-methylanisole/4-anisaldehyde mixture. In particular, the silkworm excrement, possessing a unique nanosheet structure, is converted into a graphite-like carbon by a simple calcination strategy followed by a metal-ion-doping procedure. This Fe@C composite exhibits a special nano-spongy morphology, anchoring FeC/FeC on the carbon nanosheets. Density functional theory simulations showed that 4-methylanisole presents a stronger π-π interaction and attraction forces with sp carbon nanosheets in Fe@C composites than 4-anisaldehyde. The selective adsorption experiments further confirmed that the Fe@C composites exhibited a 4-methylanisole capacity of 7.3 mmol/g at 298 K and the highest selectivity of 17 for an equimolar 4-methylanisole/4-anisaldehyde mixture among the examined adsorbents including MOFs and commercial activated carbon materials, which demonstrates the potential of this low-cost and eco-friendly porous carbon material as a promising sustainable adsorbent.
Functionalized silk fibers from transgenic silkworms for wound healing applications: Surface presentation of bioactive epidermal growth factor.
Wöltje Michael,Böbel Melanie,Bienert Michaela,Neuss Sabine,Aibibu Dilibaier,Cherif Chokri
Journal of biomedical materials research. Part A
Growth factors play a crucial role in wound healing in general and are promising tools for the treatment of chronic wounds as they can restore the physiological wound healing process. In growth factor-loaded wound dressings, human epidermal growth factor (EGF) is released in a burst and washed out quickly. The developed matrix consists of recombinant EGF produced in transgenic silkworms as a fusion protein with the fibroin light chain. The covalent linkage prevents EGF from draining into the surrounding tissue while presenting the growth factor on the surface. EGF-functionalized silk membranes and nonwovens lead to a 2.5-fold increase in the cell number of fibroblasts, while retaining full bioactivity even after e-beam sterilization. EGF is long-term presented without burst release and significantly reduces the wound area by 15% in an in vitro wound model. Hence, the cost-effective production of a biomaterial using transgenic silkworm larvae in combination with a growth factor paves the way for a promising new multifactorial wound cover for chronic wound healing. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2643-2652, 2018.
A Novel Spiro-Heterocyclic Compound Identified by the Silkworm Infection Model Inhibits Transcription in .
Paudel Atmika,Hamamoto Hiroshi,Panthee Suresh,Kaneko Keiichi,Matsunaga Shigeki,Kanai Motomu,Suzuki Yutaka,Sekimizu Kazuhisa
Frontiers in microbiology
Synthetic compounds are a vital source of antimicrobial agents. To uncover therapeutically effective antimicrobial agents from a chemical library, we screened over 100,000 synthetic compounds for antimicrobial activity against methicillin-resistant and evaluated the therapeutic effectiveness of the hits in -infected silkworms. Three antimicrobial agents exhibited therapeutic effects in the silkworm infection model. One of these, GPI0363, a novel spiro-heterocyclic compound, was bacteriostatic and inhibited RNA synthesis in cells. GPI0363-resistant strains harbored a point mutation in the gene encoding the primary sigma factor, SigA, of RNA polymerase, and this mutation was responsible for the resistance to GPI0363. We further revealed that GPI0363 could bind to SigA, inhibit promoter-specific transcription , and prolong the survival of mice infected with methicillin-resistant . Thus, GPI0363 is an attractive candidate therapeutic agent against drug-resistant infections.
Facile Photochemical Modification of Silk Protein-Based Biomaterials.
Hardy John G,Bertin Annabelle,Torres-Rendon Jose Guillermo,Leal-Egaña Aldo,Humenik Martin,Bauer Felix,Walther Andreas,Cölfen Helmut,Schlaad Helmut,Scheibel Thomas R
Silk protein-based materials show promise for application as biomaterials for tissue engineering. The simple and rapid photochemical modification of silk protein-based materials composed of either Bombyx mori silkworm silk or engineered spider silk proteins (eADF4(C16)) is reported. Radicals formed on the silk-based materials initiate the polymerization of monomers (acrylic acid, methacrylic acid, or allylamine) which functionalize the surface of the silk materials with poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), or poly(allylamine) (PAAm). To demonstrate potential applications of this type of modification, the polymer-modified silks are mineralized. The PAA- and PMAA-functionalized silks are mineralized with calcium carbonate, whereas the PAAm-functionalized silks are mineralized with silica, both of which provide a coating on the materials that may be useful for bone tissue engineering, which will be the subject of future investigations.
Genetic engineered color silk: fabrication of a photonics material through a bioassisted technology.
Bioinspiration & biomimetics
Silk produced by the silkworm Bombyx mori is an attractive material because of its luster, smooth and soft texture, conspicuous mechanical strength, good biocompatibility, slow biodegradation, and carbon neutral synthesis. Silkworms have been domesticated and bred for production of better quality and quantity of silk, resulting in the development of sericulture and the textile industry. Silk is generally white, so dyeing is required to obtain colored fiber. However, the dyeing process involves harsh conditions and generates a large volume of waste water, which have environmentally and economically negative impacts. Although some strains produce cocoons that contain pigments derived from the mulberry leaves that they eat, the pigments are distributed in the sericin layer and are lost during gumming. In trials for production of colored silk by feeding silkworms on diets containing dyes, only limited species of dye molecules were incorporated into the silk threads. A method for the generation of transgenic silkworm was established in conjunction with the discovery of green fluorescent protein (GFP), and silkworms carrying the GFP gene spun silk threads that formed cocoons that glowed bright green and still retained the original properties of silk. A wide range of color variation of silk threads has been obtained by replacing the GFP gene with the genes of other fluorescent proteins chosen from the fluorescent protein palette. The genetically modified silk with photonic properties can be processed to form various products including linear threads, 2D fabrics, and 3D materials. The transgenic colored silk could be economically advantageous due to addition of a new value to silk and reduction of cost for water waste, and environmentally preferable for saving water. Here, I review the literature regarding the production methods of fluorescent silk from transgenic silkworms and present examples of genetically modified color silk.
Silkworms with Spider Silklike Fibers Using Synthetic Silkworm Chow Containing Calcium Lignosulfonate, Carbon Nanotubes, and Graphene.
Zhang Xiaoli,Licon Ana Laura,Harris Thomas I,Oliveira Paula F,McFarland Bailey J,Taurone Blake E,Walsh Brittney J,Bell Brianne E,Walker Caleb T,Lewis Randolph V,Jones Justin A
Silkworm silk has become increasingly relevant for material applications. However, the industry as a whole is retracting because of problems with mass production. One of the key problems is the inconsistent properties of the silk. A means by which to improve the silk material properties is through enhanced sericulture techniques. One possible technique is altering the feed of the silkworms to include single-wall carbon nanotubes (SWNTs) or graphene (GR). Recently published results have demonstrated substantial improvement in fiber mechanical properties. However, the effect of the surfactant used to incorporate those materials into the feed on the fiber mechanical properties in comparison to normal silkworm silk has not been studied or reported. Thus, the total effect of feeding the SWNT and GR in the presence of surfactants on silkworms is not understood. Our study focuses on the surfactant [calcium lignosulfonate (LGS)] and demonstrates that it alone results in appreciable improvement of mechanical properties in comparison to nontreated silkworm silk. Furthermore, our study demonstrates that mixing the LGS, SWNT, and GR directly into the artificial diet of silkworms yields improved mechanical properties without decline below the control silk at high doses of SWNT or GR. Combined, we present evidence that mixing surfactants, in this case LGS, directly with the diet of silkworms creates a high-quality fiber product that can exceed 1 GPa in tensile strength. With the addition of nanocarbons, either SWNT or GR, the improvement is even greater and consistently surpasses control fibers. However, feeding LGS alone is a more economical and practical choice to consistently improve the mechanical properties of silkworm fiber.
Nitrogen-Doped Carbon Nanoparticles Derived from Silkworm Excrement as On⁻Off⁻On Fluorescent Sensors to Detect Fe(III) and Biothiols.
Lu Xingchang,Liu Chen,Wang Zhimin,Yang Junyi,Xu Mengjing,Dong Jun,Wang Ping,Gu Jiangjiang,Cao Feifei
Nanomaterials (Basel, Switzerland)
On⁻off⁻on fluorescent sensors based on emerging carbon nanoparticles (CNPs) or carbon dots (CDs) have attracted extensive attention for their convenience and efficiency. In this study, dumped silkworm excrement was used as a novel precursor to prepare fluorescent nitrogen-doped CNPs (N-CNPs) through hydrothermal treatment. The obtained N-CNPs showed good photoluminescent properties and excellent water dispersibility. Thus, they were applied as fluorescence “on⁻off⁻on” probes for the detection of Fe(III) and biothiols. The “on⁻off” process was achieved by adding Fe(III) into N-CNP solution, which resulted in the selective fluorescence quenching, with the detection limit of 0.20 μM in the linear range of 1⁻500 μM. Following this, the introduction of biothiols could recover the fluorescence efficiently, in order to realize the “off⁻on” process. By using glutathione (GSH) as the representative, the linear range was in the range of 1⁻1000 μM, and the limit of detection was 0.13 μM. Moreover, this useful strategy was successfully applied for the determination of amounts of GSH in fetal calf serum samples.
An invertebrate infection model for evaluating anti-fungal agents against dermatophytosis.
Ishii Masaki,Matsumoto Yasuhiko,Yamada Tsuyoshi,Abe Shigeru,Sekimizu Kazuhisa
Animal models of pathogenic infection are needed to evaluate candidate compounds for the development of anti-infectious drugs. Dermatophytes are pathogenic fungi that cause several infectious diseases. We established a silkworm dermatophyte infection model to evaluate anti-fungal drugs. Injection of conidia of the dermatophyte Arthroderma vanbreuseghemii into silkworms was lethal. A. vanbreuseghemii conidia germinated in liquid culture were more potent against silkworms than non-germinated conidia. Germinated conidia of other dermatophytes, Arthroderma benhamiae, Trichophyton rubrum, and Microsporum canis, also killed silkworms. Injection of heat-treated germinated A. vanbreuseghemii conidia did not kill silkworms, suggesting that only viable fungi are virulent. Injecting terbinafine or itraconazole, oral drugs used clinically to treat dermatophytosis, into the silkworm midgut had therapeutic effects against infection with germinated A. vanbreuseghemii conidia. When silkworms were injected with A. vanbreuseghemii expressing enhanced green fluorescent protein (eGFP), mycelial growth of the fungus was observed in the fat body and midgut. Injection of terbinafine into the silkworm midgut, which corresponds to oral administration in humans, inhibited the growth of A. vanbreuseghemii expressing eGFP in the fat body. These findings suggest that the silkworm infection model with eGFP-expressing dermatophytes is useful for evaluating the therapeutic activity of orally administered anti-fungal agents against dermatophytes.
Silk fibroin produced by transgenic silkworms overexpressing the Arg-Gly-Asp motif accelerates cutaneous wound healing in mice.
Baba Atsunori,Matsushita Shigeto,Kitayama Kasumi,Asakura Tetsuo,Sezutsu Hideki,Tanimoto Akihide,Kanekura Takuro
Journal of biomedical materials research. Part B, Applied biomaterials
We investigated the effect of silk fibroin (SF) on wound healing in mice. SF or an amorphous SF film (ASFF) prepared from silk produced by the wild-type silkworm Bombyx mori (WT-SF, WT-ASFF) or by transgenic worms that overexpress the Arg-Gly-Asp (RGD) sequence (TG-SF, TG-ASFF) was placed on 5-mm diameter full-thickness skin wounds made by biopsy punch on the back of 8-12 week-old BALB/c mice. Each wound was covered with WT-ASFF and urethane film (UF), TG-ASFF plus UF, or UF alone (control). Wound closure, histological thickness, the area of granulation tissue, and neovascularization were analyzed 4, 8, and 12 days later. The effect of SF on cell migration and proliferation was examined in vitro by scratch- and MTT-assay using human dermal fibroblasts. Wound closure was prompted by TG-ASFF, granulation tissue was thicker and larger in ASFF-treated wounds than the control, and neovascularization was promoted significantly by WT-ASFF. Both assays showed that SF induced the migration and proliferation of human dermal fibroblasts. The effects of TG-ASFF and TG-SF on wound closure, granulation formation, and cell proliferation were more profound than that of WT-ASFF and WT-SF. We document that SF accelerates cutaneous wound healing, and this effect is enhanced with TG-SF. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 97-103, 2019.
A silkworm based silk gland bioreactor for high-efficiency production of recombinant human lactoferrin with antibacterial and anti-inflammatory activities.
Journal of biological engineering
BACKGROUND:Silk glands are used by silkworms to spin silk fibers for making their cocoons. These have recently been regarded as bioreactor hosts for the cost-effective production of other valuable exogenous proteins and have drawn wide attention. RESULTS:In this study, we established a transgenic silkworm strain which synthesizes the recombinant human lactoferrin (rhLF) in the silk gland and spins them into the cocoon by our previously constructed silk gland based bioreactor system. The yield of the rhLF with the highest expression level was estimated to be 12.07 mg/g cocoon shell weight produced by the transgenic silkworm strain 34. Utilizing a simple purification protocol, 9.24 mg of the rhLF with recovery of 76.55% and purity of 95.45% on average could be purified from 1 g of the cocoons. The purified rhLF was detected with a secondary structure similar with the commercially purchased human lactoferrin. Eight types of N-glycans which dominated by the GlcNAc (4) Man (3) (61.15%) and the GlcNAc (3) Man (3) (17.98%) were identified at the three typical N-glycosylation sites of the rhLF. Biological activities assays showed the significant evidence that the purified rhLF could relief the lipopolysaccharide (LPS)-induced cell inflammation in RAW264.7 cells and exhibit potent antibacterial bioactivities against the () and CONCLUSIONS:These results show that the middle silk gland of silkworm can be an efficient bioreactor for the mass production of rhLF and the potential application in anti-inflammation and antibacterial.
Applicability of biotechnologically produced insect silks.
Herold Heike M,Scheibel Thomas
Zeitschrift fur Naturforschung. C, Journal of biosciences
Silks are structural proteins produced by arthropods. Besides the well-known cocoon silk, which is produced by larvae of the silk moth Bombyx mori to undergo metamorphosis inside their silken shelter (and which is also used for textile production by men since millennia), numerous further less known silk-producing animals exist. The ability to produce silk evolved multiple independent times during evolution, and the fact that silk was subject to convergent evolution gave rise to an abundant natural diversity of silk proteins. Silks are used in air, under water, or like honey bee silk in the hydrophobic, waxen environment of the bee hive. The good mechanical properties of insect silk fibres together with their non-toxic, biocompatible, and biodegradable nature renders these materials appealing for both technical and biomedical applications. Although nature provides a great diversity of material properties, the variation in quality inherent in materials from natural sources together with low availability (except from silkworm silk) impeded the development of applications of silks. To overcome these two drawbacks, in recent years, recombinant silks gained more and more interest, as the biotechnological production of silk proteins allows for a scalable production at constant quality. This review summarises recent developments in recombinant silk production as well as technical procedures to process recombinant silk proteins into fibres, films, and hydrogels.
Super-strong and Intrinsically Fluorescent Silkworm Silk from Carbon Nanodots Feeding.
Fan Suna,Zheng Xiaoting,Zhan Qi,Zhang Huihui,Shao Huili,Wang Jiexin,Cao Chengbo,Zhu Meifang,Wang Dan,Zhang Yaopeng
Fluorescent silk is fundamentally important for the development of future tissue engineering scaffolds. Despite great progress in the preparation of a variety of colored silks, fluorescent silk with enhanced mechanical properties has yet to be explored. In this study, we report on the fabrication of intrinsically super-strong fluorescent silk by feeding Bombyx mori silkworm carbon nanodots (CNDs). The CNDs were incorporated into silk fibroin, hindering the conformation transformation, confining crystallization, and inducing orientation of mesophase. The resultant silk exhibited super-strong mechanical properties with breaking strength of 521.9 ± 82.7 MPa and breaking elongation of 19.2 ± 4.3%, improvements of 55.1% and 53.6%, respectively, in comparison with regular silk. The CNDs-reinforced silk displayed intrinsic blue fluorescence when exposed to 405 nm laser and exhibited no cytotoxic effect on cells, suggesting that multi-functional silks would be potentially useful in bioimaging and other applications.
Comparative Characterization of Protein Hydrolysates from Three Edible Insects: Mealworm Larvae, Adult Crickets, and Silkworm Pupae.
Yoon Sungwon,Wong Nathan A K,Chae Minki,Auh Joong-Hyuck
Foods (Basel, Switzerland)
A comparative characterization of proteins from three edible insects- (mealworm) larvae, (cricket), and (silkworm) pupae-was performed in this study. Proteins were extracted from edible insects and their hydrolysates were prepared through enzymatic hydrolysis with commercial enzymes (Flavourzyme: 12%; Alcalase: 3%). Solubility was significantly higher following enzymatic hydrolysis, while foamability was lower compared to those of the protein control. Angiotensin-converting enzyme was significantly inhibited after enzymatic hydrolysis, especially following Alcalase treatment, with IC values of 0.047, 0.066, and 0.065 mg/mL for , larvae, and pupae, respectively. Moreover, the Alcalase-treated group of pupae and the larvae group treated with a mixture of enzymes showed the effective inhibition of α-glucosidase activity. The anti-inflammatory activity of the insect hydrolysates was assessed via nitric oxide production from macrophages, and pupae samples exhibited significant activity regardless of the method of hydrolysis. These results indicate the functional properties of protein and hydrolysates from three species of edible insects, which may be useful in their future exploitation.
Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial.
Otsuki Ryosuke,Yamamoto Masafumi,Matsumoto Erika,Iwamoto Shin-Ichi,Sezutsu Hideki,Suzui Masumi,Takaki Keiko,Wakabayashi Keiji,Mori Hajime,Kotani Eiji
Proceedings of the National Academy of Sciences of the United States of America
Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm () with posterior silk glands (PSGs) that was designed to express the cabbage butterfly () cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in -derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.
Cationized Bombyx mori silk fibroin as a delivery carrier of the VEGF165-Ang-1 coexpression plasmid for dermal tissue regeneration.
Luo Zuwei,Li Jing,Qu Jing,Sheng Weihua,Yang Jicheng,Li Mingzhong
Journal of materials chemistry. B
The angiogenesis of an implanted construct is among the most important issues in tissue engineering. In this study, spermine was used to modify Bombyx mori silk fibroin (BSF) to synthesize cationized BSF (CBSF). BSF and CBSF were coated in sequence on the surface of polyethyleneimine (PEI)/vascular endothelial growth factor 165/angiopoietin-1 coexpression plasmid DNA (pDNA) complexes to form CBSF/BSF/PEI/pDNA quaternary complexes. BSF scaffolds loaded with carrier/pDNA complexes were prepared as dermal regeneration scaffolds by freeze-drying. In one set of experiments, scaffolds were used to cover a chick embryo chorioallantoic membrane (CAM) to investigate the influence of carrier/pDNA complexes on angiogenesis; in another set of experiments, scaffolds were implanted into dorsal full-thickness wounds in Sprague-Dawley rats to evaluate the effect of carrier/pDNA complex-loaded BSF scaffolds on neovascularization and dermal tissue regeneration. After modification with spermine, the surface zeta potential value of BSF rose to +11 mV from an initial value of -9 mV, and the isoelectric point of BSF increased from 4.20 to 9.04. The in vitro transfection of human umbilical vein endothelial cells (EA.hy926) with quaternary complexes revealed that the CBSF/BSF/PEI/pDNA complexes clearly exhibited lower cytotoxicity and higher transfection efficiency than the PEI/pDNA complexes. The CAM assay showed a more abundant branching pattern of blood vessels in BSF scaffolds loaded with CBSF/BSF/PEI/pDNA complexes than in BSF scaffolds without complexes or loaded with PEI/pDNA complexes. The in vivo experimental results demonstrated that the incorporation of CBSF/BSF/PEI/pDNA complexes could effectively enhance angiogenesis in the implanted BSF scaffolds, thereby promoting the regeneration of dermal tissue, providing a new scaffold for the regeneration of dermal tissue and other tissues containing blood vessels.
Transgenic silkworms secrete the recombinant glycosylated MRJP1 protein of Chinese honeybee, Apis cerana cerana.
You Zhengying,Qian Qiujie,Wang Yiran,Che Jiaqian,Ye Lupeng,Shen Lirong,Zhong Boxiong
Major royal jelly protein-1 (MRJP1) is the most abundant glycoprotein of royal jelly (RJ) and is considered a potential component of functional foods. In this study, we used silkworm transgenic technology to obtain five transgenic silkworm lineages expressing the exogenous recombinant Chinese honeybee, Apis cerana cerana, protein-1 (rAccMRJP1) under the control of a fibroin light chain (Fib-L) promoter in the posterior silk glands. The protein was successfully secreted into cocoons; specifically, the highest rAccMRJP1 protein content was 0.78% of the dried cocoons. Our results confirmed that the protein band of the exogenous rAccMRJP1 protein expressed in the transgenic silkworm lineages was a glycosylated protein. Therefore, this rAccMRJP1 protein could be used as an alternative standard protein sample to measure the freshness of RJ. Moreover, we also found that the overall trend between the expression of the endogenous and exogenous genes was that the expression level of the endogenous Fib-L gene declined as the expression of the exogenous rAccMRJP1 gene increased in the transgenic silkworm lineages. Thus, by employing genome editing technology to reduce silk protein expression levels, a silkworm bioreactor expression system could be developed as a highly successful system for producing various valuable heterologous proteins, potentially broadening the applications of the silkworm.
Natural Silkworm Cocoon Composites with High Strength and Stiffness Constructed in Confined Cocooning Space.
Cheng Lan,Tong Xiaoling,Li Zhi,Liu Zulan,Huang Huiming,Zhao Hongping,Dai Fangyin
In this study, using round paper tubes (PTs) and rectangular cardboard boxes (CBs) as external constraints to control the size of the cocooning space, we fabricated a series of modified silkworm cocoons (PT cocoons and CB cocoons). Their microstructures, morphologies, compositions, and mechanical properties were characterized and compared with normal silkworm cocoons. These two kinds of modified silkworm cocoons exhibit dense and homogeneous layer structures. Tensile test results indicate that above a size limit of cocooning space, their tensile strengths, Young's moduli, and strain energy densities increase with the decrease in cocooning space. Especially in comparison with the normal cocoons, the tensile strength and Young's modulus of the PT-14 cocoon increase by 44% and 100%, respectively. Meanwhile, PT cocoons and CB cocoons, except PT-12, also possess better peeling resistance than normal cocoons. Owing to the dense structure and low porosity, the modified cocoons form robust fiber networks that result in high strength and toughness. This study provides a green and efficient method to fabricate mechanically enhanced silkworm cocoons with special shapes and dense layer structures. The method can be easily subjected to further modification processes and has potential applications in the production of high-performance green cocoon composites and biomimetic materials.
Honeysuckle flowers extract loaded Bombyx mori silk fibroin films for inducing apoptosis of HeLa cells.
Li Chenlin,Yang Mingying,Zhu Liangjun,Zhu Yongqiang
Microscopy research and technique
This study aimed to prepare silk fibroin (SF) films loaded with honeysuckle flowers extract (HFE) for inducing apoptosis of HeLa cells. We mixed solution of SF and HFE by air-drying for preparing the honeysuckle flowers extract loaded silk fibroin (SFH) films. The physical properties including morphologies, contact angle, roughness, and Z range were characterized. MTS assay and fluorescence micrographs proved that SFH films inhibited the proliferation rate of HeLa cells due to induction of HFE into SF films. Furthermore, cell apoptosis assay and cell cycle analysis confirmed that the apoptosis of HeLa cells resulted from SFH films. Therefore, SFH films designed in our study might be a promising candidate material for cancer therapy.
Structure analysis of the spinneret from Bombyx mori and its influence on silk qualities.
Guo Nangkuo,Lu Kunpeng,Cheng Lan,Li Zhi,Wu Chunman,Liu Zulan,Liang Shubo,Chen Sihao,Chen Wenhao,Jiang Chenlong,Dai Fangyin
International journal of biological macromolecules
Silk is an excellent natural fiber, which has been widely used in versatile fields. Silk spinning is a complex process involving the larval spinneret. The spinneret is essential for silk spinning, but the sectional morphology of the spinneret that determines the silk monofilament, the muscular activities around the silk press as well as the relationships between the spinneret and the properties of the resulting silk remain poorly understood. We studied these factors by dissecting the spinneret and analyzing silk from different Bombyx mori strains. The sectional morphology of silk monofilament was found to be largely determined by the spinneret, especially by the silk press. Moreover, contractile activity of the muscles around the silk press is high, and the contraction frequency of the muscles was estimated to range from 11.42 to 50 HZ. A comparison of the fibroin filaments before they entered the common tube indicated that the spinneret determines both silk shape and silk size. This study provides insight into the silk spinning process, which may help develop bionic spinning in further studies and also provides a rationale to study the effect of the spinneret on silk fineness at the molecular level.
Silk fiber reinforcement modulates in vitro chondrogenesis in 3D composite scaffolds.
Singh Yogendra Pratap,Adhikary Mimi,Bhardwaj Nandana,Bhunia Bibhas Kumar,Mandal Biman B
Biomedical materials (Bristol, England)
The limited self-regenerative capacity of adult cartilage has steered the upsurge in tissue engineered replacements to combat the problem of osteoarthritis. In the present study, the potential of fiber-reinforced silk composites from mulberry (Bombyx mori) and non-mulberry (Antheraea assamensis) silk has been investigated for cartilage tissue engineering. The fabricated composites were physico-chemically characterized and analyzed for cellular viability, proliferation, extracellular matrix formation and immunocompatibility. Both mulberry and non-mulberry silk composites showed effective swelling (25%-30%) and degradation (10%-30%) behavior, owing to their interconnected porous nature. The non-mulberry fiber-reinforced composite scaffolds showed slower degradation (∼90% mass remaining) than mulberry silk over a period of 28 days. The reinforcement of silk fibers within silk solution resulted in an increased compressive modulus and stiffness (nearly eight-fold). The biochemical analysis revealed significant increase in DNA content, sulphated glycosaminoglycan (sGAG) (∼1.5 fold) and collagen (∼1.4 fold) in reinforced composites as compared to pure solution scaffolds (p ≤ 0.01). Histological and immunohistochemical (IHC) staining corroborated enhanced deposition of sGAG and localization of collagen type II in fiber-reinforced composites. This was further substantiated by real time polymerase chain reaction studies, which indicated an up-regulation (∼1.5 fold) of cartilage-specific gene markers namely collagen type II, sox-9 and aggrecan. The minimal secretion of tumor necrosis factor-α (TNF-α) by murine macrophages further demonstrated in vitro immunocompatibility of the scaffolds. Taken together, the results signified the potential of silk fiber-reinforced composite (particularly non-mulberry, A. assamensis) scaffolds as viable alternative biomaterial for cartilage tissue engineering.
Silk fibroin-chondroitin sulfate scaffold with immuno-inhibition property for articular cartilage repair.
Zhou Feifei,Zhang Xianzhu,Cai Dandan,Li Jun,Mu Qin,Zhang Wei,Zhu Shouan,Jiang Yangzi,Shen Weiliang,Zhang Shufang,Ouyang Hong Wei
The demand of favorable scaffolds has increased for the emerging cartilage tissue engineering. Chondroitin sulfate (CS) and silk fibroin have been investigated and reported with safety and excellent biocompatibility as tissue engineering scaffolds. However, the rapid degradation rate of pure CS scaffolds presents a challenge to effectively recreate neo-tissue similar to natural articular cartilage. Meanwhile the silk fibroin is well used as a structural constituent material because its remarkable mechanical properties, long-lasting in vivo stability and hypoimmunity. The application of composite silk fibroin and CS scaffolds for joint cartilage repair has not been well studied. Here we report that the combination of silk fibroin and CS could synergistically promote articular cartilage defect repair. The silk fibroin (silk) and silk fibroin/CS (silk-CS) scaffolds were fabricated with salt-leaching, freeze-drying and crosslinking methodologies. The biocompatibility of the scaffolds was investigated in vitro by cell adhesion, proliferation and migration with human articular chondrocytes. We found that silk-CS scaffold maintained better chondrocyte phenotype than silk scaffold; moreover, the silk-CS scaffolds reduced chondrocyte inflammatory response that was induced by interleukin (IL)-1β, which is in consistent with the well-documented anti-inflammatory activities of CS. The in vivo cartilage repair was evaluated with a rabbit osteochondral defect model. Silk-CS scaffold induced more neo-tissue formation and better structural restoration than silk scaffold after 6 and 12weeks of implantation in ICRS histological evaluations. In conclusion, we have developed a silk fibroin/ chondroitin sulfate scaffold for cartilage tissue engineering that exhibits immuno-inhibition property and can improve the self-repair capacity of cartilage. STATEMENT OF SIGNIFICANCE:Severe cartilage defect such as osteoarthritis (OA) is difficult to self-repair because of its avascular, aneural and alymphatic nature. Current scaffolds often focus on providing sufficient mechanical support or bio-mimetic structure to promote cartilage repair. Thus, silk has been adopted and investigated broadly. However, inflammation is one of the most important factors in OA. But few scaffolds for cartilage repair reported anti-inflammation property. Meanwhile, chondroitin sulfate (CS) is a glycosaminoglycan present in the natural cartilage ECM, and has exhibited a number of useful biological properties including anti-inflammatory activity. Thus, we designed this silk-CS scaffold and proved that this scaffold exhibited good anti-inflammatory effects both in vitro and in vivo, promoted the repair of articular cartilage defect in animal model.
Combinatory approach for developing silk fibroin scaffolds for cartilage regeneration.
Ribeiro Viviana P,da Silva Morais Alain,Maia F Raquel,Canadas Raphael F,Costa João B,Oliveira Ana L,Oliveira Joaquim M,Reis Rui L
Several processing technologies and engineering strategies have been combined to create scaffolds with superior performance for efficient tissue regeneration. Cartilage tissue is a good example of that, presenting limited self-healing capacity together with a high elasticity and load-bearing properties. In this work, novel porous silk fibroin (SF) scaffolds derived from horseradish peroxidase (HRP)-mediated crosslinking of highly concentrated aqueous SF solution (16 wt%) in combination with salt-leaching and freeze-drying methodologies were developed for articular cartilage tissue engineering (TE) applications. The HRP-crosslinked SF scaffolds presented high porosity (89.3 ± 0.6%), wide pore distribution and high interconnectivity (95.9 ± 0.8%). Moreover, a large swelling capacity and favorable degradation rate were observed up to 30 days, maintaining the porous-like structure and β-sheet conformational integrity obtained with salt-leaching and freeze-drying processing. The in vitro studies supported human adipose-derived stem cells (hASCs) adhesion, proliferation, and high glycosaminoglycans (GAGs) synthesis under chondrogenic culture conditions. Furthermore, the chondrogenic differentiation of hASCs was assessed by the expression of chondrogenic-related markers (collagen type II, Sox-9 and Aggrecan) and deposition of cartilage-specific extracellular matrix for up to 28 days. The cartilage engineered constructs also presented structural integrity as their mechanical properties were improved after chondrogenic culturing. Subcutaneous implantation of the scaffolds in CD-1 mice demonstrated no necrosis or calcification, and deeply tissue ingrowth. Collectively, the structural properties and biological performance of these porous HRP-crosslinked SF scaffolds make them promising candidates for cartilage regeneration. STATEMENT OF SIGNIFICANCE:In cartilage tissue engineering (TE), several processing technologies have been combined to create scaffolds for efficient tissue repair. In our study, we propose novel silk fibroin (SF) scaffolds derived from enzymatically crosslinked SF hydrogels processed by salt-leaching and freeze-drying technologies, for articular cartilage applications. Though these scaffolds, we were able to combine the elastic properties of hydrogel-based systems, with the stability, resilience and controlled porosity of scaffolds processed via salt-leaching and freeze-drying technologies. SF protein has been extensively explored for TE applications, as a result of its mechanical strength, elasticity, biocompatibility, and biodegradability. Thus, the structural, mechanical and biological performance of the proposed scaffolds potentiates their use as three-dimensional matrices for cartilage regeneration.
Bombyx mori derived scaffolds and their use in cartilage regeneration: a systematic review.
Fazal N,Latief N
Osteoarthritis and cartilage
For the last two decades, silk has been extensively used as scaffolds in tissue engineering because of its remarkable properties. Unfortunately, the aneural property of cartilage limits its regenerative potential which can be achieved using tissue engineering approach. A lot of research has been published searching for the optimization of silk fibroin (SF) and its blends in order to get the best cartilage mimicking properties. However, according to our best knowledge, there is no systematic review available regarding the use of Bombyx mori derived biomaterials limited to cartilage related studies. This systematic review highlights the in vitro and in vivo work done for the past 7 years on structural and functional properties of B. mori derived biomaterials together with different parameters for cartilage regeneration. PubMed database was searched focusing on in vitro and in vivo studies using the search thread "silk fibroin" and "cartilage". A total of 40 articles met the inclusion criteria. All the articles were deeply studied for cell types, scaffold types and animal models used along with study design and results. Five types of cells were used for in vitro while seven types of cells were used for in vivo studies. Three types of animal models were used for scaffold implantation purpose. Moreover, different types of scaffolds either seeded with cells or supplemented with various factors were explored and discussed in detail. Results suggest the suitability of silk as a better biomaterial because of its cartilage mimicking properties.
Polyethylenimine-functionalized silk sericin beads for high-performance remediation of hexavalent chromium from aqueous solution.
Kwak Hyo Won,Lee Ki Hoon
The enhancement of the metal adsorption and remediation performance of biomass-based adsorbents is an important challenge in heavy metal removal processes. One of the most viable and practical approaches in accomplishing a high metal removal efficiency is the surface modification of natural polymer adsorbents with functional polymeric materials. In the present study, polyethylenimine (PEI)-modified silk sericin beads were fabricated. The PEI modification process was confirmed and analyzed by Fourier transform infrared spectroscopy (FTIR), field emission-scanning electron microscopy (FE-SEM), and energy-dispersive X-ray spectroscopy (EDS)-based elemental analysis. The Cr(VI) remediation capabilities of PEI-modified sericin beads were analyzed by testing Cr(VI) removal from contaminated water. The results show that the Cr(VI) removal capacity of PEI-modified sericin beads was 365.3 mg/g, which is significantly higher than that of pristine sericin beads (34.56 mg/g). During the Cr(VI) removal process, Cr(VI) adsorption and reduction to Cr(III) occurred simultaneously. The results herein reveal that the synthesized PEI-modified sericin beads are a promising material for Cr(VI) adsorption and detoxification of aqueous solution.
Crosslinked alginate/sericin particles for bioadsorption of ytterbium: Equilibrium, thermodynamic and regeneration studies.
da Costa Talles Barcelos,da Silva Meuris Gurgel Carlos,Vieira Melissa Gurgel Adeodato
International journal of biological macromolecules
Sericin is a soluble globular protein, present in Bombyx mori silkworm cocoons. Sericin's properties can be improved to expand its application by producing blends with other substances, such as alginate polysaccharide and crosslinking agent poly(vinyl alcohol). This study evaluates the use of alginate and sericin particles chemically crosslinked with poly(vinyl alcohol) (SAPVA) for batch bioadsorption of rare-earth element ytterbium from aqueous medium. The equilibrium study showed that the maximum bioadsorption capacity for ytterbium was 0.642 mmol/g at 55 °C. Equilibrium data fit both Langmuir and Dubinin-Radushkevich models. The estimation of thermodynamic parameters showed that there was an increase in the entropy change, and that the bioadsorption process is endothermic and spontaneous. Characterization analyzes revealed that SAPVA particles, even after ytterbium bioadsorption, showed spherical shape, homogeneous composition, amorphous structure, low surface area, macropores, and low porosity. After the first regeneration cycle, the amount of captured ytterbium ions showed a slight increase (about 0.01 mmol/g) and calcium ions were completely released by SAPVA particles. Bioadsorbent particles separated selectively ytterbium from synthetic effluent containing different toxic metal ions. These results show that the SAPVA particles can be used as an effective bioabsorbent to remove and recover ytterbium from wastewater.
Bioadsorption of trivalent and hexavalent chromium from aqueous solutions by sericin-alginate particles produced from Bombyx mori cocoons.
de Andrade Júlia Resende,da Silva Meuris Gurgel Carlos,Gimenes Marcelino Luiz,Vieira Melissa Gurgel Adeodato
Environmental science and pollution research international
In this study, particles produced from sericin-alginate blend were used as non-conventional bioadsorbent for removing Cr(III) and Cr(VI) from aqueous solutions. Besides chromium mitigation, the use of sericin-alginate particles as bioadsorbent aims to offer an environmental solution of added value for sericin, which is a by-product from silk industry. Sericin-alginate particles in natura and loaded with Cr(III) and Cr(VI) were characterized using N physical adsorption analysis, optical microcopy, mercury porosimetry, helium pycnometry, scanning electron microscope coupled with energy dispersive X-ray spectrometer, Fourier transform infrared spectrometer, and X-ray diffraction. Kinetic studies on the removal of Cr(III) (at pH = 3.5) and Cr(VI) (at pH = 2) indicate the ion exchange mechanism with Ca(II) and the predominance of external mass transfer resistance. Cr(VI) uptake occurs through an adsorption-coupled reduction process, and bioadsorption equilibrium is reached after ~ 1000 min. Cr(III) bioadsorption occurs faster (~ 210 min). The Cr(VI) bioadsorption is endothermic, as bioadsorption capacity increases with temperature: 0.0783 mmol/g (20 °C), 0.1960 mmol/g (30 °C), 0.4570 mmol/g (40 °C), and 0.7577 mmol/g (55 °C). The three-parameter isotherm model of Tóth best represents the equilibrium data of total chromium. From Langmuir isotherm model, the maximum bioadsorption capacity is higher for total chromium, 0.25 mmol/g (30 °C), than for trivalent chromium, 0.023 mmol/g (30 °C). The comparison of bioadsorption capacities with different biomaterials confirms sericin-alginate particles as potential bioadsorbent of chromium.
Effects of alkyl polyglycoside (APG) on Bombyx mori silk degumming and the mechanical properties of silk fibroin fibre.
Wang Fei,Zhang Yu-Qing
Materials science & engineering. C, Materials for biological applications
Alkyl polyglycoside (APG), a nonionic surfactant, is often considered to be a green surfactant and is synthesized using glucose and long chain fatty alcohols. It is used as a degumming agent of Bombyx mori silk fibre in this study for the first time. We studied APG systematically in comparison to the traditional degumming methods, such as aqueous solutions of sodium carbonate (NaCO) and neutral soap (NS). After repeatedly boiling silk fibres in an aqueous solution of 0.25% APG three times for 30min and using a bath ratio of 1:90-120 (g/mL), sericin was completely removed from the fibre. SDS-PAGE showed that the degumming in APG did not induce an evident breakage of the silk fibroin peptide chains, including the light chain and P25 protein. The tensile properties, thermal analysis, and scanning electron microscopic (SEM) observation of the degummed fibroin fibre all show that APG is a degumming agent similar to NS and far superior to NaCO. These results indicate that APG is an environment-friendly silk degumming/refining agent in the silk textile industry and in the manufacture of silk floss quilts.
Precise correlation of macroscopic mechanical properties and microscopic structures of animal silks-using Antheraea pernyi silkworm silk as an example.
Fang Guangqiang,Tang Yuzhao,Qi Zeming,Yao Jinrong,Shao Zhengzhong,Chen Xin
Journal of materials chemistry. B
Animal silks, as one type of high performance natural material, display a unique combination of modulus, tensile strength, and extensibility that gives rise to a greater toughness than any other natural or synthetic fibers. Many previous researchers have already suggested that such excellent comprehensive mechanical properties should be closely related to their special molecular structures. In this paper, we provide more direct evidence to such an assumption by using Antheraea pernyi silkworm silk (tussah silk) as an example with synchrotron radiation FTIR microspectroscopy as a major characterization tool. Being a silkworm silk, A. pernyi silk has the same function as other silkworm silks (like common Bombyx mori silk), but on the other hand, its amino acid residue sequence is similar to that of spider dragline silk. Thus, A. pernyi silk can be a bridge between silkworm silk and spider silk that is worth investigating. Hence, in this research we designed different forcibly reeled A. pernyi silk samples by controlling the reeling rate, and subsequently tested their mechanical properties and then correlated them with their molecular structures and orientation degrees. Results show that the Young's modulus and breaking stress of forcibly reeled A. pernyi silks increased with the reeling rate, whereas the breaking strain was reduced. In the meantime, structure characterization revealed that the β-sheet content and molecular chain orientation in A. pernyi silk all increased significantly with an increase in reeling rate. In addition, the mechanical performance of A. pernyi silk can be altered from close to that of spider dragline silk to that of B. mori silkworm silk, with just a change of the reeling rate. All these phenomena clearly indicate that structural changes in A. pernyi silks contrived and controlled by reeling rate have a great effect upon their final mechanical properties. These observations further confirm that the mechanical properties of animal silks are able to be tuned by structure control during harvest time. Furthermore, the results obtained in this study may provide useful guidance when designing and producing high performance regenerated silk fibers for different applications.
High-toughness natural polymer nonwoven preforms inspired by silkworm cocoon structure.
Kwak Hyo Won,Eom Jungju,Cho Se Youn,Lee Min Eui,Jin Hyoung-Joon
International journal of biological macromolecules
As the interest in environmentally friendly materials and concerns regarding depletion of petroleum resources has increased, the research on natural polymers is being actively pursued. Among the various materials based on natural polymeric resources, the interest in using natural fibers in bio-composites has grown due to their lightweight, non-toxicity, low cost, and abundance. However, the lack of interfacial adhesion between filaments and poor water resistance make the use of natural fiber-based polymer composites less attractive. To overcome these drawbacks, formaldehyde-based synthetic binders have been used. However, this requires an additional synthesis of the binder, and potential toxicity problems exist. In this work, robust and rigid natural polymer nonwoven preforms were prepared by mixing jute fibers with silk sericin (SS). SS was employed as a natural facile binder and the strong binding between jute fibers and SS resulted in remarkable enhancements in tensile strength, elongation, and toughness, which increased up to 539.1, 385.7, and 1943.8%, respectively, compared with the pristine jute nonwoven. In addition, the dense and rigid structure obtained through SS coating ensured the structural stability of the nonwoven preforms in moisture environments. Silkworm cocoon-structured natural polymer nonwoven preforms with excellent mechanical strength and higher physical stability may have more potential utilization in the composite material fields.
The silkrose of Bombyx mori effectively prevents vibriosis in penaeid prawns via the activation of innate immunity.
Ali Muhammad Fariz Zahir,Yasin Indri Afriani,Ohta Takashi,Hashizume Atsushi,Ido Atsushi,Takahashi Takayuki,Miura Chiemi,Miura Takeshi
We previously identified novel bioactive polysaccharides from Bactrocera cucurbitae and Antheraea yamamai that activate innate immunity in RAW264 murine macrophages. However, in terms of potential applications in the cultivation of prawns, there were problems with the availability of these insects. However, we have now identified a polysaccharide from Bombyx mori that activates innate immunity in RAW264 cells and penaeid prawns. This purified polysaccharide, termed silkrose of B. mori (silkrose-BM), has a molecular weight of 1,150,000 and produces a single symmetrical peak on HPLC. Eight of nine constitutive monosaccharides of silkrose-BM are concomitant with dipterose of B. cucurbitae (dipterose-BC) and silkrose of A. yamamai (silkrose-AY). The major differences are found in the molar ratios of the monosaccharides. Silkrose-BM is approximately 500-fold less potent than silkrose-AY (EC: 2.5 and 0.0043 μg/mL, respectively) in a nitrite oxide (NO) production assay using RAW264 cells. However, the maximum NO production for silkrose-BM and AY were comparable and higher than that of the lipopolysaccharide of Escherichia coli. The survival of penaeid prawns (Litopenaeus vannamei and Marsupenaeus japonicus) after infection with Vibrio penaecida was significantly improved by both dietary silkrose-BM and B. mori pupae. This suggests that silkrose-BM effectively prevents vibriosis in penaeid prawns via the activation of innate immunity.
Straightforward extraction and selective bioconversion of high purity chitin from Bombyx eri larva: Toward an integrated insect biorefinery.
Huet Gael,Hadad Caroline,Husson Eric,Laclef Sylvain,Lambertyn Virginie,Araya Farias Monica,Jamali Arash,Courty Matthieu,Alayoubi Ranim,Gosselin Isabelle,Sarazin Catherine,Van Nhien Albert Nguyen
Chitins of different purity grades (45%, 89.7% and 93.3%) were efficiently extracted from Bombyx eri larva and fully physico-chemically characterized. Compared to commercially available and extracted α-chitin from shrimp shell, the collected data showed that insect chitins had similar characteristics in terms of crystallographic structures (α-chitin), thermal stability and degree of acetylation (>87%). The major differences lay in the crystallinity indexes (66% vs 75% for shrimp chitin) and in the morphological structures. Furthermore, low ash contents were determined for the insect chitins (1.90% vs 21.73% for shrimp chitin), making this chitin extraction and purification easier, which is highly valuable for an industrial application. Indeed, after only one step (deproteinization), the obtained chitin from Bombyx eri showed higher purity grade than the one extracted from shrimp shells under the same conditions. Insect chitins were then subjected to room temperature ionic liquid (RTIL) pretreatment prior to enzymatic degradation and presented a higher enzymatic digestibility compared to commercial one whatever their purity grade and would be thus a more relevant source for the selective production of N-acetyl-D-glucosamine (899.2 mg/g of chitin-2 stepsvs 760 mg/g of chitin com). Moreover, for the first time, the fermentescibility of chitin hydrolysates was demonstrated with Scheffersomyces stipitis used as ethanologenic microorganism.
Preparation of silk resins by hot pressing Bombyx mori and Eri silk powders.
Tuan H A,Hirai S,Tamada Y,Akioka S
Materials science & engineering. C, Materials for biological applications
We investigate the mechanical properties and structure of silk resins as potential alternatives to tortoiseshell for producing eyeglass frames and various ornaments. Silk powders are obtained from Bombyx mori and Eri silk waste fibers before the degumming process. The powders are fabricated into resins via simple hot pressing under a pressure of 31.2 MPa at temperatures in the range 150-180 °C. The results indicate that the B. mori resins have higher micro-Vickers hardness, three-point bending strength, and elastic modulus (66 Hv, 122 MPa, and 8.7 GPa, respectively) compared to the Eri silk resins (58 Hv, 95 MPa, and 8.2 GPa, respectively). The better mechanical properties of the fibroin resins are related directly to longer drying times. The optimum drying conditions are found to be at a temperature of 100 °C under a-vacuum of -0.1 MPa for a time of 7 d. ATR-FTIR and XRD results show how the fibroin structure changes after resinification and drying. The morphology and the distribution size of particle of the silk powders and the fractured surfaces of the resins are analyzed from SEM micrographs. The present findings demonstrate that silk resins are suitable materials for developing useful applications because of their favorable mechanical properties.
Impact of thermal processing on physicochemical properties of silk moth pupae (Bombyx mori) flour and in-vitro gastrointestinal proteolysis in adults and seniors.
David-Birman Tatyana,Moshe Hila,Lesmes Uri
Food research international (Ottawa, Ont.)
Elimination of insects' appearance by processing may increase their consumer acceptance in the western world. This study elucidates the outcomes of cooking and baking in presence and absence of fructose on silk worm pupae (Bombyx mori) flour (SWF). Elemental analyses of SWF reveal it is rich in lipids, protein and minerals like calcium. ζ-potential analyses revealed charge reversal at pH values below pH = 4. Particle size analyses showed heat-induced Maillard glycation significantly (p < .05) accentuated reduction in mean volume weighed (d4,3) diameters. In vitro gastrointestinal digestion of SWF and processed SWF showed different breakdown patterns evident in SDS-PAGE and LC-MS/MS analyses. Interestingly, baking with fructose significantly (α < 0.01) diminished the number of peptides liberated by pepsinolysis. Moreover, cooking or baking in the presence of fructose induced a reduction of bioaccessible peptides that are homologous to arthropoda phylum bioactive peptides (>70% homology), such as antimicrobial and Acyl-Co-A binding peptides. Predictive software (PeptideRanker) analysis showed a decrease in the total number of potentially novel bioactive peptides (bioactivity probability >80%) in baked SWF with fructose. Comparison of SWF and processed SWF proteolytic breakdown in adults and seniors highlighted seniors may be less apt to digest SWF products, yet, 14 potentially novel bioactive peptides were uniquely liberated in the elderly gut. Overall, this study shows process-related Maillard glycation may interfere with SWF potential to generate bioactive peptides during digestion in adults and seniors.
Recombinant Spider Silk Functionalized Silkworm Silk Matrices as Potential Bioactive Wound Dressings and Skin Grafts.
Chouhan Dimple,Thatikonda Naresh,Nilebäck Linnea,Widhe Mona,Hedhammar My,Mandal Biman B
ACS applied materials & interfaces
Silk is considered to be a potential biomaterial for a wide number of biomedical applications. Silk fibroin (SF) can be retrieved in sufficient quantities from the cocoons produced by silkworms. While it is easy to formulate into scaffolds with favorable mechanical properties, the natural SF does not contain bioactive functions. Spider silk proteins, on the contrary, can be produced in fusion with bioactive protein domains, but the recombinant procedures are expensive, and large-scale production is challenging. We combine the two types of silk to fabricate affordable, functional tissue-engineered constructs for wound-healing applications. Nanofibrous mats and microporous scaffolds made of natural silkworm SF are used as a bulk material that are top-coated with the recombinant spider silk protein (4RepCT) in fusion with a cell-binding motif, antimicrobial peptides, and a growth factor. For this, the inherent silk properties are utilized to form interactions between the two silk types by self-assembly. The intended function, that is, improved cell adhesion, antimicrobial activity, and growth factor stimulation, could be demonstrated for the obtained functionalized silk mats. As a skin prototype, SF scaffolds coated with functionalized silk are cocultured with multiple cell types to demonstrate formation of a bilayered tissue construct with a keratinized epidermal layer under in vitro conditions. The encouraging results support this strategy of fabrication of an affordable bioactive SF-spider silk-based biomaterial for wound dressings and skin substitutes.
Potential use of silkworm gut fiber braids as scaffolds for tendon and ligament tissue engineering.
Pagán Ana,Aznar-Cervantes Salvador D,Pérez-Rigueiro José,Meseguer-Olmo Luis,Cenis Jose L
Journal of biomedical materials research. Part B, Applied biomaterials
Tendon and ligament tissue engineering require scaffolds for the treatment of various conditions in the medical field. These must meet requirements such as high tensile strength, biocompatibility, fast and stable repair and a rate of degradation that allows the repair of the damaged tissue. In this work, we propose the use of silkworm gut fiber braids as materials to temporarily replace and repair this type of tissues. The mechanical characterization of the braids made with different number of silk gut fibers is provided, as well as a descriptive analysis of the proliferation and adhesion of cultures of adult human mesenchymal stem cells from bone marrow and fibroblasts (L929) on the braids. As expected, the breaking force increases linearly in the scaffold with the number of fibers, thus being a parameter adaptable to the specific requirements of the tissue to repair and the animal model of study. On the other hand, in all of the cases studied, the values obtained for the elastic modulus of the hydrated fibers were in the range of the ones reported for various human tendons and ligaments. Moreover, the scaffold demonstrated excellent biocompatibility in vitro, allowing the adhesion and proliferation, in the same culture conditions, of the two cell types studied, therefore posing as an ideal candidate to be employed in future in vivo studies that allow elucidating its behavior in the articular environment or extra-articular tendinous areas. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res B Part B: 2019. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2209-2215, 2019.
Protective effects of silk lutein extract from Bombyx mori cocoons on β-Amyloid peptide-induced apoptosis in PC12 cells.
Singhrang Nongnuch,Tocharus Chainarong,Thummayot Sarinthorn,Sutheerawattananonda Manote,Tocharus Jiraporn
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Beta-amyloid (Aβ) peptide, the hallmark of Alzheimer's disease (AD), invokes a cascade of oxidative damage to neurons and eventually leads to neuronal death. This study evaluated the protective effects of lutein extract from yellow cocoons of Bombyx mori, and its underlying mechanisms against was investigated to assess its protective effects and the underlying mechanisms against Aβ-induced neuronal cell death in cultured rat pheochromocytoma (PC12) cells. Aβ-induced neuronal toxicity is characterized by decrease in cell viability, increase in intracellular reactive oxygen species (ROS) production, activation of mitochondrial death pathway, and activation the phospholyration of mitogen-activated protein kinase (MAPKs) pathway. Pretreatment with silk lutein extract significantly attenuated Aβ-induced loss of cell viability, apoptosis, MAPKs pathway activation and ROS production. Taken together, our present study suggests that silk lutein extract protects PC12 cells from Aβ-induced neurotoxicity via the reduction of the ROS production, and subsequent attenuation of the mitochondrial death pathway and reduces the activation of the MAPK kinase pathways. This compound might beneficial as potential therapeutic agent to prevent or retard the development and progression of AD.
Chinese Oak Tasar Silkworm Antheraea pernyi Silk Proteins: Current Strategies and Future Perspectives for Biomedical Applications.
Silva Simone S,Kundu Banani,Lu Shenzhou,Reis Rui L,Kundu Subhas C
Chinese nonmulberry temperate oak tasar/tussah, Antheraea pernyi (Ap) silk is a natural biopolymer that has attracted considerable attention as a biomaterial. The proteinaceous components of Ap silk proteins, namely fibroin and sericin may represent an alternative over mulberry Bombyx mori silk proteins. In fact, the silk fibroin (SF) of Ap is rich in Arginyl-Glycyl-Aspartic acid (RGD) peptides, which facilitate the adhesion and proliferation of various cell types. The possibility of processing Ap silk proteins into different distinct 2D- and 3D-based matrices is described in earlier studies, such as membranes, nanofibers, scaffolds, and micro/nanoparticles, contributing to a different rate of degradation, mechanical properties, and biological performance useful for various biomedical applications. This review summarizes the current advances and developments on nonmulberry Chinese oak tasar silk protein (fibroin and sericin)-based biomaterials and their potential uses in tissue engineering, regenerative medicine, and therapeutic delivery strategies.
Cytotoxic compounds against cancer cells from Bombyx mori inoculated with Cordyceps militaris.
Qiu Weitao,Wu Jing,Choi Jae-Hoon,Hirai Hirofumi,Nishida Hiroshi,Kawagishi Hirokazu
Bioscience, biotechnology, and biochemistry
Two compounds, 3'-deoxyinosine and cordycepin, were isolated from Bombyx mori inoculated with Cordyceps militaris. In the bioassay examining cytotoxicity against cancer cells, both compounds showed toxicity against A549, PANC-1, and MCF-7 cancer cells.
Purification and functional characterization of tomato mosaic virus 130K protein expressed in silkworm pupae using a baculovirus vector.
Kobayashi Chihoko,Kato Masahiko,Nagaya Hidekazu,Shimizu Nobutaka,Ishibashi Kazuhiro,Ishikawa Masayuki,Katoh Etsuko
Protein expression and purification
Tomato mosaic virus (ToMV; genus, Tobamovirus) is a member of the alpha-like virus superfamily of positive-strand RNA viruses, which includes many plant and animal viruses of agronomical and clinical importance. The genomes of alpha-like viruses encode replication-associated proteins that contain methyltransferase, helicase and/or polymerase domains. The three-dimensional structure of the helicase domain fragment of ToMV has been determined, but the structures of the other domains of alpha-like virus replication proteins are not available. In this study, we expressed full-length ToMV replication-associated protein 130 K, which contains the methyltransferase and helicase domains, using the baculovirus-silkworm expression system and purified the recombinant protein to near homogeneity. Purified 130 K, which was stable in phosphate buffer containing magnesium ions and ATP, formed a dimer in solution and hydrolyzed nucleoside 5'-triphosphates.
Chichen type III interferon produced by silkworm bioreactor induces ISG expression and restricts ALV-J infection in vitro.
Feng Min,Zhang Nan,Xie Tingting,Ren Feifei,Cao Zhenming,Zeng Xiaoqun,Swevers Luc,Zhang Xiquan,Sun Jingchen
Applied microbiology and biotechnology
Type III interferon (IFN-λ) has recently been shown to exert a significant antiviral impact against viruses in vertebrates. Avian leukosis virus subgroup J (ALV-J), which causes tumor disease and immunosuppression in infected chicken, is a retrovirus that is difficult to prevent and control because of a lack of vaccines and drugs. Here, we obtained chicken IFN-λ (chIFN-λ) using a silkworm bioreactor and demonstrated that chIFN-λ has antiviral activity against ALV-J infection of both chicken embryo fibroblast cell line (DF1) and epithelial cell line (LMH). We found that chIFN-λ triggered higher levels of particular type III interferon-stimulated genes (type III ISGs) including myxovirus resistance protein (Mx), viperin (RSAD2), and interferon-inducible transmembrane protein 3 (IFITM3) in DF1 and LMH cells. Furthermore, over-expression of Mx, viperin, and IFITM3 could inhibit ALV-J infection in DF1 and LMH cells. Therefore, these results suggested that the anti-ALV-J function of chIFN-λ was specifically implemented by induction of expression of type III ISGs. Our data identified chIFN-λ as a critical antiviral agent of ALV-J infection and provides a potentially and attractive platform for the production of commercial chIFN-λ.
Traditional Uses, Origins, Chemistry and Pharmacology of Bombyx batryticatus: A Review.
Hu Meibian,Yu Zhijie,Wang Jiaolong,Fan Wenxiang,Liu Yujie,Li Jianghua,Xiao He,Li Yongchuan,Peng Wei,Wu Chunjie
Molecules (Basel, Switzerland)
(), a well-known traditional animal Chinese medicine, has been commonly used in China for thousands of years. The present paper reviewed advances in traditional uses, origin, chemical constituents, pharmacology and toxicity studies of . The aim of the paper is to provide more comprehensive references for modern study and application. In Traditional Chinese Medicine (TCM) culture, drugs containing have been used to treat convulsions, headaches, skin prurigo, scrofula, tonsillitis and fever. Many studies indicate contains various compounds, including protein and peptides, fatty acids, flavonoids, nucleosides, steroids, coumarin, polysaccharide and others. Numerous investigations also have shown that extracts and compounds from exert a wide spectrum of pharmacological effects both in vivo and in vitro, including effects on the nervous system, anticoagulant effects, antitumor effects, antibacterial and antifungal effects, antioxidant effects, hypoglycemic effects, as well as other effects. However, further studies should be undertaken to investigate bioactive compounds (especially proteins and peptides), toxic constituents, using forms and the quality evaluation and control of . Furthermore, it will be interesting to study the mechanism of biological activities and structure-function relationships of bioactive constituents in .
In silico study of Bombyx mori fibroin enhancement by graphene in acidic environment.
Tran Duy Phuoc,Lam Vi Toan,Tran Tan Lien,Nguyen Thuy Nhu Son,Thi Tran Hanh Thu
Physical chemistry chemical physics : PCCP
Bombyx mori fibroin has been widely used since a long time ago and has become a popular material. Here, we carry out a molecular dynamics simulation-based docking simulation of a small fragment of graphene in order to seek the best binding position on the N-termini domain of Bombyx mori fibroin. We report the best binding position, of which binding free energy falls at -54.8 kJ mol-1, indicating the strong binding. The further analysis of the binding pathway shows that this position is selective for single layered graphene rather than multi-layered graphene within our limited simulation times. Via comparing the RAMAN spectra of the corresponding binding pose of atomic clusters, we report the change in the bands compared with free standing graphene fragments, implying the change in molecular orbitals.
Structural and Mechanical Properties of Silk from Different Instars of Bombyx mori.
Peng Zhangchuan,Yang Xi,Liu Chun,Dong Zhaoming,Wang Feng,Wang Xin,Hu Wenbo,Zhang Xia,Zhao Ping,Xia Qingyou
Silkworm silk has excellent mechanical properties, biocompatibility, and promising applications in the biomedical sector. Silkworms spin silk at the beginning and end of each of their five instar stages, as well as spinning mature silk after the fifth instar. We evaluated the mechanical properties and structure of 10 kinds of silk fibers from different stages. A tensile test showed that instar beginning silk, instar end silk, and mature silk possess distinct properties. Attenuated total reflectance Fourier-transform infrared spectroscopy and X-ray diffraction results showed that the excellent mechanical properties of instar end silk are attributed to higher β-sheet content and suitable crystallinity. Liquid chromatography-tandem mass spectrometry showed that P25 protein content in IV-E silk is 2.9× higher than that of cocoon silk. This study can offer guidelines for further biomimetic investigations into the design and manufacture of artificial silk protein fibers with novel function.
Uncovering the Molecular Mechanism of Anti-Allergic Activity of Silkworm Pupa-Grown Cordyceps militaris Fruit Body.
Wu Ting-Feng,Chan Yu-Yi,Shi Wan-Yin,Jhong Meng-Ting
The American journal of Chinese medicine
Cordyceps militaris has been widely used as an herbal drug and tonic food in East Asia and has also been recently studied in the West because of its various pharmacological activities such as antitumoral, anti-inflammatory and immunomodulatory effects. In this study, we examined the molecular mechanism underlying the anti-allergic activity of ethanol extract prepared from silkworm pupa-cultivated Cordyceps militaris fruit bodies in activated mast cells. Our results showed that ethanol extract treatment significantly inhibited the release of [Formula: see text]-hexosaminidase (a degranulation marker) and mRNA levels of tumor necrosis factor-[Formula: see text] as well as interleukin-4 in RBL-2H3 cells. The cells were sensitized with 2,4-dinitrophenol specific IgE and then stimulated with human serum albumin conjugated with 2,4-dinitrophenol. Oral administration of 300[Formula: see text]mg/kg ethanol extract significantly ameliorated IgE-induced allergic reaction in mice with passive cutaneous anaphylaxis. Western immunoblotting results demonstrated that ethanol extract incubation significantly inhibited Syk/PI3K/MEKK4/JNK/c-jun biochemical cascade in activated RBL-2H3 cells, which activated the expression of various allergic cytokines. In addition, it suppressed Erk activation and PLC[Formula: see text] evocation, which would respectively evoke the synthesis of lipid mediators and Ca[Formula: see text] mobilization to induce degranulation in stimulated RBL-2H3 cells. A compound, identified as [Formula: see text]-sitostenone, was shown to inhibit [Formula: see text]-hexosaminidase secretion from activated mast cells. Our study demonstrated that ethanol extract contained the ingredients, which could inhibit immediate degranulation and de novo synthesis of allergic lipid mediators and cytokines in activated mast cells.
Quantitative analysis of the yield of avian H7 influenza virus haemagglutinin protein produced in silkworm pupae with the use of the codon-optimized DNA: A possible oral vaccine.
Nerome Kuniaki,Matsuda Sayaka,Maegawa Kenichi,Sugita Shigeo,Kuroda Kazumichi,Kawasaki Kazunori,Nerome Reiko
In this study, we aimed to quantitatively compare the increased production of three H7 influenza virus-like particle (VLP) haemagglutinin (HA) with the use of a codon-optimized single HA gene in silkworm pupae. Recombinant baculovirus (Korea H7-BmNPV) could produce 0.40 million HA units per pupa, corresponding to 1832μg protein. The yield of the HA produced in larva was estimated to be approximately 0.31 million HA units per larva, and there were no significant differences between the three HA proteins. We could establish efficient recovery system of HA production in larvae and pupae with the use of three cycles sonication methods. Next, we compared yields of HA proteins from three different H7 and two H5 recombinant baculoviruses based on the amount of mRNA synthesized in BmN cells, suggesting that mRNA synthesis may be also a useful indicator for the production of HA. Based on HA titres from four recombinants, the yield of HA had a great influence on the codon-optimized effect and the characteristics of the viral HA gene. The recombinant containing codon optimized HA DNA of A/tufted duck/Fukushima/16/2011 (H5N1) did produce more than one million HA units, although another recombinant including of the wild H5N1 strain failed to show HA activity. Electron microscopy revealed the presence of large VLP and small HA particle in the heavy and light fractions. The purified VLPs reacted with the authentic anti-H7 antibodies and the antibodies prepared after immunization with the VLP H7 antigen. Also H5 and H7VLPs could produce HI antibody in chickens and mice with oral immunization. The antibodies elicited with oral immunization were confirmed in fluorescent antibody analysis and western blotting in Korea H5-BmNPV and H7HA-BmNPV recombinant infected BmN cells. Taken together, these findings provided important insights into future oral vaccine development.
A construction strategy for a baculovirus-silkworm multigene expression system and its application for coexpression of type I and type II interferons.
Liu Xingjian,Yang Xin,Mehboob Arslan,Hu Xiaoyuan,Yi Yongzhu,Li Yinü,Zhang Zhifang
The Bombyx mori nucleopolyhedrovirus (BmNPV) baculovirus expression system (BES) is a eukaryotic expression system. It possesses great capability for post-translation modification in expression of foreign proteins. With the counterselection cassette rpsL-neo and phage λ-Red recombinase, the defective-rescue BmNPV BES reBmBac can be employed for efficient heterologous multigene coexpression at different gene sites in one baculovirus genome. In the present study, a recombinant baculovirus, reBm-Cαγ, carrying two types of chicken interferon (IFN) genes (chIFN-α and chIFN-γ) was constructed using the reBmBac system. The chIFN-α and chIFN-γ genes were inserted into the same baculovirus genome at the polyhedron and p10 gene sites, respectively. The recombinant baculovirus was capable of coexpressing both chIFN-α and chIFN-γ. The expression levels of the two types of IFN in the coexpression product were exponentially high, at approximately 1.7 and 2.5 times higher, respectively, than those in the corresponding single-expression products. The increase in expression level corresponds to replacement of the nonessential p10 gene in the reBm-Cαγ recombinant baculovirus. This coexpression of recombinant chicken IFNs showed superior antiviral activity.
Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system.
Kinoshita Yurie,Xu Jian,Masuda Akitsu,Minamihata Kosuke,Kamiya Noriho,Mon Hiroaki,Fujita Ryosuke,Kusakabe Takahiro,Lee Jae Man
Protein expression and purification
Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost.
Encapsulation and Systemic Delivery of 5-Fluorouracil Conjugated with Silkworm Pupa Derived Protein Nanoparticles for Experimental Lymphoma Cancer.
Reneeta Naveengandhi Paulin,Thiyonila Berchmans,Aathmanathan Veeranarayanan Surya,Ramya Thangaraj,Chandrasekar Ponnusamy,Subramanian Natesan,Prajapati Vijay Kumar,Krishnan Muthukalingan
Protein-based drug delivery systems have an edge over conventional drug delivery systems due to their biodegradability, non-antigenicity, and excellent biocompatibility to improve the therapeutic properties of anticancer drugs. This study describes the increased anticancer efficacy of 5-fluorouracil (5-FU) conjugated with silkworm Bombyx mori pupal biowaste derived nanoparticles. Here, we have checked the toxicity of pupa-protein nanoparticles (PpNps) and their potential as a carrier for anticancer drugs. PpNps were prepared by a desolvation method which resulted in a uniform particle size of 162.7 ± 2.9 nm. The 5-FU loaded PpNps were formulated and characterized. The drug content of the developed 5-FU conjugated nanoparticles was evaluated by HPLC analysis. The entrapment efficiency and loading capacity of 5-FU were analyzed by HPLC and determined to be 93% and 88.6%, respectively. The release studies showed the biphasic release of 5-FU at pH 7.4 where rapid drug release was achieved for first 30 min, followed by a sustained release of 5-FU from the developed Nps achieved for the next 8 h. Mice with developed ascites tumors were intraperitoneally treated with 5-FU-PpNps and sacrificed. There was a significant increase in total red blood cells and hemoglobulin in 5-FU-PpNps treated mice, whereas a significant decrease in white blood cells which indicated the reduced inflammation of cancer. Subsequently, 5-FU-PpNps decreased the tumor volume and tumor cell viability, which proved its cytotoxic property to cancer cells. This study presents a novel approach to derive B. mori pupal protein nanoparticles, which can be safely used for cancer drug delivery.
Enhanced diuron remediation by microorganism-immobilized silkworm excrement composites and their impact on soil microbial communities.
Liu Jie,Yang Menrang,Wang Yutai,Qu Liwen,Zhong Guohua
Journal of hazardous materials
In response to the potential threats stemming from the constantly increasing consumption of herbicides, bioremediation offers a beneficial technology for reducing the widespread herbicide contamination. In order to facilitate the in-situ degradation of diuron, Arthrobacter globiformis D47 is captured onto a biocompatible carrier to assemble the microorganism-immobilized silkworm excrement (MSE) composites. By characterization, bacterial cells are intensively entrapped in/onto the carriers, showing high survival and stable catalytic degradation of target pollutants. Meanwhile, MES composites display excellent adaptiveness and feasibility under different conditions, and the average half-life of diuron is shortened to 7.69 d in sugarcane field where diuron is regularly sprayed for weed management. Importantly, we assess that the use of MSE may generally boost the overall xenobiotic-degrading ability, likely due to the slight alternation of the diversity and composition of soil microbial communities. Taking together, the presented MSE provides an attractive in situ approach for the efficient diuron removal as well as for the more feasible utilization of various pollutant-degrading microorganisms.
Bombyx mori hemocyte extract has anti-inflammatory effects on human phorbol myristate acetate-differentiated THP‑1 cells via TLR4-mediated suppression of the NF-κB signaling pathway.
Kim Young Il,Choi Kwang Ho,Kim Seong Ryul,Goo Tae-Won,Park Seung-Won
Molecular medicine reports
Hemolymph is the circulating fluid of insects and is a key component of their immune system. However, little is known concerning hemocyte identification, development, differentiation and related cellular immune responses. The present study aimed to determine whether a hemocyte extract prepared from Bombyx mori larvae had anti‑inflammatory effects; THP‑1 (a human monocytic leukemia cell line) cells that had been differentiated into macrophage‑like cells by treatment with phorbol myristate acetate (PMA) were used. THP‑1 cells were cultured with different concentrations of a B. mori hemocyte extract prior to exposure to lipopolysaccharide (LPS) to induce an inflammatory response. The effects of the B. mori hemocyte extract on anti‑inflammatory pathways were determined using reverse transcription‑quantitative polymerase chain reaction and western blotting to assess the expression of pro‑inflammatory molecules. The B. mori hemocyte extract inhibited the LPS‑induced mRNA expression of Toll‑like receptor 4 in addition to LPS‑induced interleukin (IL)‑1β, IL‑6, IL‑8 and tumor necrosis factor‑α. Treatment of PMA‑differentiated THP‑1 cells with B. mori hemocyte extract also inhibited inducible nitric oxide synthase and cyclooxygenase‑2 transcription and translation. Nuclear factor‑κB activation and phosphorylation also decreased. Further in‑depth functional studies are required to understand the mechanism underlying the anti‑inflammatory effects of silkworm hemocyte extract.
Sero-diagnostic potential of Plasmodium falciparum recombinant merozoite surface protein (MSP)-3 expressed in silkworm.
Deo Vipin Kumar,Inagaki Yu,Murhandarwati Elsa Herdiana,Asmara Widya,Miyazaki Takatsugu,Kato Tatsuya,Park Enoch Y
Plasmodium falciparum is a blood protozoan parasite, transmitted by Anopheles mosquitoes vectors, that can cause morbidity and even leads to mortality in tropical countries. Strategies are directed to combat malaria including development of diagnostic tools, serological markers and vaccinations. A target under intensive studies is Merozoite Surface Protein (MSP)-3. The aim of this study is to express and purify recombinant MSP3 of P. falciparum (rPfMSP3) using silkworm expression system as a host for its large-scale production and to investigate its potential effectiveness for sero-diagnosis. The rPfMSP3 formed oligomers in a blue-native PAGE and its N-glycosylation was confirmed by periodic acid-Schiff staining and PNGase F treatment. The amyloid-like morphology of the rPfMSP3 oligomers was observed. Enzyme-linked immunosorbent assay showed that 60-70% of human samples from subjects living in malaria endemic areas in Indonesia detected the rPfMSP3. Western blot results showed that the rPfMSP3 was recognized by a malaria infected human serum but not by an uninfected human serum. The rPfMSP3 was successfully expressed in silkworm as a soluble protein and has the potential to be used in serological measurement for detecting PfMSP3-specific antibodies in sera from individuals living in endemic areas.
Use of a Silkworm Larva Model in Phage Therapy Experiments.
Uchiyama Jumpei,Takemura-Uchiyama Iyo,Matsuzaki Shigenobu
Methods in molecular biology (Clifton, N.J.)
Antibiotic-resistant bacteria can cause intractable infections in humans and animals, with damaging effects to health care and economics. Phage therapy is considered a possible alternative to chemotherapy for treating infections, but still requires laborious in vivo experiments before its introduction into society and its further development. Recently, silkworm larvae have been recognized as highly convenient and useful model animals, and an alternative to higher animals. We describe the procedure for experimental phage therapy to treat Staphylococcus aureus infections in silkworm larvae.
Animal infection models using non-mammals.
Kaito Chikara,Murakami Kanade,Imai Lina,Furuta Kazuyuki
Microbiology and immunology
The use of non-human animal models for infection experiments is important for investigating the infectious processes of human pathogenic bacteria at the molecular level. Mammals, such as mice and rabbits, are also utilized as animal infection models, but large numbers of animals are needed for these experiments, which is costly, and fraught with ethical issues. Various non-mammalian animal infection models have been used to investigate the molecular mechanisms of various human pathogenic bacteria, including Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. This review discusses the desirable characteristics of non-mammalian infection models and describes recent non-mammalian infection models that utilize Caenorhabditis elegans, silkworm, fruit fly, zebrafish, two-spotted cricket, hornworm, and waxworm.
Regulation mechanism of silkworm pupa oil PUFAs on cholesterol metabolism in hepatic cell L-02.
Luo Ying,Wang Lifang,Lv Yongzhong,Wu Xiaoxia,Hou Chen,Li Jianke
Journal of the science of food and agriculture
BACKGROUND:Silkworm pupa oil polyunsaturated fatty acid (SPO PUFA) has been confirmed to have a cholesterol-lowering function. METHODS AND RESULTS:The effect of SPO PUFA and its main component, α-linolenic acid (ALA), on the metabolism of cholesterol and its regulation was investigated. The model of lipid denatured cells were constructed to carry out lipid accumulation, cholesterol metabolism and transformation. Real-time PCR and western blots were also used to analyze the expression levels of related genes and proteins to investigate the cholesterol efflux regulation mechanism. The data indicated that SPO PUFA and ALA dose-dependently decreased intracellular total cholesterol (TC) and enhanced total bile acid (TBA). They could also promote cholesterol removal by enhancing bile acid secretion and by upregulating genes LXRα, PPARγ, ABCA1, ABCG1, and CYP7A1, which were regulated by LXRα/PPARγ-ABCA1/ABCG1-CYP7A1 nuclear receptor signal pathways. CONCLUSIONS:This study is of great significance in maintaining the balance of cholesterol and lipid metabolism, and in reducing the risk of steatohepatitis. © 2019 Society of Chemical Industry.
A Crude 1-DNJ Extract from Home Made Bombyx Batryticatus Inhibits Diabetic Cardiomyopathy-Associated Fibrosis in db/db Mice and Reduces Protein -Glycosylation Levels.
Zhao Qing,Jia Tian Zhu,Cao Qi Chen,Tian Fang,Ying Wan Tao
International journal of molecular sciences
The traditional Chinese drug Bombyx Batryticatus (BB), which is also named the white stiff silkworm, has been widely used in Chinese clinics for thousands of years. It is famous for its antispasmodic and blood circulation-promoting effects. Cardiomyocyte hypertrophy, interstitial cell hyperplasia, and myocardial fibrosis are closely related to the -glycosylation of key proteins. To examine the alterations of -glycosylation that occur in diabetic myocardium during the early stage of the disease, and to clarify the therapeutic effect of 1-Deoxynojirimycin (1-DNJ) extracted from BB, we used the db/db (diabetic) mouse model and an approach based on hydrophilic chromatography solid-phase extraction integrated with an liquid Chromatograph Mass Spectrometer (LC-MS) identification strategy to perform a site-specific -glycosylation analysis of left ventricular cardiomyocyte proteins. Advanced glycation end products (AGEs), hydroxyproline, connective tissue growth factor (CTGF), and other serum biochemical indicators were measured with enzyme-linked immunosorbent assays (ELISA). In addition, the α-1,6-fucosylation of -glycans was profiled with lens culinaris agglutinin (LCA) lectin blots and fluorescein isothiocyanate (FITC)-labelled lectin affinity histochemistry. The results indicated that 1-DNJ administration obviously downregulated myocardium protein -glycosylation in db/db mice. The expression levels of serum indicators and fibrosis-related cytokines were reduced significantly by 1-DNJ in a dose-dependent manner. The glycan α-1,6-fucosylation level of the db/db mouse myocardium was elevated, and the intervention effect of 1-DNJ administration on -glycan α-1,6-fucosylation was significant. To verify this result, the well-known transforming growth factor-β (TGF-β)/Smad2/3 pathway was selected, and core α-1,6-fucosylated TGF-β receptor II (TGFR-βII) was analysed semi-quantitatively with western blotting. The result supported the conclusions obtained from LCA lectin affinity histochemistry and lectin blot analysis. The expression level of α-1,6-fucosyltransferase (FUT8) mRNA was also detected, and the results showed that 1-DNJ administration did not cause obvious inhibitory effects on FUT8 expression. Therefore, the mechanism of 1-DNJ for relieving diabetic cardiomyopathy (DCM)-associated fibrosis can be concluded as the inhibition of -acetylglucosamine (-GlcNAc) formation and the reduction of substrate concentration.
Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.
Xxxx Patmawati,Minamihata Kosuke,Tatsuke Tsuneyuki,Lee Jae Man,Kusakabe Takahiro,Kamiya Noriho
Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.
Functional horseradish peroxidase-streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes.
Patmawati ,Minamihata Kosuke,Tatsuke Tsuneyuki,Lee Jae Man,Kusakabe Takahiro,Kamiya Noriho
Journal of biotechnology
Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP)-Stav or Stav-(HRP), respectively) using a baculovirus-silkworm expression system. Both (HRP)-Stav and Stav-(HRP) were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP)-Stav and Stav-(HRP) could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP)-Stav was twofold higher than that of Stav-(HRP), and the sensitivity of (HRP)-Stav in ELISA was higher than that of a commercial HRP-Stav chemical conjugate. The successful use of (HRP)-Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme-Stav conjugates to substitute for those prepared by chemical methods.
Heparinized silk fibroin hydrogels loading FGF1 promote the wound healing in rats with full-thickness skin excision.
He Sirong,Shi Dan,Han Zhigang,Dong Zhaoming,Xie Yajun,Zhang Fengmei,Zeng WenXin,Yi Qiying
Biomedical engineering online
BACKGROUND:Silk fibroin hydrogel, derived from Bombyx mori cocoons, has been shown to have potential effects on wound healing due to its excellent biocompatibility and less immunogenic and biodegradable properties. Many studies suggest silk fibroin as a promising material of wound dressing and it can support the adhesion and proliferation of a variety of human cells in vitro. However, lack of translational evidence has hampered its clinical applications for skin repair. Herein, a heparin-immobilized fibroin hydrogel was fabricated to deliver FGF1 (human acidic fibroblast growth factor 1) on top of wound in rats with full-thickness skin excision by performing comprehensive preclinical studies to fully evaluate its safety and effectiveness. The wound-healing efficiency of developed fibroin hydrogels was evaluated in full-thickness wound model of rats, compared with the chitosan used clinically. RESULTS:The water absorption, swelling ratio, accumulative FGF1 releasing rate and biodegradation ratio of fabricated hydrogels were measured. The regenerated fibroin hydrogels with good water uptake properties rapidly swelled to a 17.3-fold maximum swelling behavior over 12 h and a total amount of 40.48 ± 1.28% hydrogels was lost within 15 days. Furthermore, accumulative releasing data suggested that heparinized hydrogels possessed effective release behavior of FGF1. Then full-thickness skin excision was created in rats and left untreated or covered with heparinized fibroin hydrogels-immobilized recombinant human FGF1. The histological evaluation using hematoxylin and eosin (HE) and Masson's trichrome (MT) staining was performed to observe the dermic formation and collagen deposition on the wound-healing site. To evaluate the wound-healing mechanisms induced by fibroin hydrogel treatment, wound-healing scratch and cell proliferation assay were performed. it was found that both fibroin hydrogels and FGF1 can facilitate the migration of fibroblast L929 cells proliferation and migration. CONCLUSION:This study provides systematic preclinical evidence that the silk fibroin promotes wound healing as a wound-healing dressing, thereby establishing a foundation toward its further application for new treatment options of wound repair and regeneration.
Intrinsic antimicrobial properties of silk spun by genetically modified silkworm strains.
Saviane Alessio,Romoli Ottavia,Bozzato Andrea,Freddi Giuliano,Cappelletti Chiara,Rosini Elena,Cappellozza Silvia,Tettamanti Gianluca,Sandrelli Federica
The domesticated silkworm, Bombyx mori, is a fundamental insect for silk industry. Silk is obtained from cocoons, protective envelopes produced during pupation and composed of single raw silk filaments secreted by the insect silk glands. Currently, silk is used as a textile fibre and to produce new materials for technical and biomedical applications. To enhance the use of both fabrics and silk-based materials, great efforts to obtain silk with antimicrobial properties have been made. In particular, a convincing approach is represented by the enrichment of the textile fibre with antimicrobial peptides, the main effectors of the innate immunity. To this aim, silkworm-based transgenic techniques appear to be cost-effective strategies to obtain cocoons in which antimicrobial peptides are integrated among the silk proteins. Recently, cocoons transgenic for a recombinant silk protein conjugated to the silkworm Cecropin B antimicrobial peptide were obtained and showed enhanced antibacterial properties (Li et al. in Mol Biol Rep 42:19-25, https://doi.org/10.1007/s11033-014-3735-z , 2015a). In this work we used the piggyBac-mediated germline transformation to generate several transgenic B. mori lines able to overexpress Cecropin B or Moricin antimicrobial peptides at the level of the silk gland. The derived cocoons were characterised by increased antimicrobial properties and the resulting silk fibre was able to inhibit the bacterial growth of the Gram-negative Escherichia coli. Our results suggest that the generation of silkworm overexpressing unconjugated antimicrobial peptides in the silk gland might represent an additional strategy to obtain antimicrobial peptide-enriched silk, for the production of new silk-based materials.
Genetically engineered bi-functional silk material with improved cell proliferation and anti-inflammatory activity for medical application.
Wang Yuancheng,Wang Feng,Xu Sheng,Wang Riyuan,Chen Wenjing,Hou Kai,Tian Chi,Wang Fan,Yu Ling,Lu Zhisong,Zhao Ping,Xia Qingyou
Functional silk is a promising material for future medical applications. These include fabrication of diverse silk fiber and silk protein-regenerated biomaterials such as silk sutures, hydrogel, films, and 3D scaffolds for wound healing and tissue regeneration and reconstruction. Here, a novel bi-functional silk with improved cell proliferation and anti-inflammatory activities was created by co-expressing the human basic fibroblast growth factor (FGF2) and transforming growth factor-β1 (TGF_β1) genes in silkworm. First, both FGF2 and TGF_β1 genes were confirmed to be successfully expressed in silk thread. The characterization of silk properties by SEM, FTIR, and mechanical tests showed that this new silk (FT silk) had a similar diameter, inner molecular composition, and mechanical properties as those of normal silk. Additionally, expressed FGF2 and TGF_β1 proteins were continuously and slowly released from FT silk for one week. Most importantly, the FGF2 and TGF_β1 contained in FT silk not only promoted cell proliferation by activating the ERK pathway but also significantly reduced LPS-induced inflammation responses in macrophages by mediating the Smad pathway. Moreover, this FT silk had no apparent toxicity for cell growth and caused no cell inflammation. These properties suggest that it has a potential for medical applications. STATEMENT OF SIGNIFICANCE: Silk spun by domestic silkworm is a promising material for fabricating various silk protein regenerated biomaterials in medical area, since it owes good biocompatibility, biodegradability and low immunogenicity. Recently, fabricating various functional silk fibers and regenerated silk protein biomaterials which has ability of releasing functional protein factor is the hot point field. This study is a first time to create a novel bi-functional silk material with the improved cell proliferation and anti-inflammatory activity by genetic engineered technology. This novel silk has a great application potential as new and novel medical material, and this study also provides a new strategy to create various functional or multifunctional silk fiber materials in future.
Safe utilization of polluted soil by arsenic, cadmium and lead through an integrated sericultural measure.
Feng Renwei,Zhu Qihong,Xu Yingming,Li Wushuang,Ding Yongzhen,Han Lei,Rensing Christopher,Wang Ruigang
The Science of the total environment
Effective remediation technologies to remediate multiple heavy metal contaminated farmlands are lacking. To make full use of farmlands and control its consequent health risks, we planted mulberry trees in arsenic (As)-cadmium (Cd)-lead (Pb) co-contaminated soils at four different sites; then reared silkworms on leaves harvested from these mulberry trees; and finally used the silkworm excrement to in situ remediate the As, Cd, and Pb polluted paddy soil. Mulberry leaves and stalks showed weak abilities to accumulate As, Cd, and Pb. As and Pb tended to accumulate in silkworm pupae and silkworm excrement, respectively, posing a potential health risk when they were used as pharmaceutical materials or foods. However, using the leaves of mulberry trees planted in Cd-contaminated soils to rear silkworms had a low health risk. Silkworm excrement significantly reduced the As, Cd and Pb concentrations in rice grains, with As and Cd concentration being lower but for Pb being higher their respective national limit standards. In conclusion, based on the rational utilization of resources such as silkworm excrement, pupae, and cocoons, the integrated measure in this study could effectively reduce the environmental health risks resulting from multiple As, Cd, and Pb contamination.
Application of economic plant for remediation of cadmium contaminated soils: Three mulberry (Moms alba L.) varieties cultivated in two polluted fields.
Lei Ming,Pan Yuqi,Chen Congying,Du Huihui,Tie Baiqing,Yan Xinpei,Huang Renzhi
In order to study the role of mulberry (Moms alba L) as an economic crop for remediation of cadmium (Cd) contaminated soil, the transport of Cd from mulberry to silkworm were investigated. Three varieties of mulberry (Yuesang-11, Nongsang-14, and Qiangsang-1) with three planting densities were cultivated in two heavy metal-contaminated fields named Dongkou in Shaoyang city and Linxiang in Yueyang city in Hunan province respectively. The both field soils were contaminated by heavy metals, especially by Cd. The potential risks of heavy metals in Linxiang's soil were higher than those in Dongkou's because of higher concentrations of Cd. Since the promotion of Cd concentrations in aerial parts (stem, branch and leaf) resulted from the increase of planting density, the method of high planting density is beneficial to improve the efficiency of the remediation of Cd contaminated soil. The percentages of average Cd contents of mulberry in Dongkou accounted for 44%, 20%, 18% and 16% in roots, stems, branches and leaves respectively, while the Cd contents were 38%, 27%, 19% and 16% distributed in roots, stems, branches and leaves respectively. Mulberry leaves from contaminated soils was applied in food source of silkworms in this study. Although there is Cd uptake occurred in silkworm growth and its products (cocoons and chrysalis), Cd contents in cocoons are lower than the national standard (100 μg*kg-1) for textile industry of China. Therefore, mulberry can be regarded as an economical crop to control soil contamination with Cd.
The mobility of cadmium and lead in the soil-mulberry-silkworm system.
Jiang Yongbing,Jiang Shimeng,Yan Xinpei,Qin Zhixiong,Jia Chaohua,Li Zhangbao,Zhang Jun,Huang Renzhi
Evaluation of the transportation of heavy metals in food chain has received a great deal of attention. In this study, the mobility of cadmium (Cd) and lead (Pb) in soil-mulberry-silkworm system was assessed. The results showed that bioconcentration factors for Cd and Pb were lower than 1 for all the three mulberry cultivars. Higher translocation factors (TFs) were observed in the levels from branch to leaf, larvae to excrement. The BCFs of Pb in root and silkworm excrement were higher than those in the other parts. Meanwhile, most of Cd accumulated from soils located in the root (48.00-54.40%) and only about 10% was in the leaf. But the Cd and Pb had significant effects on the ratios of cocoon shell with Yuesang 11 under different planting densities. For Yuesang 11 and Qiangsang 1, the Pb percentages were roots > branches > leaves > stems. The rates of ingestion (IR) and digestion (DR) were a little higher than those in the control at first and then decreased gradually with time. The IR reached the lowest values on 8th day while the DR arrived at the highest. Planting mulberry and raising silkworm could be a reasonable method for the utilization of heavy metal contaminated paddy soils.
Metabolomic Analysis of Biochemical Changes in the Serum and Urine of Freund's Adjuvant-Induced Arthritis in Rats after Treatment with Silkworm Excrement.
Zheng Tianyao,Su Shulan,Dai Xinxin,Zhang Liwen,Duan Jin-Ao,Ou-Yang Zhen
Molecules (Basel, Switzerland)
Silkworm excrement (SE), is used as a traditional antirheumatic medicine in China. The present study was designed to investigate the therapeutic efficacy of water fraction of SE (ST) and ethanol fraction of SE (CT) at two different doses on adjuvant induced arthritis (AA) rats. Arthritis severity was evaluated by body weight, paw thickness, histological changes and index of paws oedema and spleen. Serum samples were collected for estimation of biochemical indicators and cytokines. In addition, a metabonomic method based on the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) had been established to investigate the holistic efficacy of SE by serum and urine. Multivariate statistical approaches, such as partial least-squares discriminant analysis (PLS-DA) and orthogonal projection to latent structures squares-discriminant analysis (OPLS-DA) were built to evaluate the therapeutic effects of SE and find potential biomarkers and metabolic pathways. Administration with SE significantly ameliorated the AA severity, including body weight loss, paw swelling, histological changes and the levels of biochemical index. 33 endogenous metabolites had been identified (10 in serum and 23 in urine) in the AA rats. Urinary and serum metabolic profiling revealed that the metabolites underpin the metabolic pathway including nicotinate and nicotinamide metabolism; pentose and glucuronate interconversions; TCA cycle; beta-Alanine metabolism; purine metabolism and glycolysis or gluconeogenesis. The altered metabolites could be regulated closer to normal level after SE intervention. The results suggested SE possesses substantial anti-arthritic activity and demonstrated that metabonomics is a powerful tool to gain insight in the mechanism of SE formula in therapy.
Pharmacokinetics of anti-infectious reagents in silkworms.
Hamamoto Hiroshi,Horie Ryo,Sekimizu Kazuhisa
Silkworm microorganism infection models are useful for screening novel therapeutically effective antimicrobial agents. In this study, we used silkworms to investigate the pharmacokinetics and metabolism of antimicrobial agents, in which cytochrome P450 plays a major role. The pharmacokinetic parameters of the antimicrobial agents were determined based on their concentrations in the hemolymph after administration. The parameters, such as half-lives and distribution volumes, in silkworm were consistent with those in mammalian models. In addition, antifungal agents with reduced therapeutic effectiveness due to high protein-binding capacities in mammalian serum exhibited similar features in silkworm hemolymph. Cytochrome P450 enzymes, which metabolize exogenous compounds in mammalian liver, were distributed mainly in the silkworm midgut. Most of the compounds metabolized by cytochrome P450 in humans are also metabolized in the silkworm midgut. These findings suggest that the pharmacokinetics of antimicrobial agents are fundamentally similar between silkworms and mammals, and that therapeutic effects in the silkworm infection model reflect the pharmacokinetics of the test samples.
Sanguinarine caused larval lethality and growth inhibition by suppressing energy metabolism in silkworms, Bombyx mori.
Li Ping,Hu Jing-Wei,Wen Chao-Wei,Hang Yang,Zhou Zhuo-Hua,Xie Min,Lv Jia-Cheng,Wang Chun-Meng,Huang Ying-Hao,Xu Jia-Ping,Deng Ming-Jie
Pesticide biochemistry and physiology
Sanguinarine (Sang) is a natural alkaloid and distributed in several plants of Papaveraceae. The antitumor, antioxidant, antimicrobial and anti-inflammatory effects of Sang were extensively reported, but its speciality and mechanism against Lepidoptera insects were still unknown. In this study, detailed toxicological parameters of Sang against silkworms, Bombyx mori (B. mori), were determined by a toxicological test. Then, a nuclear magnetic resonance-based (NMR) metabolomics method was adopted to analyze the changes in hemolymph metabolites of silkworms after feeding Sang. The growth of fourth-instar larvae was significantly ceased by the oral administration of 0.05-0.3% Sang and vast deaths appeared in 0.3% Sang group on Day 4 and Day 5. The quantitative analysis of metabolites indicated that trehalose and citrate levels in hemolymph were increased after 24 h of feeding 0.3% Sang, whereas the concentrations of pyruvate, succinate, malate and fumarate were decreased. In addition, the enzymatic determination and reverse transcription quantitative PCR (RT-qPCR) showed that the trehalase (THL) activity and the transcriptional level of one gene coding THL were uniformly weakened by 0.3% Sang. One of the important mechanisms of Sang against silkworms might be interpreted as follows. Sang impaired trehalose hydrolysis, reduced THL activity and transcription, and led to the inhibition of energy metabolism, consequent antigrowth and high lethality in larvae of B. mori. Our findings offered new insights into the insecticidal effect of Sang from the perspective of energy metabolism and provided the basis for the application of Sang in the control of Lepidoptera pests.
Silkworm dropping extract regulates food allergy symptoms via inhibition of Th2-related responses in an ovalbumin-induced food allergy model.
Jung Sun Young,See Hye-Joeng,Kwon Da-Ae,Choi Dae-Woon,Lee So-Young,Shon Dong-Hwa,Shin Hee Soon
Journal of the science of food and agriculture
BACKGROUND:Silkworm droppings have long been used in traditional medicine to remedy allergic itching, palsy, blood circulation problems, and arthritis in Asian countries. To investigate the anti-allergic effect of silkworm dropping extract (SDE) and its mechanism, we used a mouse model of food allergy induced by ovalbumin (OVA). RESULTS:SDE ameliorated the symptoms of OVA-induced food allergies, and the levels of T helper 2 (Th2)-related cytokines [such as interleukin (IL)-4, IL-5, IL-10, and IL-13] were found to be significantly decreased in both the spleen and mesenteric lymph nodes by SDE. Furthermore, SDE treatment directly inhibited OVA permeation, IL-4 production, and degranulation of mast cells; in contrast, immunoglobulin E (IgE) production from B cells was not affected. CONCLUSION:These results suggest that SDE has potential anti-allergic activities, and SDE may be useful in the treatment/prevention of allergic disorders such as food allergies, serving as therapeutic agents. © 2019 Society of Chemical Industry.
Tea silkworm droppings as an enriched source of tea flavonoids.
Chou Tzu-Yun,Yang Meei-Ju,Tseng Shih-Kung,Lee Shoei-Sheng,Chang Chia-Chuan
Journal of food and drug analysis
Andraca droppings is the waste excreted from the tea biter Andraca theae. Its chemical constituents and potential medical use, unlike those of the traditional Chinese medicine silkworm droppings, have not been reported yet. To explore new nutraceuticals, the chemical constituents of this substance were investigated. Since the bioactive ingredients are generally present in the EtOAc-soluble fraction, this fraction, obtained from the ethanolic extract of the dried Andraca droppings by liquid-liquid partitioning, was separated by chromatographic methods, including Sephadex LH-20, centrifugal partition chromatography, and RP-18 columns, to produce 14 compounds (1-14). They were characterized as 1,7-dimethyl xanthine (1), three benzoic acids (2, 3, and 5), and 10 flavonoids (4, 6-14). The amount of compounds 6, 7, 10, 13, and 14 in the droppings were 1.7-15.5-fold compared to those of tea leaves. In addition, 1,7-dimethyl xanthine (1) was found present only in the Andraca droppings but absent in tea leaves. Therefore, except for compound 1, which might be transformed from caffeine by microflora in the insect, the compounds were believed not to be absorbed by the worm gut and excreted directly. The present study suggests the Andraca droppings are an enriched source of the bioactive flavonoids from tea leaves and are potential as a useful nutraceutical.
N-glycan sialylation in a silkworm-baculovirus expression system.
Suganuma Masatoshi,Nomura Tsuyoshi,Higa Yukiko,Kataoka Yukiko,Funaguma Shunsuke,Okazaki Hironobu,Suzuki Takeo,Fujiyama Kazuhito,Sezutsu Hideki,Tatematsu Ken-Ichiro,Tamura Toshiki
Journal of bioscience and bioengineering
A silkworm-baculovirus system is particularly effective for producing recombinant proteins, including glycoproteins. However, N-glycan structures in silkworm differ from those in mammals. Glycoproteins in silkworm are secreted as pauci-mannose type N-glycans without sialic acid or galactose residues. Sialic acid on N-glycans plays important roles in protein functions. Therefore, we developed pathways for galactosylation and sialylation in silkworm. Sialylated N-glycans on proteins were successfully produced in silkworm by co-expressing galactosyltransferase and sialyltransferase and providing an external supply of a sialylation-related substrate. α2,3/α2,6 Sialylation to N-glycans was controlled by changing the type of sialyltransferase expressed in silkworm. Furthermore, the co-expression of N-acetylglucosaminyltransferase II facilitated the formation of additional di-sialylated N-glycan structures. Our results provide new information on the control of N-glycosylation in silkworm.
Characterization of glycoengineered anti-HER2 monoclonal antibodies produced by using a silkworm-baculovirus expression system.
Egashira Yuriko,Nagatoishi Satoru,Kiyoshi Masato,Ishii-Watabe Akiko,Tsumoto Kouhei
Journal of biochemistry
Silkworm-baculovirus expression systems are efficient means for the production of recombinant proteins that provide high expression levels and post-translational modifications. Here, we characterized the stability, glycosylation pattern and antibody-dependent cell-mediated cytotoxicity activity of anti-HER2 monoclonal antibodies containing native or glycoengineered mammalian-like N-glycans that were produced by using a silkworm-baculovirus expression system. Compared with a monoclonal antibody produced by using a Chinese hamster ovary cell expression system, the glycoengineered monoclonal antibody had comparable thermal stability and a higher antibody-dependent cell-mediated cytotoxicity activity. These results suggest that silkworm-baculovirus expression systems are next-generation expression systems potentially useful for the cost-effective production of therapeutic antibodies.
Insect symbionts as valuable grist for the biotechnological mill: an alkaliphilic silkworm gut bacterium for efficient lactic acid production.
Liang Xili,Sun Chao,Chen Bosheng,Du Kaiqian,Yu Ting,Luang-In Vijitra,Lu Xingmeng,Shao Yongqi
Applied microbiology and biotechnology
Insects constitute the most abundant and diverse animal class and act as hosts to an extraordinary variety of symbiotic microorganisms. These microbes living inside the insects play critical roles in host biology and are also valuable bioresources. Enterococcus mundtii EMB156, isolated from the larval gut (gut pH >10) of the model organism Bombyx mori (Lepidoptera: Bombycidae), efficiently produces lactic acid, an important metabolite for industrial production of bioplastic materials. E. mundtii EMB156 grows well under alkaline conditions and stably converts various carbon sources into lactic acid, offering advantages in downstream fermentative processes. High-yield lactic acid production can be achieved by the strain EMB156 from renewable biomass substrates under alkaline pretreatments. Single-molecule real-time (SMRT) sequencing technology revealed its 3.01 Mbp whole genome sequence. A total of 2956 protein-coding sequences, 65 tRNA genes, and 6 rRNA operons were predicted in the EMB156 chromosome. Remarkable genomic features responsible for lactic acid fermentation included key enzymes involved in the pentose phosphate (PP)/glycolytic pathway, and an alpha amylase and xylose isomerase were characterized in EMB156. This genomic information coincides with the phenotype of E. mundtii EMB156, reflecting its metabolic flexibility in efficient lactate fermentation, and established a foundation for future biotechnological application. Interestingly, enzyme activities of amylase were quite stable in high-pH broths, indicating a possible mechanism for strong EMB156 growth in an alkaline environment, thereby facilitating lactic acid production. Together, these findings implied that valuable lactic acid-producing bacteria can be discovered efficiently by screening under the extremely alkaline conditions, as exemplified by gut microbial symbionts of Lepidoptera insects.
Establishment of a gnotobiotic silkworm model.
Nakajima Hiroto,Matsumoto Yasuhiko,Sekimizu Kazuhisa
Drug discoveries & therapeutics
Gnotobiotic animals are useful for investigation of the effects of specific lactic acid bacteria on individual animals. Here we report that lactic acid bacteria colonize and proliferate in the intestinal tract of germ-free silkworms. When silkworms hatching from formalin-treated eggs were reared to fifth-instar larvae with an artificial diet containing antibiotics, bacteria and fungi were not observed in their intestines. An antibiotic-free diet supplemented with viable lactic acid bacteria, such as Enterococcus faecalis 0831-07, Lactococcus lactis 11/19-B1, or Leuconostoc carnosum #7-2, was fed to the germ-free silkworms for 1 day. After feeding the larvae on a diet without lactic acid bacteria for 5 days, each type of lactic acid bacterium was found in the intestine. Moreover, an increase in the number of Enterococcus faecalis 0831-07 was observed in the intestine 2-5 days after ingestion. These findings suggest that we successfully established a method to construct a gnotobiotic silkworm model.
Construction of a simple evaluation system for the intestinal absorption of an orally administered medicine using Bombyx mori larvae.
Ichino Fumika,Bono Hidemasa,Nakazato Takeru,Toyoda Atsushi,Fujiyama Asao,Iwabuchi Kikuo,Sato Ryoichi,Tabunoki Hiroko
Drug discoveries & therapeutics
Human intestinal absorption is estimated using a human colon carcinoma cell line (Caco-2) cells from human colorectal adenocarcinoma, intestinal perfusion, or a mammalian model. These current evaluation systems are limited in their ability to estimate human intestinal absorption. In addition, in vivo evaluation systems using laboratory animals such as mice and rats entail animal ethics problems, and it is difficult to screen compounds on a large scale at the drug discovery stage. Thus, we propose the use of Bombyx mori larvae for evaluation of intestinal absorption of compounds as an alternative system in this study. First, to compare the characteristics among Caco-2 cells, human intestine, and B. mori larval midgut, we analyzed their RNA-seq data, and we found 26 drug transporters common to humans and B. mori. Next, we quantitatively developed an oral administration technique in B. mori and established a method using silkworm B. mori larvae that can easily estimate the intestinal permeability of compounds. Consequently, we could determine the dose and technique for oral administration in B. mori larvae. We also developed a B. mori model to evaluate the intestinal permeability of orally administered. Our constructed evaluation system will be useful for evaluating intestinal permeability in medical drug development.
Advantages of the Silkworm As an Animal Model for Developing Novel Antimicrobial Agents.
Panthee Suresh,Paudel Atmika,Hamamoto Hiroshi,Sekimizu Kazuhisa
Frontiers in microbiology
The demand for novel antibiotics to combat the global spread of multi drug-resistant pathogens continues to grow. Pathogenic bacteria and fungi that cause fatal human infections can also kill silkworms and the infected silkworms can be cured by the same antibiotics used to treat infections in the clinic. As an invertebrate model, silkworm model is characterized by its convenience, low cost, no ethical issues. The presence of conserved immune response and similar pharmacokinetics compared to mammals make silkworm infection model suitable to examine the therapeutic effectiveness of antimicrobial agents. Based on this, we utilized silkworm bacterial infection model to screen the therapeutic effectiveness of various microbial culture broths and successfully identified a therapeutically effective novel antibiotic, lysocin E, which has a novel mode of action of binding to menaquinone, thus leading to membrane damage and bactericidal activity. The similar approach to screen potential antibiotics resulted in the identification of other therapeutically effective novel antibiotics, such as nosokomycin and ASP2397 (VL-2397). In this regard, we propose that the silkworm antibiotic screening model is very effective for identifying novel antibiotics. In this review, we summarize the advantages of the silkworm model and propose that the utilization of silkworm infection model will facilitate the discovery of novel therapeutically effective antimicrobial agents.
Development of Novel Antibiotic Lysocin E Identified by Silkworm Infection Model.
Hamamoto Hiroshi,Sekimizu Kazuhisa
Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan
In this symposium, we reported the identification and mechanistic analysis of a novel antibiotic named lysocin E. Lysocin E was identified by screening for therapeutic effectiveness in a silkworm Staphylococcus aureus infection model. The advantages of the silkworm infection model for screening and purification of antibiotics from the culture supernatant of soil bacteria are: 1) low cost; 2) no ethical issues; 3) convenient for evaluation of the therapeutic effectiveness of antibiotics; and 4) pharmacokinetics similar to those of mammals. Lysocin E has remarkable features compared with known antibiotics such as a novel mechanism of action and target. Here, we summarize our reports presented in this symposium.
Silkworm fungal infection model for identification of virulence genes in pathogenic fungus and screening of novel antifungal drugs.
Ishii Masaki,Matsumoto Yasuhiko,Nakamura Ikuko,Sekimizu Kazuhisa
Drug discoveries & therapeutics
The silkworm infection model has the potential to replace conventional animal models for evaluation of the efficacy and toxicity of investigational antifungal agents. Silkworms are relatively inexpensive, can be simply grown in large numbers and can be easily infected with pathogenic fungi, including mutant strains. Antifungal agents can then be injected into the silkworm either via the hemolymph to mimic intravenous administration or directly into the gut for oral administration, and their antifungal effect can be evaluated. Common features regarding the mechanisms of pharmacokinetics between the silkworm and mammals result in consistent therapeutic effectiveness of antifungal agents. ASP2397, a promising new antifungal agent, was discovered using the silkworm model. The conclusion is that silkworms can be a more ethical and less expensive alternative to standard animal models, particularly for the identification and testing of new antifungal agents.
Effects of amino acid substitutions on the biological activity of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori).
Aoyama Michihiko,Tada Minoru,Tatematsu Ken-Ichiro,Hashii Noritaka,Sezutsu Hideki,Ishii-Watabe Akiko
Biochemical and biophysical research communications
Recombinant monoclonal antibodies (mAbs) have been used in various therapeutic applications including cancer therapy. Fc-mediated effector functions play a pivotal role in the tumor-killing activities of some tumor-targeting mAbs, and Fc-engineering technologies with glyco-engineering or amino acid substitutions at the antibody Fc region have been used to enhance cytotoxic activities including antibody-dependent cellular cytotoxicity (ADCC). We previously reported that the mAbs produced using transgenic silkworms showed stronger ADCC activity and lower complement-dependent cytotoxicity (CDC) activity than mAbs derived from Chinese hamster ovary (CHO) cells due to their unique N-glycan structure (lack of core-fucose and non-reducing terminal galactose). In this study, we generated anti-CD20 mAbs with amino acid substitutions using transgenic silkworms and analyzed their biological activities to assess the effect of the combination of glyco-engineering and amino acid substitutions on the Fc-mediated function of mAbs. Three types of amino acid substitutions at the Fc region (G236A/S239D/I332E, L234A/L235A, and K326W/E333S) modified the Fc-mediated biological activities of silkworm-derived mAbs as in the case of CHO-derived mAbs, resulting in the generation of Fc-engineered mAbs with characteristic Fc-mediated functions. The combination of amino acid substitutions at the Fc region and glyco-engineering using transgenic silkworm made it possible to generate Fc-engineered mAbs with suitable Fc-mediated biological functions depending on the pharmacological mechanism of their actions. Transgenic silkworms were shown to be a promising system for the production of Fc-engineered mAbs.
In situ biomineralization by silkworm feeding with ion precursors for the improved mechanical properties of silk fiber.
Guo Zhenhu,Xie Wensheng,Gao Qin,Wang Dan,Gao Fei,Li Shengrong,Zhao Lingyun
International journal of biological macromolecules
Possessing excellent biocompatibility, biodegradability and good reactive activity, silk fiber has been attracting great attention in biomedicine including surgical suture, drug delivery and tissue engineering. So far, several protocols have been developed to further improve the mechanical properties of the silk fiber. In current study, a novel in suit biomineralization strategy was developed to produce nano-hydroxyapatite (HA) strengthened silk fiber based on the natural alkaline condition in the body of silkworm by feeding the silkworm with ion precursors of Ca and PO ions. Our observation proved that nanocomposite silk fiber contained more α-helix and random coil structures and fewer β-sheets. Tensile test showed that such obtained silk fiber has superior mechanical properties compared to normal silk fiber. To the best of our knowledge, no attempts have been made to fabricate the nanocomposite SF by in situ biomineralization and such protocol established in the current study may shed light on the investigation of nanoparticles reinforced silk fiber organisms of Bombyx mori.
Accelerated wound-healing capabilities of a dressing fabricated from silkworm cocoon.
Yu Kun,Lu Fei,Li Qing,Zou Yini,Xiao Yang,Lu Bitao,Liu Jiawei,Dai Fangying,Wu Dayang,Lan Guangqian
International journal of biological macromolecules
Silk fibroin materials have shown some success in wound dressing applications; however, their use for this purpose remains limited by a complex production process and wasted sericin. In the present study, Bombyx mori cocoon materials are used because the protective function of the silkworm cocoon resembles the manner in which the skin protects the human body. A series of silkworm cocoon sol-gel film (SCSF) wound dressings are prepared by immersion in a CaCl-ethanol-HO solution for different treatment times. The accelerated wound-healing capabilities of SCSFs are systematically evaluated. Among them, the SCSF sample immersed for 90min exhibits stronger biocompatibility and antibacterial performance compared to other SCSFs. SCSF-90 also exhibits excellent transparency, a high swelling ratio, and good extensibility. Furthermore, in vivo experiments indicate that SCSF-90 can significantly accelerate the healing rate of wounds in New Zealand white rabbits, compared to the standard Mepitel dressing, and histological examinations reveal that SCSF-90 aided in the successful reconstruction of intact and thickened epidermis. These results demonstrate that the proposed approach may be utilized in the design of antibacterial materials with promising applications in wound dressing.
Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System.
Yamashita Mami,Xu Jian,Morokuma Daisuke,Hirata Kazuma,Hino Masato,Mon Hiroaki,Takahashi Masateru,Hamdan Samir M,Sakashita Kosuke,Iiyama Kazuhiro,Banno Yutaka,Kusakabe Takahiro,Lee Jae Man
The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.
Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm-Baculovirus Expression System.
Xu Jian,Lee Jae Man,Tatsuke Tuneyuki,Ebihara Takeru,Masuda Akitsu,Hino Masato,Morokuma Daisuke,Fujita Ryosuke,Mon Hiroaki,Kusakabe Takahiro,Takahashi Masateru
Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm-baculovirus expression vector system (silkworm-BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 μg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg, Mn, Ca, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm-BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.
Production of highly immunogenic virus-like particles of bovine papillomavirus type 6 in silkworm pupae.
Watanabe Satoko,Iizuka Tetsuya,Hatama Shinichi,Kanno Toru,Mase Masaji,Shibahara Tomoyuki
Bovine papillomaviruses (BPVs) are the causative agent of bovine teat papillomatosis, which can lead to severe economic losses in dairy cattle. Among the 14 identified BPV genotypes, BPV type 6 (BPV6) is the most frequently detected in teat papilloma lesions, and is therefore thought to play a major role in teat papillomatosis. To develop an effective vaccine against BPV6 infection, we produced virus-like particles of BPV6 (BPV6-VLP) in silkworm (Bombyx mori) pupae and purified these by heparin affinity chromatography using a single column. About 0.7mg purified BPV6-VLP was obtained from one pupa. BPV6-VLP-immunized mice produced a specific IgG to BPV6 that recognized BPV6 antigen with high sensitivity in an immunohistochemical analysis. Thus, silkworm pupae are a useful bioreactor for the production of BPV6-VLP, which can potentially be used as a vaccine for bovine teat papillomatosis.
High-efficiency production of human serum albumin in the posterior silk glands of transgenic silkworms, Bombyx mori L.
Qian Qiujie,You Zhengying,Ye Lupeng,Che Jiaqian,Wang Yiran,Wang Shaohua,Zhong Boxiong
Human serum albumin (HSA) is an important biological preparation with a variety of biological functions in clinical applications. In this study, the mRNA of a fusion transposase derived from the pESNT-PBase plasmid and a pBHSA plasmid containing the HSA gene under the control of a fibroin light chain (FL) promoter were co-injected into fertilized eggs. Fifty-six transgenic silkworm pedigrees expressing theexogenous recombinant HSA (rHSA) in the posterior silk glands (PSGs) with stable inheritance were successfully obtained. The SDS-PAGE and Western blot results confirmed that the rHSA was secreted into the transgenic silkworm cocoon, and the rHSA could be easily extracted with phosphate-buffered saline (PBS). In our research, the isolated highest amount rHSA constituted up to 29.1% of the total soluble protein of the cocoon shell, indicating that the transgenic silkworm produced an average of 17.4 μg/mg of rHSA in the cocoon shell. The production of soluble rHSA in the PSGs by means of generating transgenic silkworms is a novel approach, whereby a large amount of virus-free and functional HSA can be produced through the simple rearing of silkworms.
Silkworm genetic sexing through W chromosome-linked, targeted gene integration.
Zhang Zhongjie,Niu Baolong,Ji Dongfeng,Li Muwang,Li Kai,James Anthony A,Tan Anjiang,Huang Yongping
Proceedings of the National Academy of Sciences of the United States of America
Sex separation methods are critical for genetic sexing systems in commercial insect production and sterile insect techniques. Integration of selectable marker genes into a sex chromosome is particularly useful in insects with a heterogametic sex determination system. Here, we describe targeted gene integration of fluorescent marker expression cassettes into a randomly amplified polymorphic DNA (RAPD) marker region in the W chromosome of the lepidopteran model insect using transcriptional activator-like effector nuclease (TALEN)-mediated genome editing. This silkworm strain shows ubiquitous female-specific red or green fluorescence from the embryonic to adult stages. Furthermore, we developed a binary, female-specific, embryonic lethality system combining the TALEN and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. This system includes one strain with TALEN-mediated, W-specific Cas9 expression driven by the silkworm germ cell-specific () promoter and another strain with U6-derived single-guide RNA (sgRNA) expression targeting (), an essential gene for silkworm embryonic development. Filial 1 (F1) hybrids exhibit complete female-specific lethality during embryonic stages. Our study provides a promising approach for genetic sexing and sheds light on developing sterile insect techniques in other insect species, especially in lepidopteran pests with WZ/ZZ sex chromosome systems.
Identification of Bombyx mori sericin 4 protein as a new biological adhesive.
Dong Zhaoming,Guo Kaiyu,Zhang Xiaolu,Zhang Tong,Zhang Yan,Ma Sanyuan,Chang Huaipu,Tang Muya,An Lingna,Xia Qingyou,Zhao Ping
International journal of biological macromolecules
Sericins are large proteins with molecular weights >70 kDa. Three sericin genes were reported in the silkworm, including sericin 1, sericin 2 and sericin 3. In this study, we have identified a new sericin gene and designated it as sericin 4. The sequence, exon-intron structure, alternative splicing, and translation products of this gene have been described in this study. Quantitative RT-PCR analysis indicates that sericin 4 is expressed in the middle silk gland. Immunofluorescence results show co-localization of sericin 1 and sericin 4 in the MSG. Western blot analysis revealed that sericin 4 was found in the larval silk produced from the second instar to the fourth instar. Two protein bands at approximately 280 kDa and 260 kDa, were detected by western blot for sericin 4. Two repetitive motifs that are rich in charged amino acids and glutamine have been identified, and they are likely to be responsible for the adhesiveness of sericin 4. Overall, this study identifies a novel biological adhesive protein and provides new information for understanding how sericins contribute to the adhesive properties of larval silks.
Properties of chitin and chitosan extracted from silkworm pupae and egg shells.
Battampara Prajwal,Nimisha Sathish T,Reddy Roopa,Guna Vijaykumar,Nagananda G S,Reddy Narendra,Ramesha B S,Maharaddi V H,Rao A Prabhakar,Ravikumar H N,Biradar Ashok,Radhakrishna P G
International journal of biological macromolecules
Chitin and chitosan from silkworm pupae and egg shells show distinct properties with excellent antimicrobial properties and cytocompatiblity. Spent silkworm pupae and hatched egg shells are discarded as waste but contain valuable carbohydrates, proteins and lipids. Chitosan has excellent antimicrobial properties and is widely used for food, medical and biotechnological applications. In this paper, we report the properties of chitin and chitosan from silkworm pupae and egg shells in comparison to commercially available chitosan. Defatted and deproteinated pupae and shells were demineralized and later deacetylated to form chitosan. Thermal behavior, physical structure, antimicrobial activity and ability to support the attachment and growth of NIH3T3 cells were studied. Chitin and chitosan from both pupae and shells had similar structure and composition. Crystallinity of the pupae chitosan was 48% compared to 38% for egg shell chitosan. Silkworm chitosan showed considerably higher antibacterial and antifungal activity compared to standard. Cells were viable in the presence of pupae and egg shell chitosan in all the concentrations tested. Based on these observations, it can be inferred that silkworm pupae and shells provide a renewable and sustainable source for chitosan with properties suitable for food and medical applications.
Expression, Purification, and Characterization of Recombinant Human α-Antitrypsin Produced Using Silkworm-Baculovirus Expression System.
Morifuji Yoshiki,Xu Jian,Karasaki Noriko,Iiyama Kazuhiro,Morokuma Daisuke,Hino Masato,Masuda Akitsu,Yano Takumi,Mon Hiroaki,Kusakabe Takahiro,Lee Jae Man
Human α-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~ 15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.
Silkworm dropping extract ameliorate trimellitic anhydride-induced allergic contact dermatitis by regulating Th1/Th2 immune response.
Choi Dae Woon,Kwon Da-Ae,Jung Sung Keun,See Hye-Jeong,Jung Sun Young,Shon Dong-Hwa,Shin Hee Soon
Bioscience, biotechnology, and biochemistry
Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by hapten-specific immune response. Silkworm droppings are known to exert beneficial effects during the treatment of inflammatory diseases. Here, we studied whether topical treatment and oral administration of silkworm dropping extract (SDE) ameliorate trimellitic anhydride (TMA)-induced ACD. In ACD mice model, SDE treatment significantly suppressed the increase in both ear thickness and serum IgE levels. Furthermore, IL-1β and TNF-α levels were reduced by SDE. In allergic responses, SDE treatment significantly attenuated the production of the Th2-associated cytokine IL-4 in both ear tissue and draining lymph nodes. However, it increased the production of the Th1-mediated cytokine IL-12. Thus, these results showed that SDE attenuated TMA-induced ACD symptoms through regulation of Th1/Th2 immune response. Taken together, we suggest that SDE treatment might be a potential agent in the prevention or therapy of Th2-mediated inflammatory skin diseases such as ACD and atopic dermatitis. ABBREVIATIONS:ACD: allergic contact dermatitis; AD: atopic dermatitis; APC: antigen presenting cells; CCL: chemokine (C-C motif) ligand; CCR: C-C chemokine receptor; Dex: dexamethasone; ELISA: enzyme-linked immunosorbent assay; IFN: interferon; Ig: immunoglobulin; IL: interleukin; OVA: ovalbumin; PS: prednisolone; SDE: silkworm dropping extract; Th: T helper; TMA: trimellitic anhydride; TNF: tumor necrosis factor.
The biogas production potential from silkworm waste.
Łochyńska Małgorzata,Frankowski Jakub
Waste management (New York, N.Y.)
In view of the increasing demand of organic agriculture, utilization of waste and environmental protection, sericulture focuses not only on the cocoon production, but also on other ways that can benefit the farm's economy. It is necessary to find new sources of income for small-scale farmers not only through cocoon selling, but also by the multiple uses of by-products. Insect farming technology provides a cheap source of biomass, which may be a good material in biogas production. Studies showed that the examined substrates, both silkworm breeding waste and caterpillar excreta, generate a biogas yield comparable to other substrates of agricultural origin, such as cattle, pig and chicken manures. Fermentation of silkworm excreta under mesophilic conditions produces 167.32 m/Mg TS of methane and 331.97 m/Mg TS of biogas, while fermentation of silkworm breeding waste yields 256.59 m/Mg TS of methane and 489.24 m/Mg TS of biogas. Moreover, the chemical composition of these raw materials was analyzed.
[A Silkworm Infection Model to Evaluate Antifungal Drugs for Cryptococcosis].
Matsumoto Yasuhiko,Ishii Masaki,Shimizu Kiminori,Kawamoto Susumu,Sekimizu Kazuhisa
Medical mycology journal
The development of effective drugs against fungal diseases involves performing infection experiments in animals to evaluate candidate therapeutic compounds. Cryptococcus neoformans is a pathogenic fungus that causes deep mycosis, resulting in respiratory illness and meningitis. Here we describe a silkworm system established to evaluate the safety and efficacy of therapeutic drugs against infection by Cryptococcus neoformans and the advantages of this system over other animal models. The silkworm assay system has two major advantages: 1) silkworms are less expensive to rear and their use is less problematic than that of mammals in terms of animal welfare, and 2) in vivo screenings for identifying candidate drugs can be easily performed using a large number of silkworms. The pharmacokinetics of compounds are consistent between silkworms and mammals. Moreover, the ED values of antibiotics are concordant between mammalian and silkworm infection models. Furthermore, the body size of silkworms makes them easy to handle in experimental procedures compared with other invertebrate infectious experimental systems, and accurate amounts of pathogens and chemicals can be injected fairly easily. These advantages of silkworms as a host animal make them useful for screening candidate drugs for cryptococcosis.
[Development of Antibiotics Using Silkworm Bacteria and Fungi Infection Model].
Hamamoto Hiroshi,Sekimizu Kazuhisa
Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan
The emergence of antimicrobial resistant (AMR) bacteria has become a serious threat to public health. It is important that we find a mechanistically novel antibiotic to combat AMR. However, finding compounds which are both therapeutically effective and safe is difficult in the development of antibiotics. To solve these problems, we have focused on the silkworm model, which is economical and poses fewer ethical issues, as a means to evaluate the therapeutic effectiveness of test compounds in early stages of antibiotic development. Actually, the silkworm has pharmacokinetic parameters similar to mammals, and we revealed that antibiotics showed EDs consistent with mammalian models. Thus, we screened therapeutically effective samples from natural products using the silkworm model, and found 23 candidates out of 15000 samples. We ultimately identified a novel antibiotic, lysocin E, and found that it demonstrates a potent therapeutic effect in the mouse systemic infection model. Furthermore, since the target of lysocin E is menaquinone on the bacterial membrane, it belongs to a novel class of antibiotics. In addition, we found a novel antibacterial agent named nosokomycin, GPI0363, and an antifungal agent, VL-2397 (ASP2397), using the silkworm model. In this report, we introduce the usefulness of the silkworm model in the development of antibiotics.
Transgenic Silkworm-Based Silk Gland Bioreactor for Large Scale Production of Bioactive Human Platelet-Derived Growth Factor (PDGF-BB) in Silk Cocoons.
Chen Wenjing,Wang Feng,Tian Chi,Wang Yuancheng,Xu Sheng,Wang Riyuan,Hou Kai,Zhao Ping,Yu Ling,Lu Zhisong,Xia Qingyou
International journal of molecular sciences
Human platelet derived growth factor (PDGF) is a major therapeutic protein with great demand in the clinical setting; however, its rate of supply is far from meeting needs. Here, we provide an effective strategy to produce PDGF-BB in large quantities using a transgenic silkworm. The codon-optimized gene regulated by the highly efficient expression system was integrated into the genome of a silkworm. The high transcriptional expression of the PDGF-BB gene in the transgenic silkworm competitively inhibited the transcription expression of the endogenous gene which caused a significant 37.5% decline. The PDGF-BB synthesized in the middle silk gland (MSG) of transgenic silkworms could form a homodimer through intermolecular disulfide bonds, which is then secreted into sericin lumen and finally, distributed in the sericin layer of the cocoon. In this study, a protein quantity of approximately 0.33 mg/g was found in the cocoon. Following a purification process, approximately 150.7 μg of recombinant PDGF-BB with a purity of 82% was purified from 1 g of cocoons. Furthermore, the bioactivity assays showed that the purified recombinant PDGF-BB was able to promote the growth, proliferation and migration of NIH/3T3 cells significantly. These results suggest that the silk gland bioreactor can produce active recombinant PDGF-BB as an efficient mitogen and wound healing agent.
Fabrication of the FGF1-functionalized sericin hydrogels with cell proliferation activity for biomedical application using genetically engineered Bombyx mori (B. mori) silk.
Wang Feng,Wang Yuancheng,Tian Chi,Xu Sheng,Wang Riyuan,Hou Kai,Chen Wenjing,Zhao Ping,Yu Ling,Lu Zhisong,Kaplan David L,Xia Qingyou
Sericin, as the major component of Bombyx mori silk, is a useful biomaterial for tissue engineering due to its hydrophilicity, biocompatibility and biodegradability. Here, we report the fabrication of a human acidic fibroblast growth factor (FGF1)-functionalized sericin hydrogel using a transgenic silkworm spun silk with FGF1 incorporated in its sericin layer. Sericin, together with FGF1, were simultaneously extracted from the silk fiber and then exposed to cold-induced hydrogel formation without additional crosslinking. The fabricated FGF1 sericin hydrogels demonstrated injectability, useful mechanical properties and a porous microstructure, which contributed to cell adhesion and survival. In addition, FGF1 achieved long-term storage in the sericin hydrogels over a wide range of temperatures. Further, the sericin-FGF1 demonstrated sustained release to promote cell proliferation and wound healing. Furthermore, cellular inflammatory responses showed that the FGF1 sericin hydrogels exhibited biocompatibility and no immunogenicity. This study revealed the successful exploration of FGF1-functionalized sericin hydrogels as a new protein-based biomaterial to expand applications of FGF1 and sericin in tissue and medical engineering. Further, we demonstrated a strategy for the predesign of exogenous protein-functionalized sericin hydrogels through genetically modifying silk fibers as sources for their cost effective production at a large scale. STATEMENT OF SIGNIFICANCE:Sericin from the Bombyx mori silk, is regarded as a desirable biomaterial for tissue engineering due to its hydrophilicity, biocompatibility and biodegradability. Genetically engineering the sericin with functional exogenous proteins would enhance its biofunctions and further expand its application in tissue engineering. In this study, we demonstrated a method to fabricate a human acidic fibroblast growth factor (FGF1)-functionalized sericin hydrogel using a transgenic silkworm spun silk with FGF1 incorporated in its sericin layer. The fabricated FGF1 sericin hydrogels demonstrated injectability, porous microstructure, biocompatibility and no immunogenicity which contributed to cell adhesion and survival. Remarkably, FGF1 could achieve a long-term stability in the sericin hydrogels over a wide range of temperatures and sustained release to promote cell proliferation and wound healing. This study revealed the successful exploration of FGF1-functionalized sericin hydrogels as a new protein-based biomaterial in tissue and medical engineering application, and provided a strategy for the predesign of exogenous protein-functionalized sericin hydrogels through genetically modifying silk fibers as sources for their cost effective production at a large scale.
Protective effect of silkworm pupa oil on hydrochloric acid/ethanol-induced gastric ulcers.
Long Xingyao,Zhao Xin,Wang Wei,Zhang Yu,Wang Hongwei,Liu Xinqi,Suo Huayi
Journal of the science of food and agriculture
BACKGROUND:Silkworm pupae are a traditional Chinese food, rich in various saturated and unsaturated fatty acids. Unsaturated fatty acids have a certain protective effect against oxidative damage. The present study used an animal model to determine the protective effect of silkworm pupa oil on hydrochloric acid / ethanol-induced gastric ulcer. RESULTS:Silkworm pupa oil is rich in unsaturated fatty acids, including palmitoleic acid 63.4 g kg , oleic acid 249.1 g kg , linoleic acid 47.0 g kg , and linolenic acid 337.8 g kg , whereas its unsaturated fatty acid content is 700 g kg . Compared to a gastric ulcer control group, high and low doses of pupa oil reduced gastric ulcer area and gastric secretion, whereas gastric pH increased. It also increased serum antioxidant superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) levels, somatostatin (SST), and vasoactive intestinal peptide (VIP) levels, and reduced serum interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor (TNF-α), and interferon-γ (IFN-γ), motilin (MTL), and gastrin (GT) levels. RT-qPCR and western blot analyses indicated that silkworm pupa oil significantly increased CAT, GSH-Px, epidermal growth factor (EGF), Epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), Cu/Zn-SOD, Mn-SOD, and NF-kappa-B inhibitor-α (IκB-α) expression and lowered nuclear factor-kappa B (NF-κB), B-cell lymphoma-2 (Bcl-2), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) expression. CONCLUSION:Silkworm pupa oil treatment reduced oxidative damage and inflammation in mice, and high-dose silkworm pupa oil was superior to low-dose silkworm pupa oil, following ranitidine. © 2018 Society of Chemical Industry.
[Development of Large Scale Silkworm-rearing Technologies for the GMP Production of Biologics].
Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan
There exists an increasing need to produce useful proteins in recombinant technologies. In particular, most biologics for medical purposes are produced as recombinant proteins. Various host cell/vector systems have been developed, but it remains difficult to efficiently produce large molecular weight proteins with complex structures. As a result of breeding for several thousand years, the silkworm has acquired the ability to synthesize bulk amounts of silk proteins. To utilize this capacity for the mass production of useful proteins, transgenic silkworms have been generated that synthesize recombinant proteins in the silk gland and secrete them into the silk cocoon. Using this transgenic silkworm system, various proteins, including antibodies, collagen, and fibrinogen, have been successfully produced and are being developed as materials for diagnostic or research-use reagents, as well as for cosmetics. Moreover, several silkworm-produced proteins are being developed as biologics for therapeutic use. Transgenic silkworms need to be reared under good manufacturing practices (GMP)-compliant conditions to produce biologics. Therefore, we have constructed a GMP-compliant pilot plant for producing biologics using transgenic silkworm, and are now developing silkworm-rearing technology under GMP-compliant conditions.
Solid state fermentation process with Aspergillus kawachii enhances the cancer-suppressive potential of silkworm larva in hepatocellular carcinoma cells.
Cho Hyun-Dong,Min Hye-Ji,Won Yeong-Seon,Ahn Hee-Young,Cho Young-Su,Seo Kwon-Il
BMC complementary and alternative medicine
BACKGROUND:Mulberry silkworm larvae (Bombyx mori) are known as the oldest resource of food and traditional medicine. Although silkworm larvae have been reported to treat various chronic diseases, the effect of fermentation by microorganisms improving the biological activities of silkworm larvae was not reported. In the present study, fermented silkworm larvae was developed via solid-state fermentation with Aspergillus kawachii and investigated its anti-cancer activity in human hepatocellular carcinoma cells. METHODS:We investigated the anti-cancer effects of unfermented (SEE) and fermented silkworm larva ethanol extract (FSEE) on HepG2 human hepatocellular carcinoma cells as well as compared changes in free amino acid, fatty acid, and mineral contents. Anti-cancer activities were evaluated by SRB staining, cell cycle analysis, Annexin V staining, Hoechst staining, DNA fragmentation analysis and western blot analysis. Fatty acid, free amino acid and mineral contents of SEE and FSEE were determined by gas chromatography, amino acid analyzer and flame atomic absorption spectrophotometer, respectively. RESULTS:Compared with SEE, treatment with FSEE resulted in apoptotic cell death in HepG2 cells characterized by G0/G1 phase cell cycle arrest, DNA fragmentation, and formation of apoptotic bodies. Furthermore, FSEE significantly up-regulated pro-apoptotic as well as down-regulated anti-apoptotic proteins in HepG2 cells. However, an equivalent concentration of SEE did not induce cell cycle arrest or apoptosis in HepG2 cells. Moreover, fermentation process by Aspergillus kawachii resulted in enhancement of fatty acid contents in silkworm larvae, whereas amino acid and mineral contents were decreased. CONCLUSION:Collectively, this study demonstrates that silkworm larvae solid state-fermented by Aspergillus kawachii strongly potentiates caspase-dependent and -independent apoptosis pathways in human hepatocellular carcinoma cells by regulating secondary metabolites.
[Construction of a Platform for the Development of Pharmaceutical and Medical Applications Using Transgenic Silkworms].
Sezutsu Hideki,Sumitani Megumi,Kondo Mari,Kobayashi Isao,Takasu Yoko,Suzuki Takao,Yonemura Naoyuki,Iizuka Tetsuya,Uchino Keiro,Tamura Toshiki,Tsubota Takuya,Tatematsu Ken-Ichiro
Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan
We have been constructing a platform for the development of pharmaceutical and medical applications using the domesticated silkworm, Bombyx mori, as a new animal model for drug development and evaluation. Because silkworm larvae originally have the capacity to synthesize up to 0.5 g of silk proteins, genetically modified silkworms (transgenic silkworms) are expected to have high potential in the production of recombinant silks/proteins. An innovative method for generating transgenic silkworms was established in 2000, and ever since this epoch-defining technological development, longstanding efforts have succeeded in developing novel silks that enable the manufacture of new textile materials for regenerative medical uses. Furthermore, we have succeeded in developing a new system of recombinant protein production. This recombinant protein production system is currently capable of producing a maximum of approximately 15 mg recombinant protein per silkworm larva. Transgenic silkworms have also been shown to produce a wide variety of useful proteins, including antibodies and membrane proteins. Some of these recombinant proteins have been in commercial use since 2011. In addition, we have been developing transgenic silkworms as a novel animal model for testing medicines based on metabolic similarities between silkworms and mammals. These applications show the suitability and potential of transgenic silkworms for medical use. Here, we will describe the challenges faced in creating a transgenic silkworm-based platform for pharmaceutical and medical applications.
Aluminum toxicity related to SOD and expression of presenilin and CREB in Bombyx mori.
Liu Longhai,Qian Xiaoran,Chao Mengling,Zhao Yijiao,Huang Junyi,Wang Taichu,Sun Fan,Ling Erjun,Song Hongsheng
Archives of insect biochemistry and physiology
Aluminum (Al) is an important environmental metal factor that can be potentially associated with pathological changes leading to neurotoxicity. The silkworm, Bombyx mori, is an important economic insect and has also been used as a model organism in various research areas. However, the toxicity of Al on silkworm physiology has not been reported. Here, we comprehensively investigate the toxic effects of Al on the silkworm, focusing on its effects on viability and development, superoxide dismutase (SOD) activity, and the expression of presenilin and cAMP response element-binding protein (CREB) in BmE cells and silkworm larvae. BmE cell viability decreased after treatment with aluminum chloride (AlCl ) in both dose- and time-dependent manners. When AlCl solution was injected into newly hatched fifth instar larvae, both larval weight gain and survival rate were significantly decreased in a manner correlating with AlCl dose and developmental stage. Furthermore, when BmE cells and silkworm larvae were exposed to AlCl , SOD activity decreased significantly relative to the control group, whereas presenilin expression increased more than twofold. Additionally, CREB and phosphorylated CREB (p-CREB) expression in the heads of fifth instar larvae decreased by 28.0% and 50.0%, respectively. These results indicate that Al inhibits the growth and development of silkworms in vitro and in vivo, altering SOD activity and the expressions of presenilin, CREB, and p-CREB. Our data suggest that B. mori can serve as a model animal for studying Al-induced neurotoxicity or neurodegeneration.
Silkworm silk-based materials and devices generated using bio-nanotechnology.
Huang Wenwen,Ling Shengjie,Li Chunmei,Omenetto Fiorenzo G,Kaplan David L
Chemical Society reviews
Silks are natural fibrous protein polymers that are spun by silkworms and spiders. Among silk variants, there has been increasing interest devoted to the silkworm silk of B. mori, due to its availability in large quantities along with its unique material properties. Silk fibroin can be extracted from the cocoons of the B. mori silkworm and combined synergistically with other biomaterials to form biopolymer composites. With the development of recombinant DNA technology, silks can also be rationally designed and synthesized via genetic control. Silk proteins can be processed in aqueous environments into various material formats including films, sponges, electrospun mats and hydrogels. The versatility and sustainability of silk-based materials provides an impressive toolbox for tailoring materials to meet specific applications via eco-friendly approaches. Historically, silkworm silk has been used by the textile industry for thousands of years due to its excellent physical properties, such as lightweight, high mechanical strength, flexibility, and luster. Recently, due to these properties, along with its biocompatibility, biodegradability and non-immunogenicity, silkworm silk has become a candidate for biomedical utility. Further, the FDA has approved silk medical devices for sutures and as a support structure during reconstructive surgery. With increasing needs for implantable and degradable devices, silkworm silk has attracted interest for electronics, photonics for implantable yet degradable medical devices, along with a broader range of utility in different device applications. This Tutorial review summarizes and highlights recent advances in the use of silk-based materials in bio-nanotechnology, with a focus on the fabrication and functionalization methods for in vitro and in vivo applications in the field of tissue engineering, degradable devices and controlled release systems.
Skin depigmenting action of silkworm (Bombyx mori L.) droppings in zebrafish.
Lee Yeong Ro,Park Ji-Hae,Castaneda Molina Rodrigo,Nam Youn Hee,Lee Yeong-Geun,Hong Bin Na,Baek Nam-In,Kang Tong Ho
Archives of dermatological research
The excrement of silkworms (Bombyx mori L.), referred to here as silkworm droppings (SDs), is used as a traditional drug in eastern medicine to treat skin diseases such as urticaria and atopy. However, the depigmentation effects of SDs have not previously been evaluated. We focused on the depigmentation effect of a methanol extract of SDs and isolated components of the extract using a zebrafish model system. (+)-Dehydrovomifoliol (M-1), (6R,7E,9R)-9-hydroxy-4,7-megastigmadien-3-one (M-2), (3S,5R,8R)-3,5-dihydroxymegastigma-6,7-dien-9-one (M-3), roseoside (M-4), and citroside A (M-5) were isolated from only SDs extract (SDE), and chemical structures were identified through spectroscopic methods. Toxicity of SDE was evaluated by assessing its effect on the viability of human fibroblast cells and the hatching rate of zebrafish embryos. In addition, the depigmentation ability of SDE and isolated constituents was evaluated using a zebrafish model. Binary threshold, histograms, and the size of the black spots on the dorsal region of zebrafish larvae were analyzed using image analysis tools. Finally, SDE is a non-toxic material and has a dose-dependent depigmentation effect in zebrafish larvae. Moreover, various doses of compounds isolated from SDE, namely, M-1 to M-5, had a depigmentation effect. In particular, M-5 inhibited melanin synthesis in melanocytes stimulated by α-melanocyte stimulating hormone (α-MSH). Together, our results suggest that SDs can be used for depigmentation purposes in health and/or cosmetic applications.
Investigation of immunogenic properties of Hemolin from silkworm, Bombyx mori as carrier protein: an immunoinformatic approach.
Aathmanathan Veeranarayanan Surya,Jothi Nattarsingam,Prajapati Vijay Kumar,Krishnan Muthukalingan
Infectious diseases are the major cause of high mortality among infants and geriatric patients. Vaccines are the only weapon in our arsenal to defend us ourselves against innumerable infectious diseases. Though myriad of vaccines are available, still countless people die due to microbial infections. Subunit vaccine is an effective strategy of vaccine development, combining a highly immunogenic carrier protein with highly antigenic but non-immunogenic antigen (haptens). In this study we have made an attempt to utilize the immunoinformatic tool for carrier protein development. Immunogenic mediators (T-cell, B-cell, IFN-γ epitopes) and physiochemical properties of hemolin protein of silkworm, Bombyx mori were studied. Hemolin was found to be non-allergic and highly antigenic in nature. The refined tertiary structure of modelled hemolin was docked against TLR3 and TLR4-MD2 complex. Molecular dynamics study emphasized the stable microscopic interaction between hemolin and TLRs. In-silico cloning and codon optimization was carried out for effective expression of hemolin in E. coli expression system. The overall presence of Cytotoxic T Lymphocytes (CTL), Humoral T Lymphocytes (HTL), and IFN-γ epitopes with high antigenicity depicts the potential of hemolin as a good candidate for carrier protein.
Metformin prolongs lifespan through remodeling the energy distribution strategy in silkworm, .
Song Jiangbo,Jiang Guihua,Zhang Jianfei,Guo Jieshu,Li Zheng,Hao Kaige,Liu Lian,Cheng Zilin,Tong Xiaoling,Dai Fangyin
Metformin is a hypoglycemic agent used clinically in the treatment of type 2 diabetics. In addition, metformin is being investigated as a potential geroprotector. Here, we investigated the effects of metformin silkworm lifespan and the underlying molecular pathways involved. We found that metformin prolonged the lifespan of the male silkworm without reducing body weight, which suggests metformin can increase lifespan through remodeling of the animal's energy distribution strategy. Consistent with that idea, metformin reduced silk production and thus the energy devoted to that process. Metformin also increased fasting tolerance and levels of the antioxidant glutathione, and also activated an adenosine monophosphate-activated protein kinase-p53-forkhead box class O signaling pathway in silkworm. These results suggest that activity in this pathway may contribute to metformin-induced lifespan extension in silkworm by increasing stress resistance and antioxidative capacity while reducing energy output for silk product. The results also show that the silkworm is a potential useful animal model for evaluating the effects of small molecules with potential clinical utility.
Variation of lifespan in multiple strains, and effects of dietary restriction and BmFoxO on lifespan in silkworm, Bombyx mori.
Song Jiangbo,Tang Dongmei,Li Zhiquan,Tong Xiaoling,Zhang Jianfei,Han Minjin,Hu Hai,Lu Cheng,Dai Fangyin
Established animal models have accelerated our understanding of the mechanisms involved in lifespan determination. However, more experimental animals are required to clarify the complex mechanisms behind the phenomena of aging and lifespan. In this study, we reported the variation of lifespan in nine distinct silkworm strains. Lifespan correlated significantly with BmFoxO gene expression in the representative silkworm strains tested (Xiafang, Dazao-N, and N4). In general, the female silkworm was longer lived than the male of the same strain. Dietary restriction extended the silkworm lifespan compared with that of silkworms fed ad libitum. The expression of BmFoxO was significantly elevated in the dietary restriction group on day 3 of the 4th instar and day 3 of the 5th instar, suggesting that BmFoxO contributes to dietary restriction-mediated lifespan extension. The RNA interference and overexpression of the BmFoxO gene significantly shortened and extended the silkworm adulthood, respectively. In conclusion, our findings suggest that the silkworm might serve as a promising experimental animal to explore the complex biological mechanisms of lifespan determination.
Toxic effects of acute exposure to polystyrene microplastics and nanoplastics on the model insect, silkworm Bombyx mori.
Muhammad Abrar,Zhou Xiaoxia,He Jintao,Zhang Nan,Shen Xiaoqiang,Sun Chao,Yan Bing,Shao Yongqi
Environmental pollution (Barking, Essex : 1987)
Microplastics and nanoplastics (MPs and NPs, respectively) are major contaminants of environmental concern due to their potentially detrimental effects on aquatic and terrestrial ecosystems. However, little is known about their potential toxicity in terrestrial organisms. Here, we used the model insect silkworm (Bombyx mori) to evaluate the potential hazardous effects of acute exposure (72 h) to polystyrene (PS) MPs and NPs at physiological, molecular, and biochemical levels as well as their impact on pathogen infection. Our results revealed no significant changes in larval body mass or survival. Nevertheless, exposure led to significant alterations in the expression of immunity-related genes (Cecropin A, Lysozyme, SOD, and GST) and antioxidant-mediated protective response (SOD, GST, and CAT enzymes) which differed in the PS-MP and PS-NP groups. Interestingly, PS-MPs induced a stronger immune response (higher expressions of Lysozyme, SOD, and GST genes along with increased activities of SOD, GST, and CAT enzymes) while the PS-NP response was more that of an inhibitory nature (decreased SOD activity and expression). As a result, upon infection with the natural pathogen Serratia marcescens Bm1, the PS-MP-exposed individuals survived the infection better whereas, PS-NP-exposed individuals exhibited significantly higher mortality. Thus, we infer that PS-MPs/NPs present ecological toxicity, which is closely related to their size, and that their exposure may render the organisms vulnerable or confer resistance to pathogen infections and ecotoxicants. Given the suitability of silkworm as a model organism, this study may promote its application for further investigation of the mechanism of adverse outcome pathways and in studies on bio-nano interactions.
In vivo toxicity evaluation of boron nitride nanosheets in Bombyx mori silkworm model.
Ma Lin,Andoh Vivian,Adjei Mark Owusu,Liu Haiyan,Shen Zhongyuan,Li Long,Song Jiangchao,Zhao Weiguo,Wu Guohua
Boron nitride nanosheets (BN NSs), a novel material with a structure similar to graphene, have attracted much attention due to their extraordinary properties. A deep in vivo study of the toxicity of BN NSs is indispensable, which can help to understand their potential risk and provide useful information for their safe application. However, so far as we know, the systematic in vivo toxicity evaluation of BN NSs hasn't been reported. In this study, silkworm (Bombyx mori) was used as a model to investigate the toxicity of BN NSs, by continuously feeding silkworm larvae with BN NSs at various mass concentrations (1%, 2%, 3%, 4%). The toxicity was evaluated from the levels of animal entirety (mortality, silkworm growth, cocoons and silk properties), tissues (pathological examination) and genes (transcriptomic profiling). The results show that the exposure to BN NSs causes no obvious adverse effects on the growth, silk properties or tissues of silkworm, but the expressions of genes in midgut concerned with some specific functions and pathways are significantly changed, indicating that BN NSs may have potential danger to lead to dysfunction. This study has performed in vivo toxicity evaluation of BN NSs and provided useful safety information for the application of BN NSs.
Accumulation and translocation of food chain in soil-mulberry (Morus alba L.)-silkworm (Bombyx mori) under single and combined stress of lead and cadmium.
Si Liqing,Zhang Jiatong,Hussain Azfar,Qiao Yong,Zhou Jinxing,Wang Xiaoping
Ecotoxicology and environmental safety
In recent years, heavy metal pollution has caused immeasurable harm to the environment. As an emerging technology, phytoremediation technology has gained a place in the treatment of heavy metal pollution with its unique advantages. This study analyzes the toxic effects of mulberry (Morus alba) seeds, seedling growth and silkworm under heavy metal stress of lead (Pb) and cadmium (Cd), and explore the accumulation and migration of Pb and Cd in the soil-mulberry tree-silkworm system. The main results were as follows: (1) Seed germination and potted seedling experiments were conducted under heavy metal Pb and Cd stresses, and it was found that Pb and Cd had inhibitory effects on mulberry seed germination, growth and photosynthesis of mulberry seedlings, and as the concentration of heavy metals increased, the stronger the inhibitory effect. Moreover, Pb and Cd have a synergistic effect under compound stress. (2) The accumulation and transfer rules of Pb and Cd ions in mulberry were different. The content of Pb in mulberry was root > leaf > stem and the content of Cd was root > stem > leaf. The combined stress promoted the transfer of Pb and Cd from the underground part to the aerial portion of mulberry. (3) The silkworm feeds on mulberry leaves contaminated with heavy metals in this experiment and found that: with the increase of silkworm feeding, the heavy metal content in the silkworm body increased significantly, but the content remained in the silkworm body was less, most of it was excreted with silkworm excrement. Combined stress has no significant effect on the detoxification mechanism of silkworm. It is indispensable to think of the synergistic effect of heavy metals on plants germination when seeds are used for phytoremediation.