Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers of cytokines for tissue engineering. Here we show the complete healing in critical-sized bone defects by bone morphogenetic protein-2 (BMP-2) encapsulated polyhedra. Although absorbable collagen sponge (ACS) safely and effectively delivers recombinant human BMP-2 (rhBMP-2) into healing tissue, the current therapeutic regimens release rhBMP-2 at an initially high rate after which the rate declines rapidly. ACS impregnated with BMP-2 polyhedra had enough osteogenic activity to promote complete healing in critical-sized bone defects, but ACS with a high dose of rhBMP-2 showed incomplete bone healing, indicating that polyhedral microcrystals containing BMP-2 promise to advance the state of the art of bone healing.
Moth-eye nanostructures are a well-known example of biological antireflective surfaces formed by pseudoregular arrays of nipples and are often used as a template for biomimetic materials. Here, we provide morphological characterization of corneal nanostructures of moths from the Bombycidae family, including strains of domesticated Bombyx mori silk-moth, its wild ancestor Bombyx mandarina, and a more distantly related Apatelodes torrefacta. We find high diversification of the nanostructures and strong antireflective properties they provide. Curiously, the nano-dimple pattern of B. mandarina is found to reduce reflectance as efficiently as the nanopillars of A. torrefacta. Access to genome sequence of Bombyx further permitted us to pinpoint corneal proteins, likely contributing to formation of the antireflective nanocoatings. These findings open the door to bioengineering of nanostructures with novel properties, as well as invite industry to expand traditional moth-eye nanocoatings with the alternative ones described here.
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.
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.
Silkworm silk is gaining significant attention from both the textile industry and research society because of its outstanding mechanical properties and lustrous appearance. The possibility of creating tougher silks attracts particular research interest. Carbon nanotubes and graphene are widely studied for their use as reinforcement. In this work, we report mechanically enhanced silk directly collected by feeding Bombyx mori larval silkworms with single-walled carbon nanotubes (SWNTs) and graphene. We found that parts of the fed carbon nanomaterials were incorporated into the as-spun silk fibers, whereas the others went into the excrement of silkworms. Spectroscopy study indicated that nanocarbon additions hindered the conformation transition of silk fibroin from random coil/α-helix to β-sheet, which may contribute to increased breaking elongation and toughness modules. We further investigated the pyrolysis of modified silk, and a highly developed graphitic structure with obviously enhanced electrical conductivity was obtained through the introduction SWNTs and graphene. The successful generation of these SWNT- or graphene- embedded silks by in vivo feeding is expected to open up possibilities for the large-scale production of high strength silk fibers.
During larval-pupal transformation, the anterior silk glands (ASGs) of the silkworm Bombyx mori undergo programmed cell death (PCD) triggered by 20-hydroxyecdysone (20E). Under standard in vitro culture conditions (0.3ml of medium with 1μM 20E), ASGs of the fourth-instar larvae do not undergo PCD in response to 20E. Similarly, larvae of the fifth instar do not respond to 20E through day 5 of the instar (V5). However, ASGs of V6 die when challenged by 20E, indicating that the glands might be destined to die before V6 but that a death commitment is not yet present. When we increased the volume of culture medium for one gland from 0.3 to 9ml, V5 ASGs underwent PCD. We examined the response of ASGs to 20E every day by culturing them in 9ml of medium and found that ASGs on and after V2 were fully responsive to 20E. Because pupal commitment is associated with juvenile hormone (JH), the corpora allata (a JH secretory organ) were removed on day 3 of the fourth larval instar (IV3), and their ASGs on V0 were cultured with 20E. Removal of the corpora allata allowed the V0 larval ASGs to respond to 20E with PCD. In contrast, topical application of a JH analogue inhibited the response to 20E when applied on or before V5. We conclude that the acquisition of responsiveness to 20E precedes the loss of JH sensitivity, and that the death commitment in ASGs occurs between V5 and 6.
Silk fibroin-based matrices of non-mulberry silkworm Antheraea mylitta and mulberry Bombyx mori had demonstrated good applicability in regenerative medicine. However, the cocoons of Antheraea mylitta are underutilized in part due to their lack of solubility in traditional organic solvents. Therefore, the present work investigates the solubilization and processing of the degummed fibers obtained from the cocoons of both silk species into hydrogels using ionic liquids (ILs). The developed hydrogels exhibited a rubbery consistency, viscoelastic behavior and a rapid degradation in the presence of protease XIV. SEM and confocal microscopy images suggest that human adipose stem cells (hASCs) are able to adhere and migrate at different levels within the hydrogels structure. Moreover, MTS assay demonstrates the maintenance of cells metabolic activity up to 28 days, while, DNA quantification shows that hASCs are able to proliferate on the seeded hydrogels. The finding indicates the complete IL removal from the fabricated hydrogels resulting in positive hASCs cellular response. Therefore, the present approach provides a unique opportunity to broaden mainly the processability and application of A. mylitta fibroin obtained from cocoons for regenerative medicine, namely cartilage regeneration.
Beauveria bassiana is an important entomopathogenic fungus that not only often causes infection and epidemics of wild insects but some strains also show pathogenicity to the silkworm, Bombyx mori. The present study is about diversity of B. bassiana isolated from the silkworm in southwest China. Five strains of B. bassiana were isolated from infected silkworm. Two isolates, GXtr1009 and GXtr1010, were isolated from infected silkworms treated with two kinds of biological pesticides applied in Guangxi province, and three isolates, SCsk1006, YNsk1106 and GXsk1011, were collected from naturally infected silkworms from different geographical locations in Yunnan and Sichuan. All of the isolates showed highly similar conidia and conidial fructification, but the colony characteristics demonstrated great differences among the isolates. The ITS and 18S rDNA sequence analysis was sufficient to identify all five isolates as B. bassiana. However, the dendrogram, based on the ISSR data, produced two large genetic groups. GXtr1009 and GXtr1010 comprised one group, and SCsk1006, YNsk1106 and GXsk1011 converged in a different large group. The results suggested that, although all of these five B. bassiana strains were pathogenic to silkworms, strains of biological pesticides could be differentiated from strains of naturally infected silkworm via ISSR analysis.
Serine proteases and serine protease homologs are involved in the prophenoloxidase (proPO)-activating system leading to melanization. The Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization. Here, we show the dual role of BmSPH-1 in the development and immunity of B. mori. BmSPH-1 was expressed in hemocytes after molting and during the larval-pupal transformation in normal development. In contrast, following infection, BmSPH-1 was expressed in hemocytes and cleaved in the hemolymph, which resulted in the induction of PO activity. Moreover, BmSPH-1 was cleaved in the cuticle during the larval-pupal transformation and early pupal stages. In BmSPH-1 RNAi-treated silkworms, the reduced BmSPH-1 mRNA levels during the spinning stage or the prepupal stage resulted in the arrest of pupation or pupal cuticular melanization, respectively. The binding assays revealed that BmSPH-1 interacts with B. mori immulectin, proPO, and proPO-activating enzyme. Our findings demonstrate that BmSPH-1 paticipates larval-pupal transformation, pupal cuticular melanization and innate immunity of silkworms, illustrating the dual role of BmSPH-1 in development and immunity.
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, two-dimensional fabrics, and three-dimensional 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.