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Bioengineered Silkworm for Producing Cocoons with High Fibroin Content for Regenerated Fibroin Biomaterial-Based Applications.

ABSTRACT: Silk fibroin exhibits high biocompatibility and biodegradability, making it a versatile biomaterial for medical applications. However, contaminated silkworm-derived substances in remnant sericin from the filature and degumming process can result in undesired immune reactions and silk allergy, limiting the widespread use of fibroin. Here, we established transgenic silkworms with modified middle silk glands, in which sericin expression was repressed by the ectopic expression of cabbage butterfly-derived cytotoxin pierisin-1A, to produce cocoons composed solely of fibroin. Intact, nondegraded fibroin can be prepared from the transgenic cocoons without the need for sericin removal by the filature and degumming steps that cause fibroin degradation. A wide-angle X-ray diffraction analysis revealed low crystallinity in the transgenic cocoons. However, nondegraded fibroin obtained from transgenic cocoons enabled the formation of fibroin sponges with varying densities by using 1-5% (v/v) alcohol. The effective chondrogenic differentiation of ATDC5 cells was induced following their cultivation on substrates coated with intact fibroin. Our results showed that intact, allergen-free fibroin can be obtained from transgenic cocoons without the need for sericin removal, providing a method to produce fibroin-based materials with high biocompatibility for biomedical uses.

SUBMITTER: Yamano M

PROVIDER: S-EPMC9267247 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial.

Project description: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 (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) 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 B. mori-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.

| S-EPMC5495258 | biostudies-literature

Protein composites from silkworm cocoons as versatile biomaterials.

Project description:Silk is a naturally occurring biopolymer formed into fibers composed primarily of fibroin and sericin proteins. The outstanding mechanical properties of silk fibroin (SF) provides numerous applications for silk-based biomaterials. However, the canonical approaches for fabricating silk-based biomaterials typically involve degumming to remove the silk sericin (SS) to avoid adverse biological effects. Meanwhile, sericin has multiple biological functions including outstanding hydrophilicity, promoting cell attachment that are useful to exploit in new materials, inspiring the use of sericin-based biomaterials for biomedical applications. However, compared to fibroin, sericin is not a structural protein, thus sericin-based materials do not provide robust mechanical properties. To address this problem, we report an effective method for fabricating silk fibroin-sericin protein (SS-SF) composites directly from whole cocoons, negating the traditional extraction step to remove the sericin. This approach combines the material features from both fibroin as a structural unit and sericin as a biological functional unit, to achieve advantages regarding processing and materials properties, not only simplifying processing and maintaining the mechanical properties of the fibroin by avoiding degumming, but also endowing these SS-SF composite materials with enhanced hydrophilicity and cell adhesion performance to promote cell growth and proliferation. In addition, these protein composites could be fabricated into a variety of materials formats (e.g. films, sponges, monoliths) to fit different biomedical applications.

| S-EPMC8268066 | biostudies-literature

Hydrogelation of Regenerated Silk Fibroin via Gamma Irradiation.

Project description:Gamma irradiation, which is one of the more conventional sterilization methods, was used to induce the hydrogelation of silk fibroin in this study. The physical and chemical characteristics of the irradiation-induced silk fibroin hydrogels were investigated. Silk fibroin solution with a concentration greater than 1 wt% formed hydrogel when irradiated by gamma rays at a dose of 25 or 50 kGy. The hydrogel induced by 50 kGy of radiation was more thermally stable at 80 °C than those induced by 25 kGy of radiation. When compared to the spontaneously formed hydrogels, the irradiated hydrogels contained a greater fraction of random coils and a lower fraction of β-sheets. This finding implies that gelation via gamma irradiation occurs via other processes, in addition to crystalline β-sheet formation, which is a well-established mechanism. Our observation suggests that crosslinking and chain scission via gamma irradiation could occur in parallel with the β-sheet formation. The irradiation-induced hydrogels were obtained when the solution concentration was adequate to support the radiation crosslinking of the silk fibroin chains. This work has, therefore, demonstrated that gamma irradiation can be employed as an alternative method to produce chemical-free, random coil-rich, and sterilized silk fibroin hydrogels for biomedical applications.

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High content imaging in the screening of biomaterial-induced MSC behavior

Project description:Upon contact with a biomaterial, cells and surrounding tissues respond in a manner dictated by the physicochemical and mechanical properties of the material. Traditionally, cellular responses are monitored using invasive analytical methods that report the expression of genes or proteins. These analytical methods involve assessing commonly used markers for a predefined readout, masking the actual situation induced in the cells. Hence, a broader expression profile of the cellular response should be envisioned, which technically limits up scaling to higher throughput systems. However, it is increasingly recognized that morphometric readouts, obtained non-invasively, are related to gene expression patterns. Here, we introduced distinct surface roughness to three PLA surfaces, by exposure to oxygen plasma of different duration times. The response of mesenchymal stromal cells was compared to smooth untreated PLA surfaces without the addition of differentiation agents. Morphological and genome wide expression profiles revealed underlying cellular changes which was hidden for the commonly used gene markers for osteo-, chondro- and adipogenesis. Using 3 morphometric parameters, obtained by high content imaging, we were able to build a classifier and discriminate between oxygen plasma-induced modified sheets and non-modified PLA sheets where evaluating classical candidates missed this effect. This approach shows the feasibility to use noninvasive morphometric data in high-throughput systems to screen biomaterial surfaces indicating the underlying genetic biomaterial-induced changes.

2018-11-10 | E-MTAB-7352 | biostudies-arrayexpress

3D Printing of Monolithic Proteinaceous Cantilevers Using Regenerated Silk Fibroin.

Project description:Silk fibroin, regenerated from Bombyx mori, has shown considerable promise as a printable, aqueous-based ink using a bioinspired salt-bath system in our previous work. Here, we further developed and characterized silk fibroin inks that exhibit concentration-dependent fluorescence spectra at the molecular level. These insights supported extrusion-based 3D printing using concentrated silk fibroin solutions as printing inks. 3D monolithic proteinaceous structures with high aspect ratios were successfully printed using these approaches, including cantilevers only supported at one end. This work provides further insight and broadens the utility of 3D printing with silk fibroin inks for the microfabrication of proteinaceous structures.

| S-EPMC9000323 | biostudies-literature

Fractionation of Regenerated Silk Fibroin and Characterization of the Fractions.

Project description:The molecular weight (MW) of regenerated silk fibroin (RSF) decreases during degumming and dissolving processes. Although MW and the MW distribution generally affect polymer material processability and properties, few reports have described studies examining the influences of MW and the distribution on silk fibroin (SF) material. To prepare different MW SF fractions, the appropriate conditions for fractionation of RSF by ammonium sulfate (AS) precipitation process were investigated. The MW and the distribution of each fraction were found using gel permeation chromatography (GPC) and SDS-polyacrylamide electrophoresis (SDS-PAGE). After films of the fractionated SFs formed, the secondary structure, surface properties, and cell proliferation of films were evaluated. Nanofiber nonwoven mats and 3D porous sponges were fabricated using the fractionated SF aqueous solution. Then, their structures and mechanical properties were analyzed. The results showed AS precipitation using a dialysis membrane at low temperature to be a suitable fractionation method for RSF. Moreover, MW affects the nanofiber and sponge morphology and mechanical properties, although no influence of MW was observed on the secondary structure or crystallinity of the fabricated materials.

| S-EPMC8540890 | biostudies-literature

Non-mulberry Silk Fibroin Biomaterial for Corneal Regeneration.

Project description:Successful repair of a damaged corneal surface is a great challenge and may require the use of a scaffold that supports cell growth and differentiation. Amniotic membrane is currently used for this purpose, in spite of its limitations. A thin transparent silk fibroin film from non-mulberry Antheraea mylitta (Am) has been developed which offers to be a promising alternative. The silk scaffolds provide sufficient rigidity for easy handling, the scaffolds support the sprouting, migration, attachment and growth of epithelial cells and keratocytes from rat corneal explants; the cells form a cell sheet, preserve their phenotypes, express cytokeratin3 and vimentin respectively. The films also support growth of limbal stem cell evidenced by expression of ABCG2. The cell growth on the silk film and the amniotic membrane is comparable. The implanted film within the rabbit cornea remains transparent, stable. The clinical examination as well as histology shows absence of any inflammatory response or neovascularization. The corneal surface integrity is maintained; tear formation, intraocular pressure and electroretinography of implanted eyes show no adverse changes. The silk fibroin film from non-mulberry silk worms may be a worthy candidate for use as a corneal scaffold.

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The effect of sterilization on silk fibroin biomaterial properties.

Project description:The effects of common sterilization techniques on the physical and biological properties of lyophilized silk fibroin sponges are described. Sterile silk fibroin sponges were cast using a pre-sterilized silk fibroin solution under aseptic conditions or post-sterilized via autoclaving, ? radiation, dry heat, exposure to ethylene oxide, or hydrogen peroxide gas plasma. Low average molecular weight and low concentration silk fibroin solutions could be sterilized via autoclaving or filtration without significant loses of protein. However, autoclaving reduced the molecular weight distribution of the silk fibroin protein solution, and silk fibroin sponges cast from autoclaved silk fibroin were significantly stiffer compared to sponges cast from unsterilized or filtered silk fibroin. When silk fibroin sponges were sterilized post-casting, autoclaving increased scaffold stiffness, while decreasing scaffold degradation rate in vitro. In contrast, ? irradiation accelerated scaffold degradation rate. Exposure to ethylene oxide significantly decreased cell proliferation rate on silk fibroin sponges, which was rescued by leaching ethylene oxide into PBS prior to cell seeding.

| S-EPMC4456215 | biostudies-literature

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Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons.

Project description:With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a "safe harbor" locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

| S-EPMC4644950 | biostudies-literature

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