Impacts of Blended Bombyx mori Silk Fibroin and Recombinant Spider Silk Fibroin Hydrogels on Cell Growth.
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ABSTRACT:
SUBMITTER: Laomeephol C
PROVIDER: S-EPMC8659740 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:1. A solution of Bombyx mori silk fibroin was digested with chymotrypsin. Amino acid analyses of the chymotryptic precipitate showed in addition to the main constituents Gly, Ala, Ser and Tyr, very small amounts of Lys, His, Arg, Asp, Thr, Glu, Pro, Cys, Val, Met, Ile, Leu, Phe and Trp. 2. A stable solution of the chymotryptic precipitate in 6m-urea was obtained by dialysing a solution in 50% (w/v) lithium thiocyanate against 6m-urea. 3. The dinitrophenylated chymotryptic precipitate in 6m-urea was fractionated by gel filtration and by ion-exchange chromatography. On Dowex 1 (X2), a main fraction I(d) and three further fractions with different amino acid compositions and molecular weights were obtained. 4. Specific rearrangement and fission of the bonds involving the serine nitrogen atoms of fraction I(d) and fractionation of the resulting mixture by gel filtration yielded five fractions. Two of these fractions had the compositions DNP-Ser-(Gly(6),Ala(4),Ser) and DNP-Ser-(Gly(4),Ala(2) or Ala(3),Ser) and are presumably double repeating units according to the proposed formula of Lucas, Shaw & Smith (1957), namely [Ser-Gly-(Ala-Gly)(n)](2), for n values of 2 and 1 respectively.
| S-EPMC1271246 | biostudies-other
Project description:Silk fibroin produced by the domesticated silkworm, Bombyx mori, has been studied widely as a substrate for tissue engineering applications because of its mechanical robustness and biocompatibility. However, it is often difficult to precisely tune silk fibroin's biological properties due to the lack of easy, reliable, and versatile methodologies for decorating it with functional molecules such as those of drugs, polymers, peptides, and enzymes necessary for specific applications. In this study we applied an azido-functionalized silk fibroin, AzidoSilk, produced by a state-of-the-art biotechnology, genetic code expansion, to produce silk fibroin decorated with cell-repellent polyethylene glycol (PEG) chains for controlling the cell adhesion property of silk fibroin film. Azido groups can act as selective handles for chemical reactions such as a strain-promoted azido-alkyne cycloaddition (SPAAC), known as a click chemistry reaction. We found that azido groups in AzidoSilk film were selectively decorated with PEG chains using SPAAC. The PEG-decorated film demonstrated decreased cell adhesion depending on the lengths of the PEG chains. Azido groups in AzidoSilk can be decomposed by UV irradiation. By partially decomposing azido groups in AzidoSilk film in a spatially controlled manner using photomasks, cells could be spatially arranged on the film. These results indicated that SPAAC could be an easy, reliable, and versatile methodology to produce silk fibroin substrates having adequate biological properties.
| S-EPMC7570510 | biostudies-literature
Project description:Bombyx mori silk fibroin (SF) is a biopolymer that can be processed into materials with attractive properties (e.g., biocompatibility and degradability) for use in a multitude of technical and medical applications (including textiles, sutures, drug delivery devices, tissue scaffolds, etc.). Utilizing the information from experimental and computational SF studies, a simplified SF model has been produced (alanine-glycine [Ala-Gly] n crystal structure), enabling the application of both molecular dynamic and density functional theory techniques to offer a unique insight into SF-based materials. The secondary structure of the computational model has been evaluated using Ramachandran plots under different environments (e.g., different temperatures and ensembles). In addition, the mean square displacement of water incorporated into the SF model was investigated: the diffusion coefficients, activation energies, most and least favorable positions of water, and trajectory of water diffusion through the SF model are obtained. With further computational study and in combination with experimental data, the behavior/degradation of SF (and similar biomaterials) can be elucidated. Consequently, greater control of the aforementioned technologies may be achieved and positively affect their potential applications.
| S-EPMC8717555 | biostudies-literature
Project description:Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, Bombyx mori, has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in B. mori by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) in the spider Nephila clavipes We successfully replaced the ∼16-kb endogenous FibH gene with a 1.6-kb MaSp1 gene fused with a 1.1-kb partial FibH sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted MaSp1 integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.
| S-EPMC6126722 | biostudies-literature
Project description:The goals of this study are to develop a high purity patented silk fibroin (SF) film and test its suitability to be used as a slow-release delivery for insulin-like growth factor-1 (IGF-1). The release rate of the SF film delivering IGF-1 followed zero-order kinetics as determined via the Ritger and Peppas equation. The release rate constant was identified as 0.11, 0.23, and 0.09% h-1 at 37 °C for SF films loaded with 0.65, 6.5, and 65 pmol IGF-1, respectively. More importantly, the IGF-1 activity was preserved for more than 30 days when complexed with the SF film. We show that the IGF-1-loaded SF films significantly accelerated wound healing in vitro (BALB/3T3) and in vivo (diabetic mice), compared with wounds treated with free IGF-1 and an IGF-1-loaded hydrocolloid dressing. This was evidenced by a six-fold increase in the granulation tissue area in the IGF-1-loaded SF film treatment group compared to that of the PBS control group. Western blotting analysis also demonstrated that IGF-1 receptor (IGF1R) phosphorylation in diabetic wounds increased more significantly in the IGF-1-loaded SF films group than in other experimental groups. Our results suggest that IGF-1 sustained release from SF films promotes wound healing through continuously activating the IGF1R pathway, leading to the enhancement of both wound re-epithelialization and granulation tissue formation in diabetic mice. Collectively, these data indicate that SF films have considerable potential to be used as a wound dressing material for long-term IGF-1 delivery for diabetic wound therapy.
| S-EPMC8230471 | biostudies-literature
Project description:Silk Fibroin (SF) obtained from Bombyx mori is a very attractive biopolymer that can be useful for many technological applications, from optoelectronics and photonics to biomedicine. It can be processed from aqueous solutions to obtain many scaffolds. SF dissolution is possible only with the mediation of chaotropic salts that disrupt the secondary structure of the protein. As a consequence, recovered materials have disordered structures. In a previous paper, it was shown that, by modifying the standard Ajisawa's method by using a lanthanide salt, CeCl3, as the chaotropic agent, it is possible to regenerate SF as a fibrous material with a very ordered structure, similar to that of the pristine fiber, and doped with Ce+3 ions. Since SF exhibits a moderate fluorescence which can be enhanced by the incorporation of organic molecules, ions and nanoparticles, the possibility of doping it with lanthanide ions could be an appealing approach for the development of new photonic systems. Here, a systematic investigation of the behavior of degummed SF in the presence of all lanthanide ions, Ln+3, is reported. It has been found that all lanthanide chlorides are chaotropic salts for solubilizing SF. Ln+3 ions at the beginning and the end of the series (La+3, Pr+3, Er+3, Tm+3, Yb+3, Lu+3) favor the reprecipitation of fibrous SF as already found for Ce+3. In most cases, the obtained fiber preserves the morphological and structural features of the pristine SF. With the exception of SF treated with La+3, Tm+3, and Lu+3, for all the fibers re-precipitated a concentration of Ln+3 between 0.2 and 0.4% at was measured, comparable to that measured for Ce+3-doped SF.
| S-EPMC8222627 | biostudies-literature
Project description:Bombyx mori silk fibroin-based materials have good biocompatibility and biodegradability. In order to maximize their utility while maintain appropriate features, silk fibroin (SF) films were modified with reduced glutathione (GSH) (NH2)-ECG-(COOH), using the carbodiimide chemistry method, for the introduction of thiol groups onto surfaces. The effects of this modification on SF films' chemical and physical properties, and cytotoxicity were assessed. The chemical and elemental composition analysis results suggested that reduced glutathione (GSH) was covalently coupled onto the surface of silk fibroin films. Atomic force microscopy (AFM) results indicated the surface roughness of silk fibroin film was increased after the modification by GSH. The GSH-modified silk fibroin films also showed the smaller contact angle due to the hydrophilic peptides coupled on the film surface. Through MTT assay, it was shown that the chemically modified SF film was not cytotoxic to HEK293 cells, and it had no adverse influence on the growth of HEK293 cells. Our approach provides a new option to engineer SF-based material surface with thiol groups in order to allow for secondary reactions and holds great promise for applications of SF-based materials in the biomedical field.
| S-EPMC6661838 | biostudies-literature
Project description:Protein-based hydrogels with distinct conformations which enable encapsulation or differentiation of cells are of great interest in 3D cancer research models. Conformational changes may cause macroscopic shifts in the hydrogels, allowing for its use as biosensors and drug carriers. In depth knowledge on how 3D conformational changes in proteins may affect cell fate and tumor formation is required. Thus, this study reports an enzymatically crosslinked silk fibroin (SF) hydrogel system that can undergo intrinsic conformation changes from random coil to ?-sheet conformation. In random coil status, the SF hydrogels are transparent, elastic, and present ionic strength and pH stimuli-responses. The random coil hydrogels become ?-sheet conformation after 10 days in vitro incubation and 14 days in vivo subcutaneous implantation in rat. When encapsulated with ATDC-5 cells, the random coil SF hydrogel promotes cell survival up to 7 days, whereas the subsequent ?-sheet transition induces cell apoptosis in vitro. HeLa cells are further incorporated in SF hydrogels and the constructs are investigated in vitro and in an in vivo chick chorioallantoic membrane model for tumor formation. In vivo, Angiogenesis and tumor formation are suppressed in SF hydrogels. Therefore, these hydrogels provide new insights for cancer research and uses of biomaterials.
| S-EPMC4971568 | biostudies-literature
Project description:Bombyx mori (B. mori) silk fibroin (SF) microcapsules have acted as a great candidate in delivering drugs. However, it is difficult to fabricate SF nanocapsules using the present layer-by-layer (LBL) technique. In addition, the current SF microcapsules have limits in loading negatively charged drugs. Here, we invent a novel LBL method by introducing silane (APTES) as a structure indicator to produce SF nanocapsules that can load drugs with negative or positive charge. LBL assembly was completed by alternately coating SF and APTES on the template of polystyrene (PS) nanospheres by electrostatic attraction. SF nanocapsules were obtained after removal of the PS templates. Zeta potential analysis proved LBL assembly was indeed driven by the interaction between negative charge of SF and positive charge of APTES. Fluorescence images and electric microscope images indicated that SF nanocapsules had a hollow and stable structure with diameter at nearly 250 nm. The highest encapsulation rate of DOX or Ce6 were up to 80% and 90%, respectively, indicating SF nanocapsules have a high loading capability for both cationic and anionic drugs. In vitro cell experiments proved the biocompatibility of SF nanocapsules and their burst drug release in response to acidic environment. Furthermore, chemotherapy and photodynamic therapy proved SF nanocapsules loaded with DOX or Ce6 had significant inhibition on tumor cells. Our results suggested that this LBL technique is a facile method for polymers with negative charge to fabricate nanocapsules for antitumor drug carrier.
| S-EPMC9055660 | biostudies-literature
Project description:Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. An important phase of wound healing is the generation of a basal epithelium able to wholly replace the epidermis of the wound. A broad range of products derived from fibroin and sericin from Bombyx mori silk are used to stimulate wound healing. However, so far the molecular mechanism underlying this phenomenon has not been elucidated. The aim of this work was to determine the molecular basis underlying wound healing properties of silk proteins using a cell model. For this purpose, we assayed fibroin and sericin in a wound healing scratch assay using MDA-MB-231 and Mv1Lu cells. Both proteins stimulated cell migration. Furthermore, treatment with sericin and fibroin involved key factors of the wound healing process such as upregulation of c-Jun and c-Jun protein phosphorylation. Moreover, fibroin and sericin stimulated the phosphorylation of ERK 1/2 and JNK 1/2 kinases. All these experiments were done in the presence of specific inhibitors for some of the cell signalling pathways referred above. The obtained results revealed that MEK, JNK and PI3K pathways are involved in fibroin and sericin stimulated cells migration. Inhibition of these three kinases prevented c-Jun upregulation and phosphorylation by fibroin or sericin. Fibroin and sericin were tested in the human keratinocyte cell line, HaCaT, with similar results. Altogether, our results showed that fibroin and sericin initiate cell migration by activating the MEK, JNK and PI3K signalling pathways ending in c-Jun activation.
| S-EPMC3409175 | biostudies-literature