Effect of Degumming Methods on Structure and Properties of Regenerated Antheraea Pernyi Silk Fibroin Films
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The extraction of fibroin (degumming) from the silk fibers impacts the properties of silk fibroin. However, the effect of the degumming method on structures and properties of regenerated Antheraea pernyi silk fibroin (ASF) biomaterials has not been investigated in detail. In this study, Na2CO3 and NaHCO3 are selected to remove sericin of Antheraea pernyi silks. The results show that the silk fibroin prepared by NaHCO3 degumming possessed a higher molecular weight, demonstrated by SDS-PAGE and viscosity testing. Na2CO3 degumming produced more low molecular weight silk fibroin protein below 40 kDa, whereas NaHCO3 processing produced more silk fibroin protein above 70 kDa. The increased molecular weight significantly improved the mechanical properties of ASF film. The enzyme degradation results indicated that the degradation rate of ASF film prepared by Na2CO3 degumming was faster in the early stage due to the presence of low molecular weight silk fibroin. After 21 days, the two silk fibroin films showed approximate degradation ratios. These results showed that the degumming conditions have a significant effect on the structure and properties of ASF biomaterials, and provide options for regulating the mechanical properties and degradation rate of ASF biomaterials.Keywords:
Fibroin
Antheraea pernyi
Sericin
Degradation
The regenerated Antheraea pernyi silk fibroin (RASF) was harvested by dissolving the silk fibers in a calcium nitrate solution. XRD result demonstrated that both native and regenerated Antheraea pernyi silk fibroin involved the α-helix conformation, and DTG curves showed that their thermal decompositions were quite similar and proceeded three steps. However, rheological measurements figured out that the molecule weight of RASF decreased from 246 kDa to 199 kDa, comparing with native Antheraea pernyi silk fibroin. Also, the tensile properties of the RASF film, which were the same to those of regenerated Bombyx mori silk fibroin film, were observed.
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Silk is a functional protein biomaterial produced by a variety of insects like flies, silkworms, scorpions, spiders, and mites. Silk synthesized by silkworms is extensively studied for its applications in tissue engineering and wound healing. Silk is undoubtedly a natural biocompatible material with humans and has its role in medical treatments from ancient times. The silk worm protein comprises two types of proteins namely fibroin and sericin. Silk fibroin makes up approximately 70% of cocoon weight and has wide applications in textiles and in all biomedical applications owing to its biocompatible, nontoxic, biodegradable, less immunogenic, and noncarcinogenic nature. It possesses outstanding toughness and mechanical strength, while silk sericin possesses high defensive ability against ultraviolet light and oxidation. Silk fibroin has been known to induce wound healing by increasing cell proliferation and growth and migrating various types of cells which are involved in different stages of wound healing process. With several silk varieties like silk worm fibroin, silk sericin, recombinant silk materials, and native spider silk have been investigated for its wound healing applications over the last several decades. With an objective of harnessing the silk regenerative properties, plentiful strategies have been studied and applied to develop bioartificial skin grafts and bioactive wound dressings in recent times. This review gives a detailed insight into the structure, general properties, fibroin structure-properties relationship, and biomedical applications of silk fibroin.
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The development of a method for the quantitative determination of the sericin content ratio (SCR) is urgently needed for silk refining and the purification of silk fibroin for biomedical applications. In this work, a series of sericin/fibroin mixed samples with known SCRs were prepared by mixing initial samples of extracted sericin and fibroin from Bombyx mori silk. Significant differences were found in the contents of characteristic hydrophilic amino acids abundant in sericin and hydrophobic amino acids abundant in fibroin, and several linear relationships of SCR associated with the content ratios of Ser/Ala, Asp/Ala, Lys/Ala, Asp/Gly and Ser/Gly were established by amino acid analysis. Subsequently, the linear equation expressing SCR (%) as a function of the Asp to Ala content ratio X (%) was established as SCR = 2.5634X − 12.5587 (R2= 0.9972). The results indicated that the SCR of degummed silks calculated by the equation is more objective and effective than the results obtained by the traditional weight loss method. Our study provides a novel approach for the sensitive and quantitative detection of the sericin content within the detection limit in unknown silks, which can contribute to quality control in the silk production process.
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Sericin, one of the two main proteins of silk cocoon besides fibroin, has been wildly used as ingredient in cosmetic products and nutrition supplements; however there are considerable controversial reports on its toxicity to cells and its advantages. This work aimed to investigate cell biocompatibility of sericin both in the systems of blended silk fibroin/sericin and pure sericin films using L929 mouse fibroblasts. The effect of concentration of commercial heat extracted sericin on cell viability was first investigated in the system of silk fibroin/sericin (F/S) films using 2 types of silk fibroin, Bombyx mori Nangnoi Srisaket 1 and Bombyx mori Jul 1/1. For both types of silk fibroin, it was found the lower cell number attached and proliferated on the blended F/S films at all sericin concentrations, compared to that on glass and pure fibroin. However, proliferation rate of cells cultured on the blended F/S films was similar to that of cells cultured on glass and pure fibroin films, as confirmed by population doubling time (PDT). Cytotoxicity of sericin extracted from 4 different methods including heat, acid, alkali and urea treatments was further studied in this work in the system of pure sericin films. It could be seen that acid and urea extracted sericin films showed high percentage of cell attachment at 92% and 88%, respectively. However, number of cells proliferated on all sericin films after 48 h culture was not significantly different. This indicated that L929 cells had different proliferation rate when cultured on different types of sericin films. Among 4 extraction methods, the PDT of cells proliferated on urea extracted sericin film was lowest (27 h) and also lower than that of the blended F/S films. This study suggested that sericin extracted by urea treatment could enhance proliferation rate of L929 mouse fibroblasts.
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Understanding the interactions between fibroin and sericin is crucial in solving the mechanism of silk spinning. In this study, various commercially available dyes were used to monitor the interface between fibroin and sericin during the gelation of fibroin. The phase separation between fibroin and sericin could be observed by the addition of azo dyes over a certain molecular weight. Furthermore, the addition of the dyes to the sericin layer showed vivid phase separation over addition to the fibroin layer.
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Recently, regenerated silk fibroin is one of the most investigated silk-based materials for tissue engineering. However, it needs complex preparation process and will irresistibly denature to solid state with time, which is the biggest challenge to produce in large scale and widely use it as commercial material. In this work, industrially produced sericin, silk fibroin amino acid, and silk fibroin powder were investigated on its physico–chemical and biological properties compared with laboratory prepared regenerated silk fibroin, in order to excavate the potential application in biomedical and therapeutical fields. The results showed that sericin may be the most suitable silk materials to use as an alternative in tissue engineering compared with regenerated silk fibroin. It was found that sericin can absorb UV in the range of 223–300 nm, inhibit tyrosinase, resist to free radicals, and retain water capacity similar to hyaluronic acid. Importantly, cell experiment on human fibroblast showed that sericin (0.05 wt%) could stimulate cell proliferation and obviously up-regulate the expression of collagen type I, collagen type III, and hyaluronidase I compared with the control at seven days, which is the closest equivalent to regenerated silk fibroin. These results suggested that sericin might be a valuable ingredient for skin repair on tissue engineering.
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The extraction of fibroin (degumming) from the silk fibers impacts the properties of silk fibroin. However, the effect of the degumming method on structures and properties of regenerated Antheraea pernyi silk fibroin (ASF) biomaterials has not been investigated in detail. In this study, Na2CO3 and NaHCO3 are selected to remove sericin of Antheraea pernyi silks. The results show that the silk fibroin prepared by NaHCO3 degumming possessed a higher molecular weight, demonstrated by SDS-PAGE and viscosity testing. Na2CO3 degumming produced more low molecular weight silk fibroin protein below 40 kDa, whereas NaHCO3 processing produced more silk fibroin protein above 70 kDa. The increased molecular weight significantly improved the mechanical properties of ASF film. The enzyme degradation results indicated that the degradation rate of ASF film prepared by Na2CO3 degumming was faster in the early stage due to the presence of low molecular weight silk fibroin. After 21 days, the two silk fibroin films showed approximate degradation ratios. These results showed that the degumming conditions have a significant effect on the structure and properties of ASF biomaterials, and provide options for regulating the mechanical properties and degradation rate of ASF biomaterials.
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Event Abstract Back to Event Silk protein as a cell additive You-Young Jo1, Haeyong Kweon1 and Kwang-Gill Lee1 1 National Academy of Agricultureal Science, Department of Agricultural BIology, Korea Introduction: A substance for the cell proliferation, additives such as cell cytokine is a very expensive. It is known that silk protein supports effectively cell proliferation[1]-[4]. Although there are many varieties of Bombyx mori silkworm, the effect of silkworm varieties on cell proliferation has not been considered in detail. To develop the cell additives using silk proteins, we studied that effects on cell proliferation and characteristics of silk obtained from Korea various silkworm varieties. Materials and Methods: Silk sericin was prepared under high temperature high pressure condition. Silk fibroin was prepared using CaCl2 : H2O : ethanol (1:8:2) with different dissolution time(1, 3, 5 hour). The characteristics of each silk protein were analyzed by performing Tensile strength, amino acid composition, porosity, XRD and gel electrophoresis. We investigated the attachment, proliferation, morphology of the cells and the expression levels of genes related to cell growth. Results: There are differences in silk cocoon from different silkworm varieties. The porosity of cocoons was 73~85%. The crystal structure of each cocoon was different by XRD analysis[5]. Also amino acid composition was different. But the molecular weight of silk proteins was same. The proliferation was accelerated in the presence all of silk sericin and silk fibroin. Especially, this was the best Baekokjam silk sericin in cell proliferation. The Baekokjam silk serin was upregulated the EGF gene expression up to 5 times. Conclusion: We expect that silk proteins could be a preferable culture medium supplement for stimulating the proliferation of cell. Then, these results suggest silk as a new material for medium supplement. This study was carried out with the support of "Research Program for Agricultural Science & Technology Development (Project No. PJ010006)", National Academy of Agricultural Science, Rural Development Administration, Republic of KoreaReferences:[1] Aramwit P, Kanokpanont S, De-Eknamkul W, Kamei K, Srichana T (2009) The effect of sericin with variable amino-acid content from different silk strains on the production of collagen and nitric oxide. Journal of Biomaterials Science-Polymer Edition 20 1295~1306.[2] Bisceglie V (1933) Uber die antineoplasticsche immunitat: heterologe Einplflanzung von tumoren in Huhner-embryonen. Ztschr. Krebsforsch 40 122~140.[3] Cha HM, Kim SM, Choi YS (2015) Serum-free media supplement from silkworm gland for the expansion of mesenchymal stem cells. Tissue Engineering and Regenerative Medicine 12, 53~59.[4] Chen F, David P, Vollrath F (2012) Morphology and structure of silkworm cocoons. Materials Science and Engineering C 32, 772~778.[5] Um IC, Kwon HY, Lee KG, Park YH (2003) The role of formic acid in solution stability and crystallization of silk protein polymer. International Journal of Biological Macromolecules 33, 203~213. Keywords: Cell Proliferation, Gene Expression, Biocompatibility Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Cellular migration and biomaterials Citation: Jo Y, Kweon H and Lee K (2016). Silk protein as a cell additive. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00401 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers You-Young Jo Haeyong Kweon Kwang-Gill Lee Google You-Young Jo Haeyong Kweon Kwang-Gill Lee Google Scholar You-Young Jo Haeyong Kweon Kwang-Gill Lee PubMed You-Young Jo Haeyong Kweon Kwang-Gill Lee Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.
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