Cytocompatibility of silkworm cocoon layer extracts

Abstract

Recently silk polymer produced by Bombyx mori silkworm has been considered as biological macromolecules. Silk polymer was extracted in PBS solution at 37 o C for 72 h or 72 o C for 24 h. The effect of EtOH treatment on the cocoon extraction was also examined. The extraction yield of cocoon was less than 1 wt% regardless of extraction conditions. UV spectroscopy showed that the experimental extracts have absorption bands at 280 nm. There is no cytotoxicity effect on the mouse fibroblast L929 cell. The phenotype of L929 cell was not changed under the experimental conditions. The proliferation behavior of L929 cell was not affected by the addition of cocoon extract. Therefore, cocoon extract might be cytocompatible and can be used as promising biomaterials. IntroductionA silkworm cocoon of Bombyx mori provides silk fiber for textile industries for more than 4,000 years. Recently, silk polymer has been revealed several advantages for biological materials and then applied various functional materials such as cosmetics, suture and artificial eardrum (Kundu et al., 2013;Ju et al., 2014;Vepari and Kaplan, 2007; Kim et al., 2010), and so on.In the view point of material, silk is a natural protein polymer composed of two proteins: one is a central protein called silk fibroin and another is gum-like protein called silk sericin. Silk fibroin is a natural fibrous protein with a semicrystalline structure and it consists of heavy chain (390 kDa), light chain (25 kDa) and glycoprotein. Each polypeptides linked by disulfide bond (Zhou et al., 2001).Especially, fibroin as the main component of silk protein has found diverse applications in the biomedical field, which has a strong tensile strength, biodegradability and biocompatibility (Horan et al., 2005;Meinel et al., 2005). Hence, many researchers select silk fibroin as one of the materials for biomedical application.Silk sericin is a protein polymer, which acts as an adhesive binder to maintain the structure of the fibers. Sericin is an albuminoid protein composed of serine (30%) and other amino acids (Teramoto and Miyazawa, 2005;Garel et al., 1997). Sericin was removed easily from Bombyx mori cocoon using a thermo or chemical process called degumming (Mo et al., 2009) Preparation of cocoon extractsEach cocoon was washed with tap water and then dried absolutely with room temperature in the dark. The cocoon was sliced into 5 mm ⅹ 5 mm pieces. Each cocoon slices were divided into two groups; one group was treated with absolute ethyl alcohol (EtOH) for 24 h at R.T. and another group was not treated EtOH. After treatment, cocoon was dried with R.T. for overnight. The extraction condition were followed; weigh 1 g of cocoon and add into 10 mL of 1X PBS buffer at 37 o C for 72 h and/or 72 o C for 24 h. Save the cocoon extracts and then filtered it with 0.22 um syringe filter (Sartorius Ltd., Epsom, UK) and protein concentration of all extracts was determined by bicinchronic acid (BCA) assay. Forty ug of each extracts used for cell cytotoxicity and proliferation assay. UV vis sp...