Mineralization And Characterization Of Antheraea Pernyi Silk Protein

Silks are naturally occurring polymers that have been used clinically as sutures for centuries. Bombyx mori (B.mori) cocoon produces a delicate twin thread of silk fibroin, which is coated by a protective cover named as sericin. Sericin constitutes20-30%of total cocoon weight and it envelops the fibroin fiber with successive sticky layers that help in the formation of a cocoon. Silk sericin is a kind of water-soluble natural protein produced by B.mori, and represents a family of proteins whose molecular mass ranges from20to310kDa. Sericin resists oxidation, and has capability of anti-bacterial activity and UV resistantance as well as biodegradation.The absorbance and release of sericin in moisture is easily controllable when it is processed into scaffolds. The fibroin is a giant molecule comprising a crystalline portion of about two-thirds and an amorphous region of about one-third. The crystalline portion composed of repetitive amino acids (-Gly-Ala-Gly-Ala-Gly-Ser-) forms an anti-parallel β-sheet and contributes to the stability and strength of the fiber. Compared to other biomaterials, silk fibroins have excellent mechanical properties such as remarkable strength and toughness. Furthermore, it has excellent properties like biocompatibility and biodegradability. Hence, silk proteins can potentially be used in biomaterials.Biomineralization is a natural and widespread phenomenon. Bones and teeth are examples of mineralized biocomposites with unique aligned structures of nanocrystalline hydroxyapatite on an organic matrix of collagen fibers. Bone tissue engineering is becoming increasingly of interest as a regeneration strategy in clinical orthopedics. Hydroxyapatite,(HAp, Ca10(PO4)6(OH)2), has received much attention due to its unique features such as exceptional biocompatibility and bioactivity desired in the biomedical application. In addition, sericin can be used as a template mediating the nucleation of hydroxyapatite in the forms of film and aqueous solution. SF can also be used as template for HAp nucleation due to its strong chemical interactions between HA and fibroin protein.Antherea pernyi (A. pernyi) is a wild species of silkworms. It is produced mass in north-east China for silk fiber production. The silkworms are living on leaves of oak trees. A. pernyi sericin is biochemically different from that of B. mori with lower percentages of serine and tyrosine. There is little report on sericin derived from A. pernyi. According to the previous reports on apatite nucleation of B. mori sericin, we investigated the mineralization of A. pernyi sericin to promote its application in bone tissue engineering. The amino acid composition of A. pernyi silk fibroin is different from that of B. mori fibroin by having high content of Ala, Asp and Arg and low content of Gly.The abundance of alkaline amino acids and the presence of tripeptide sequence Arg-Gly-Asp (RGD) of A. pernyi silk fibroin contribute to its special interactions with mammal cells. The production of A. pernyi silk is large in quantity10,000tons per year. The dissolution of A. pernyi silk fibroin by using low-cost and ecofriendly techniques is important for A. pernyi fibroin as non-textile materials such as wound dressing, artificial skin, and matrix for cell culture, and drug delivery. Therefore, this study aimed to explore A. pernyi silk based scaffolds for bone regeneration and implants. In order to achieve this goal, we attempted to identify the minerlazation of A.pernyi sericin and fibroin, and then invent a green and ecofriendly aquoues solvent for dissolving A.pernyi fibroin for processing into forms of scaffold such as films.Firstly, mineralization of A.pernyi sericin was performed by alternative soaking in calcium and phosphate. The inhibition of precipitation of calcium carbonate and von Kossa staining on A. pernyi sericin were tested. From the above experiment it was proved that the A. pernyi sericin has Ca binding activity. Scanning electron microscope (SEM) observation showed that the spherical crystals could be nucleated on the A. pernyi sericin film confirmed byFT-IR and XRD spectra. The cell adhesion and growth activity assay provedthat the A. pernyi sericin has excellent biocompatibility for the growth of MG-63cells.Secondly, the nucleation of hydroxyapatite (HAp) on A. pernyi fibroin film was carried out. Von Kossa staining proved that A. pernyi fibroin had Ca binding activity. The A. pernyi fibroin film was mineralized with HAp crystals by alternative soaking in calcium and phosphate solutions. Spherical crystals were nucleated on the A. pernyi fibroin film according to scanning electron microscope imaging results. The FT-IR and X-ray diffraction spectra confirmed that these spherical crystals were HAp. The results of in vitro cell culture using MG-63cells demonstrated that the mineralized A. pernyi fibroin film showed excellent cytocompatibility indicated by the improvement of the MG-63cell viability.Thirdly, A. pernyi fibroin fiber is hardly soluble in common solvents. Combined with production cost and environmental protection, in this study, two kinds of alkaline solvent systems were prepared, including8mol/L urea to regenerate AF powder to facilitate application of AF in the field of biomedical material. The results showed that AF fiber could be dissolved and decomposed in two solvent systems at normal temperature. In urea/NaOH solvent system decomposition of AF occurred in the non-crystalline region, while in urea/LiOH solvent system, the decomposition occured in both the crystalline region and non-crystalline regions. Different urea solvent systems can be selected based on different purposes to use regenerated AF in production. In summary, it has been proved that both of A.pernyi sericin and A.pernyi fibroin can direct the nucleation of hydroxyapatite. The hydroxyapatite coating can improve the biocompability of A.pernyi sericin and A.pernyi fibroin. Two green and friendly solvents including urea/NaOH and urea/LiOH aquous solution were invented for regeneration of A.pernyi fibroin. This study will provde implication for extend A.pernyi silk application in the field of bone tissue enginerring.