Pernyi Silk Fibroin Research Articles

Assessment of freestanding membranes prepared from Antheraea pernyi silk fibroin as a potential vehicle for corneal epithelial cell transplantation

Freestanding membranes created from Bombyx mori silk fibroin (BMSF) offer a potential vehicle for corneal cell transplantation since they are transparent and support the growth of human corneal epithelial (HCE) cells. Fibroin derived from the wild silkworm Antheraea pernyi (APSF) might provide a superior material by virtue of containing putative cell-attachment sites that are absent from BMSF. Thus we have investigated the feasibility of producing transparent, freestanding membranes from APSF and have analysed the behaviour of HCE cells on this material. No significant differences in cell numbers or phenotype were observed in short term HCE cell cultures established on either fibroin. Production of transparent freestanding APSF membranes, however, proved to be problematic as cast solutions of APSF were more prone to becoming opaque, displayed significantly lower permeability and were more brittle than BMSF-membranes. Cultures of HCE cells established on either membrane developed a normal stratified morphology with cytokeratin pair 3/12 being immuno-localized to the superficial layers. We conclude that while it is feasible to produce transparent freestanding membranes from APSF, the technical difficulties associated with this biomaterial, along with an absence of enhanced cell growth, currently favour the continued development of BMSF as a preferred vehicle for corneal cell transplantation. Nevertheless, it remains possible that refinement of techniques for processing APSF might yet lead to improvements in the handling properties and performance of this material.

Nucleation of Hydroxyapatite on Antheraea pernyi (A. pernyi) Silk Fibroin Film

Antheraea pernyi (A. pernyi) silk fibroin, which is spun from a wild silkworm, has increasingly attracted interest in the field of tissue engineering. The aim of this study was to investigate the nucleation of hydroxyapatite (HAp) on A. pernyi fibroin film. 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 microscopeimaging 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-63 cells demonstrated that the mineralized A. pernyi fibroin film showed excellent cytocompatibility and sound improvement of the MG-63 cellviability.

Study on Antheraea Pernyi Silk Fibroin Microspheres Carried Drug

A new method was developed to prepare the Antheraea Pernyi silk fibroin (ASF) microspheres and rhodamine was as a model drug. The structure of ASF microsphere carried drug was β-sheet structure characterized by X-ray diffraction and FTIR. The morphology of microspheres was observed by scanning electron microscope. The diameter of the ASF particles was not changed before and after drug carried. It was in the range of 1 μm to 5μm with a narrow size distribution. The encapsulation rate, drug content, and the drug release were tested. The resulted showed that encapsulation rate was increased and drug content was decreased with the addition of rhodamine increasing. The drug released 70% after 12 days and there was no significant difference with different pH value.

Incorporation of Exogenous RGD Peptide and Inter-Species Blending as Strategies for Enhancing Human Corneal Limbal Epithelial Cell Growth on Bombyx mori Silk Fibroin Membranes.

While fibroin isolated from the cocoons of domesticated silkworm Bombyx mori supports growth of human corneal limbal epithelial (HLE) cells, the mechanism of cell attachment remains unclear. In the present study we sought to enhance the attachment of HLE cells to membranes of Bombyx mori silk fibroin (BMSF) through surface functionalization with an arginine-glycine-aspartic acid (RGD)-containing peptide. Moreover, we have examined the response of HLE cells to BMSF when blended with the fibroin produced by a wild silkworm, Antheraea pernyi, which is known to contain RGD sequences within its primary structure. A procedure to isolate A. pernyi silk fibroin (APSF) from the cocoons was established, and blends of the two fibroins were prepared at five different BMSF/APSF ratios. In another experiment, BMSF surface was modified by binding chemically the GRGDSPC peptide using a water-soluble carbodiimide. Primary HLE were grown in the absence of serum on membranes made of BMSF, APSF, and their blends, as well as on RGD-modified BMSF. There was no statistically significant enhancing effect on the cell attachment due to the RGD presence. This suggests that the adhesion through RGD ligands may have a complex mechanism, and the investigated strategies are of limited value unless the factors contributing to this mechanism become better known.

Calcium Nitrate/에탄올 혼합 용액에 의한 작잠견사(繭絲)의 용해

작잠은 가잠과는 종속이 다른 섬유 곤충으로 우수한 섬유용 재료를 만든다. 최근 비섬유용 소재로 작잠 견단백질을 활용하기 위한 기초 연구의 일환으로 작잠 견단백질을 용해 조건에 대하여 연구 결과 작잠견사의 연감율은 16.8%로 나타났으며, 작잠견사는 염화칼슘 에탄올 혼합 용매에는 용해되지 않으나 질산칼슘수화물의 용융액에는 용해되었다. 질산칼슘과 에탄올의 조성비를 조정하여 낮은 농도의 질산칼슘 농도에서도 작잠견사가 용해되었다. 작잠 실크단백질의 주요 아미노산 조성은 알라닌, 글리신, 세리신으로 작잠 정련사와 유사한 조성을 보였다. 따라서, 금후 작잠 견단백질의 적절한 용해시스템 확보 및 소재개발을 위한 기초 자료로 활용될 수 있을 것으로 사료된다. Antheraea pernyi silkworm is one of typical wild silkworms, which spins a tawny color cocoon. The cocoon has been used as a resource for textile material due to strong chemical stability and good mechanical properties. In this study, to increase the solubility efficiency of A. pernyi silk fibroin, the composition of dissolution solvent were examined. Calcium chloride tertiary system, normally used for dissolution of Bombyx mori silk fibroin, does not act on A. pernyi silk fibroin. Calcium nitrate system dissolves A. pernyi silk fibroin, and calcium nitrate ethanol system do more easily than calcium nitrate system. Amino acid composition of A. pernyi silk fibroin obtained after dissolution is mainly composed of alanine, glycine, and serine. A. pernyi silk fibroin would be used for non-textile applications near future.

Acylation Modification of Antheraea pernyi Silk Fibroin Using Succinic Anhydride and Its Effects on Enzymatic Degradation Behavior

The degradation rate of tissue engineering scaffolds should match the regeneration rate of new tissues. Controlling the degradation behavior of silk fibroin is an important subject for silk‐based tissue engineering scaffolds. In this study, Antheraea pernyi silk fibroin was successfully modified with succinic anhydride and then characterized by zeta potential, ninhydrin method, and FTIR. In vitro, three‐dimensional scaffolds prepared with modified silk fibroin were incubated in collagenase IA solution for 18 days to evaluate the impact of acylation on the degradation behavior. The results demonstrated that the degradation rate of modified silk fibroin scaffolds was more rapid than unmodified ones. The content of the β‐sheet structure in silk fibroin obviously decreased after acylation, resulting in a high degradation rate. Above all, the degradation behavior of silk fibroin scaffolds could be regulated by acylation to match the requirements of various tissues regeneration.

Gelation of <i>Antheraea pernyi</i> Silk Fibroin Accelerated by Shearing

The rapid manufacture of silk fibroin gels in mild conditions is an important subject in the field of silk-based biomaterials. In this study, the gelation of Antheraea pernyi silk fibroin (ASF) aqueous solution was induced by shearing, without chemical cross-linking agents. Simple shearing controlled and accomplished the steady and rapid conformational transition to β-sheets with ease. The conformational transformation and rapid gelation mechanisms of ASF induced by shearing were tracked and analyzed by circular dichroism spectrometry, Fourier transform infrared spectroscopy and X-ray diffractometry, then compared with Bombyx mori silk fibroin (BSF). ASF quickly formed hydrogels within 24 - 48 h after shearing under different shearing rates for 30 - 90 min, resulting in sol-gel transformation when the β-sheet content reached nearly 50%, which is the minimum content needed to maintain a stable hydrogel system in ASF. The gel structures remained stable once formed. The rapid gelation of ASF through shearing compared with BSF was achieved because of ASF’s alternating polyalanine-containing units, which tend to form α-helix structures spontaneously. Further, the entropic cost during the conformational transition from the α-helix to the β-sheet structure is less than the cost of the transition from the random coil structure. This method is a simple, non-chemical cross-linking approach for the promotion of rapid gelation and the protection of the biological properties of ASF, and it may prove useful for application in the field of biomedical materials.

Preparation of Water-Insoluble Antheraea Pernyi Silk Fibroin Films

Antheraea pernyi silk fibroin (ASF) solution was prepared by dissolving Antheraea pernyi silk fiber in lithium thiocyanate solution. The ASF/glycol blend films were prepared by casting aqueous solution of ASF mixed with glycol. The structure of blend film was investigated by the X-ray diffraction method and infrared spectroscopy. The result showed that the structure of regenerated ASF film was α-helix and random coil conformation. After mixing with glycol, it resulted in significant increase in β-sheet structure with the improvement of water resistance of the films. This effect was strongly dependent on glycol content in the blend film. When the glycol content was more than 45 wt%, the structure of ASF changed to β-sheet and the film became water-insoluble. The breaking strength and elongation of ASF/glycol blend film were 30 Mpa and 50 %, respectively. In summary, the ASF/glycol blend film provided a great potential as a biological material.

Preparation and characterization of electrospun Antheraea pernyi silk fibroin nanofibers from aqueous solution

AbstractAntheraea pernyi silk fibroin nanofibers were prepared from aqueous solution with electrospinning. The viscosity and conductivity values of the A. pernyi silk fibroin aqueous solutions with different concentrations were measured, and the morphologies of the prepared electrospun nanofibers were observed by scanning electron microscopy. The morphologies of the electrospun nanofibers changed from beadlike to uniform cylindrical and then to beltlike. The A. pernyi silk fibroin aqueous solution with a concentration of 31% exhibited good spinnability, in which uniform and regular nanofibers with an average diameter of 422 nm were obtained. The secondary structure of the electrospun A. pernyi silk fibroin nanofibers was studied by solid 13C‐NMR cross‐polarization/magic angle spinning spectroscopy, X‐ray diffraction, and differential scanning calorimetry. The as‐spun A. pernyi silk fibroin nanofibers presented a complete α‐helix/random coil structure, and no β‐sheet structure was observed. After insoluble treatment with water vapor, α‐helix and β‐sheet conformations coexisted in the A. pernyi silk fibroin nanofibers, and the β‐sheet conformation was predominant. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Regenerated <i>Antheraea pernyi </i>Silk Fibroin Nanofiber Film

Regenerated Antheraea Pernyi silk fibroin (ASF) solution was prepared by dissolving Antheraea Pernyi (A. pernyi) silk fiber in lithium thiocyanate solution. Atomic force microscopy (AFM) examination showed that there were many short nanofibers in regenerated ASF solution, which were made up of 10-20 nm nanospheres. In this paper, we discussed the formation mechanism of these nanofibers. The results of the surface tension showed that ASF had surface active and can significantly decline the surface tension of water from 73 mN/m to 51 mN/m. The hydrophobic side chains of ASF molecular would try to break away from water environment and thereby undergo self-assembly into nanospheres. And then, these nanospheres arrange into a short nanofiber. Then, the ASF nanofiber film was prepared by casting regenerated ASF solution blended with glycol. The structure of ASF was β-sheet and the film was water-insoluble. This blend film not only had excellent mechanical properties, but also can be biodegradated by enzyme in vitro. All of this indicated that this new regenerated ASF nanofiber film not only provided excellent biocompatibility, but also had large surface as extracellular matrix (ECM) to cell adhesion. The film made of nanofiber was similar to ECM on the nanoscale, which promoted cell migration and proliferation. In summary, it provided a great potential as a biological material.

Comparison of Gelation Time and Polyalcohol Effect on Hydrogels from Domestic and Wild Silk Fibroins

Silk fibroin (SF) hydrogels were obtained from both domestic (Bombyx mori) and wild (Antheraea pernyi) silkworms from aqueous silk fibroin solutions at room temperature. The gelation time of theAntheraea pernyi(A. pernyi) SF solution was significantly shorter than that of theBombyx mori(B. mori) SF solution. The secondary structures of the two kinds of hydrogels were also compared. In order to further reduce the gelation time, various amounts of polyethylene glycol (PEG) were blended with the silk fibroins ofA. pernyiandB. mori. The gelation time of bothA. pernyiSF andB. moriSF decreased with the increased amount of PEG. After freeze-drying, the hydrogels were characterized through X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy. Results showed that the addition of polyalcohol did not change the main secondary structure of the hydrogels. However, the addition of polyalcohol did reduce the gelation time and triggered additional formation ofβ-sheets.

Preparation of <i>Antheraea pernyi</i> Silk Fibroin/Chitosan Blend Scaffolds

In this work, Antheraea pernyi silk fibroin (ASF) /chitosan (CS) blend scaffolds were prepared by freeze-drying. The relationship between preparation conditions and morphological structures of blend scaffolds was also studied. The results indicated that the porosity and pore diameter of the ASF /CS scaffolds decreased with increasing of the proportion of chitosan and decreasing of the freezing temperature. By changing the process conditions, the average pore diameter and porosity of ASF/CS scaffolds could be adjusted in the range of 134 - 527 μm and 71 - 91% respectively. Compared with the pure ASF or CS materials, the ASF / CS (60/40) blend materials were more favor of cell adhesion.

Study on Antheraea Pernyi Silk Fibroin Porous Materials

Antheraea pernyi silk fibroin (ASF) solution was prepared by dissolving Antheraea pernyi silk fiber in lithium thiocyanate solution. The ASF/1,4-butanediol (ASF/BDO) blend porous materials were prepared with freeze-drying method. The structure of ASF was investigated by the X-ray diffraction method. The result showed that the structure of regenerated ASF scaffold was α-helix and random coil conformation. After ASF mixed with 5 wt% BDO, the structure of ASF was changed to β-sheet and the ASF scaffolds became water-insoluble. There were some small pores and fibrous structure in the big pores and the surface of pores was rough with a great many raised particles. The scaffolds with the average pore size of 300-1000μm and the porosity of 82-92% can efficiently be produced in this paper. Due to avoid the use of organic solvents or harsh chemicals, these new ASF based porous materials provide much more excellent biocompatibility and is expected to be applied to tissue regeneration scaffolds.

Enhancement of Lipid Metabolism and Antioxidant Defense Status in Mice Fed with High Fat Diet Supplemented with Antheraea pernyi Silk Fibroin Powder

The effect of diet supplementation of Antheraea pernyi (A. pernyi) silk fibroin on the lipid metabolism and antioxidant defense status in high fat-fed mice was investigated. The animals were given normal control diet (NC group), high fat diet (HF group), or high fat diet supplemented with A. pernyi silk fibroin powder (HFS group) for 7 weeks. After the experimental period, the HF group showed significant increase in body weight, plasma and hepatic total cholesterol levels, and hepatic triglyceride concentration, and decreased activities of hepatic antioxidant enzymes relative to NC group. However, the HFS group exhibited marked reduction in body weight, plasma cholesterol and hepatic triglyceride levels, hepatic lipogenic enzyme activities, and lipid peroxidation rate, and higher high-density lipoprotein (HDL)-cholesterol level, fecal triglyceride content, and antioxidant enzymes activities compared with that of HF group. These findings demonstrate that dietary feeding of A. pernyi silk fibroin could improve the lipid metabolism and antioxidant defense system via regulation of hepatic antioxidant and lipogenic enzymes activities. Hence, this silk fibroin may be beneficial as a functional biomaterial for the development of therapeutic agent against high fat diet-induced hyperlipidemia and its related diseases.

Characterization and Properties of <i>Antheraea pernyi</i> Silk Finished with Reactive Quaternary Ammonium Salt of Chitosan

In order to obtain a water soluble and reactive chitosan derivative, quaternary ammonium salt groups and acrylamidomethyl groups were introduced to chitosan by two steps. The chitosan derivative, reactive quaternary ammonium salt of chitosan, was then applied to Antheraea pernyi (A. pernyi) silk by padding and curing method. FT-IR, XRD and SEM were used to characterize the structure of A. pernyi silk before and after treatment. The peak in FT-IR spectra at 1480 cm-1 and 1650 cm-1 revealed the crosslinking reactions of A. pernyi silk fibroin and reactive quaternized chitosan. The XRD results indicated the crystallinity of the treated silk fiber increase a little in comparison with the untreated ones. SEM images clearly showed the reactive quaternary ammonium salt of chitosan attached on the surface of the treated A. pernyi silk fiber, while the surface of the untreated sample was relatively smooth. The thermal stability of the treated silk fiber was enhanced compared to the untreated one by DSC analysis. It was also found that the antibacterial activity of the treated A. pernyi silk fabric against E. coli was more than 99%.

Enzymatic degradation of Antheraea pernyi silk fibroin 3D scaffolds and fibers

In this study, the in vitro enzymatic degradation behavior of the regenerated Antheraea pernyi silk fibroin ( Ap-SF) three-dimensional (3D) scaffolds and the natural Ap-SF fibers exposed to enzyme solutions of α-chymotrypsin, collagenase IA and protease XIV were investigated. The results indicated that all three proteases could degrade the Ap-SF 3D scaffolds, and the degradation ability was in the order protease XIV > collagenase IA > α-chymotrypsin. The regenerated Ap-SF 3D scaffold could be degraded completely in 18 days when exposed to 1.0 U/ml protease XIV at 37 °C, whereas under the same condition, the natural Ap-SF fiber only lost 5.6% of its weight, revealing its long-term degradation characteristics. There were abundant peptides and some free amino acids in the Ap-SF degradation products, but no free alanine. We suggested that the polyalanine block in the regenerated Ap-SF 3D scaffolds had strong resistance to enzyme attack. The proteolytic attack occurred in the non-polyalanine block of Ap-SF. The degradation rate of Ap-SF materials depended on the molecular conformation of Ap-SF, which could be controlled in the manufacturing process.

Characterization of tyrosine-rich Antheraea pernyi silk fibroin hydrolysate

Antheraea pernyi silk fibroin (SF) hydrolysate were characterized using UV–VIS spectrometer, amino acid composition and heavy metal contents to explore its potential sources for food or cosmetic additives. The hydrolyzed A. pernyi SF was separated into two parts: (a) SFA, alanine-rich fraction and (b) SFB, tyrosine-rich fraction. SFB exhibited strong absorption peaks at 210 and 280 nm due to the presence of the tyrosine. Heavy metal analysis showed that arsenic and mercury did not detect. Other heavy metals, which includes lead, cadmium, etc., were recorded only a trace amount. Therefore, A. pernyi SF hydrolysate could be safely used as sources of food, cosmetic and pharmaceuticals.

Gelation behavior of Antheraea pernyi silk fibroin

The sol-gel transition behavior of Antherae pernyi silk fibroin (Ap-SF) has not been systematically investigated. In this work, the influence of environmental temperature, pH, the concentration of Ap-SF, K+ and Ca2+ on the gelation time, and the structural changes of Ap-SF in sol-gel transformation were studied. The results indicated that the gelation time of the Ap-SF aqueous solution decreased with the increase of the Ap-SF concentration and environmental temperature. The sol-gel transformation of Ap-SF was much more rapid than that of Bombyx mori silk fibroin under the same conditions. The Ap-SF was sensitive to changes in the concentration of Ca2+ and K+. Upon gelation, the random coil structure of the Ap-SF was significantly transformed into the β-sheet structure.

Antheraea pernyi silk fibroin maintains the immunosupressive properties of human bone marrow mesenchymal stem cells

We reported previously that regenerated Antheraea pernyi silk fibroin (A. pernyi SF) could support the attachment and growth of human bone marrow mesenchymal stem cells (hBMSCs). In this work, the immunosupressive effects of hBMSCs cultured on the A. pernyi SF films on T-cells were investigated in vitro. The production of IL-6, CD80, CD86 and HLA-DR by the hBMSCs was also observed. The study showed that hBMSCs cultured on the regenerated A. pernyi SF films still kept their immunosupression on T-cell proliferation and IL-2 secretion. Moreover, regenerated A. pernyi SF like regenerated Bombyx mori SF and collagen did not elicit T-cell proliferation but it could support the expression of IL-6 and surface antigen of hBMSCs. Regenerated A. pernyi SF can maintain the function of hBMSCs in immunomodulation and cytokines production, which has the potential utility of hBMSCs combined with A. pernyi SF in tissue replacement and repair.

Condensed Structure of Regenerated Antheraea Pernyi Silk Fibroin Porous Materials Prepared by Freeze-drying

Condensed Structure of Regenerated Antheraea Pernyi Silk Fibroin Porous Materials Prepared by Freeze-drying