Regenerated Antheraea Pernyi Silk Fibroin Research Articles

Effect of Degumming Methods on Structure and Properties of Regenerated Antheraea Pernyi Silk Fibroin Films

ABSTRACT 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.

Comparison of Native and Regenerated Antheraea Pernyi Silk Fibroin

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.

Fabrication and characterization of regenerated Antheraea pernyi silk fibroin scaffolds for Schwann cell culturing

Antheraea pernyi silk, which is from a kind of non-mulberry silkworm produced in China, has better mechanical properties and cytocompatibility than the extensively studied silk from mulberry silkworm (Bombyx mori). In this work, the regenerated Antheraea pernyi silk fibroin (RASF) scaffolds were prepared from RASF aqueous solutions with different concentrations by a freeze-drying method. The morphologies, structures and properties of the obtained RASF scaffolds were investigated. It was found that with the decrease of RASF solution concentration and the increase of freezing temperature, the porosity of the scaffold increased while its apparent density decreased. The micro-morphology of the scaffold from the 1 wt% RASF solution was irregular. When the solution concentration was increased to the range of 4–13 wt%, the micro-morphologies of the obtained scaffolds mainly showed lamellar-like structure. And with the increase of solution concentration, the lamellar gap of the scaffold decreased while the lamellar thickness of the scaffold increased. However, with the further increase of solution concentration, the morphology of scaffold could gradually transfer into porous structure. In addition, the scaffold micro-morphology could be also affected by freezing temperature. Moreover, with the increase of RASF solution concentration or (and) the decrease of freezing temperature, the obtained scaffold had higher content of β-sheet conformation and higher crystallinity, which could improve its mechanical properties. It was further found that the swelling ratio and degradation rate of RASF scaffold prepared for tissue engineering increased with the decrease of solution concentration. The results of MTT indicated that the obtained RASF scaffold had no cytotoxicity. Moreover, the results of field emission scanning electron microscopy and laser confocal scanning microscopy revealed that the viability, cytocompatibility and migration ability of Schwann cells cultured on RASF scaffold were much better than those cultured on regenerated Bombyx mori silk fibroin (RBSF) scaffold. The RASF scaffold prepared in this work may have great potential applications in the area of nerve tissue engineering.

<i>Antheraea pernyi</i> Silk Fibroin Nanoparticles for Drug Delivery

Silk nanoparticles were easily obtained from regenerated Antheraea Pernyi Silk Fibroin (ASF). The morphology and average size of the silk particles was sensitive to pH value of fibroin solution. The diameter of nanoparticles prepared was in the range of 30 nm to 1000 nm with a narrow size distribution. On this process, the molecular conformation of regenerated ASF changed from α-helix to β-sheet structure. The shape of prepared nanoparticles were regular spherical structure when the pH value was about 4.3 (pI) in ASF solution. Doxorubicin hydrochloride (DOX) was loaded in the ASF particles as drug release model and the drug-loading ratio was 3.4 %. The release rate of DOX from ASF nanoparticles was pH sensitive. After 23 days release, there was still 84% DOX in the ASF nanoparticles. The result suggested that the ASF nanoparticles might be suitable microcarriers for drug delivery.

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.

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.

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

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, 13C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol–gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were α-helix and random coil in solution. There also existed a little β-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no α-helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol–gel transformation. The velocity increased obviously when the temperature was above 30 °C. During the sol–gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to β-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.

Attachment and growth of human bone marrow derived mesenchymal stem cells on regenerated antheraea pernyi silk fibroin films

Silk fibroin of the silkworm Bombyx mori has been studied extensively, while the research on Antheraea pernyi silk fibroin (A. pernyi SF) in biomaterials is only at an early stage. In this study, the attachment, morphology, growth and phenotype of human bone marrow derived mesenchymal stem cells (hBMSCs) cultured on the regenerated A. pernyi SF films were studied in vitro. The results indicated that the attachment of hBMSCs on the regenerated A. pernyi SF films was almost the same as that on the collagen films. MTT and cell counting analyses demonstrated that the growth of hBMSCs on the regenerated A. pernyi SF films was better than that on controls. Moreover, electron scanning microscopy and fluorescence-activated cell sorting assays showed that the regenerated A. pernyi SF supported hBMSCs growth and functional maintenance compared with the controls. These data suggest that the regenerated A. pernyi SF, like Bombyx mori silk fibroin (B. mori SF) and collagen, can support hBMSCs attachment, growth and phenotypic maintenance, and has better biocompatibilities for hBMSCs in vitro culture.

Controlling molecular conformation of regenerated wild silk fibroin by aqueous ethanol treatment

AbstractRegenerated Antheraea pernyi silk fibroin film was prepared by dissolution of native silk fiber in aqueous lithium thiocyanate. The influence of aqueous ethanol treatment of the dried regenerated film on molecular conformation was studied by X‐ray diffraction, infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analysis. While the initial regenerated film consisted of α‐helix and random coil components, aqueous ethanol treatment of the film resulted in significant increase in β‐sheet component and improvement of water resistance of the film. This effect was strongly dependent on ethanol concentration, and 40–60% (w/w) ethanol was most effective due to balance of hydrophilic/hydrophobic action of the solvent. Copyright © 2003 John Wiley & Sons, Ltd.

Compliant film of regenerated Antheraea pernyi silk fibroin by chemical crosslinking

A compliant film was prepared by chemical crosslinking of fibroin from silk fiber of wild silkworm, Antheraea pernyi. The silk fiber was dissolved in concentrated aqueous lithium thiocyanate and desalinated by dialysis. The film was cast from the regenerated aqueous solution, and crosslinked by polyethylene glycol diglycidyl ether (PEG-DE). This film showed high water resistively while maintaining random coil and α-helix structure, unlike films prepared by organic solvent treatment that causes β-sheet formation. The films containing about 20 wt.% crosslinker were remarkably compliant and tenacious. These features, combined with the living-cell affinity of the wild silkworm fibroin, are expected to be useful in biomedical applications.

Effect of heat treatment on the structural and conformational changes of regenerated Antheraea pernyi silk fibroin films

Effect of heat treatment on the structural and conformational changes of regenerated Antheraea pernyi silk fibroin films

Thermal behavior of regenerated Antheraea pernyi silk fibroin film treated with aqueous methanol

The regenerated Antheraea pernyi silk fibroin films prepared from calcium nitrate solution were treated with 80% aqueous methanol. The thermal and dynamic thermomechanical behaviors of the films were investigated through TGA, DSC and DMTA. IR spectroscopy was used for the conformational changes with methanol treatments. The treatment time of aqueous methanol highly influenced the thermal behavior of regenerated films. As the methanol treatment time increased up to 60min, the thermal decomposition behavior did not differ significantly compared with untreated films while the endo/exo transition was gradually decreased, with the exotherm finally disappearing. The dynamic mechanical thermal behavior was also affected by the treatment time of aqueous methanol. The onset temperature of storage modulus drop or damping peak shifted to a higher temperature due to the increase in crystallinity induced by the methanol treatment. The thermal transition was strongly dependent on the formation of β-sheet crystal conformation of the regenerated films upon methanol treatment.

Structural and conformational changes of regeneratedAntheraea pernyi silk fibroin films treated with methanol solution

The regenerated Antheraea pernyi silk fibroin films were prepared from calcium nitrate solution and crystallized with aqueous methanol solution. The structural and conformational changes were investigated by the X-ray diffraction method and infrared spectroscopy upon methanol treatment. The concentration and treatment time of aqueous methanol solution have a great influence on the conformation of regenerated films. The conformational change from a random coil to β-sheet structure was occurred within 5 min by the treatment of 40–60% methanol solution. As the methanol concentration increases up to 100%, the transition time of conformational change is delayed, and the structural change is not occurred in the case of 100% methanol used. The content of a α-helix, β-sheet, and random coil conformation was calculated and examined to find out the effect of methanol treatment on the conformation changes. The β-sheet structure can be transformed from a random coil, while the content of α-helix structure is not changed, regardless of methanol treatment. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 2887–2894, 1999