Preparation of porous scaffolds from silk fibroin extracted from the silk gland of Bombyx mori (B. mori).

In order to use a simple and ecofriendly method to prepare porous silk scaffolds, aqueous silk fibroin solution (ASF) was extracted from silk gland of 7-day-old fifth instar larvae of Bombyx mori (B. mori). SDS-page analysis indicated that the obtained fibroin had a molecular weight higher than 200 kDa. The fabrication of porous scaffolds from ASF was achieved by using the freeze-drying method. The pore of porous scaffolds is homogenous and tends to become smaller with an increase in the concentration of ASF. Conversely, the porosity is decreased. The porous scaffolds show impressive compressive strength which can be as high as 6.9 ± 0.4 MPa. Furthermore, ASF has high cell adhesion and growth activity. It also exhibits high ALP activity. This implies that porous scaffolds prepared from ASF have biocompatibility. Therefore, the porous scaffolds prepared in this study have potential application in tissue engineering due to the impressive compressive strength and biocompatibility.

Expression, purification, and refolding of a recombinant human bone morphogenetic protein 2 in vitro.

In this work, the recombinant human bone morphogenetic protein 2 (rhBMP-2) gene was cloned from MG-63 cells by RT-PCR, and the protein was expressed in Escherichia coli expression system, purified by Ni-NTA column under denaturing conditions and refolded at 4°C by urea gradient dialysis. We found that the protein refolding yield was increased with the increase of pH value from pH 6.0 to pH 9.0. The yield was 42% and 96% at pH 7.4 and pH 9.0, respectively, while that at pH 6.0 was only 3.4%. The cell culture results showed that the rhBMP-2 refolded at pH 7.4 urea gradient dialysis had higher biological activity for MG-63 cell proliferation and differentiation than that refolded at pH 9.0 since pH 7.4 is closer to the conditions in vivo leading to the formation of dimers through the interchain disulfide bond. Moreover, the biological activity for MG-63 was promoted with the increase of rhBMP-2 concentration in the cell culture medium. This work may be important for the in vitro production and biomedical application of rhBMP-2 protein.

Enhancing effect of glycerol on the tensile properties of Bombyx mori cocoon sericin films.

An environmental physical method described herein was developed to improve the tensile properties of Bombyx mori cocoon sericin films, by using the plasticizer of glycerol, which has a nontoxic effect compared with other chemical crosslinkers. The changes in the tensile characteristics and the structure of glycerolated (0-40 wt% of glycerol) sericin films were investigated. Sericin films, both in dry and wet states, showed enhanced tensile properties, which might be regulated by the addition of different concentrations of glycerol. The introduction of glycerol results in the higher amorphous structure in sericin films as evidenced by analysis of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra, thermogravimetry (TGA) and differential scanning calorimetry (DSC) curves. Scanning Electron Microscopy (SEM) observation revealed that glycerol was homogeneously blended with sericin molecules when its content was 10 wt%, while a small amount of redundant glycerol emerged on the surface of sericin films when its content was increased to 20 wt% or higher. Our results suggest that the introduction of glycerol is a novel nontoxic strategy which can improve the mechanical features of sericin-based materials and subsequently promote the feasibility of its application in tissue engineering.

[Research on cytotoxicity of silk fibroin gel materials prepared with polyepoxy compound].

Two kinds of gel materials were prepared from silk fibroin with polyepoxy compound at subfreezing temperature and higher temperature. In order to evaluate the feasibility of their application in biomaterials, we tested the cytotoxicity of silk fibroin gels by detecting the effect of the extracted liquid on the cell relative proliferation rate of L-929 mouse fibroblasts. The results indicated that both of the gel materials displayed high relative proliferation rate and grade 1 cytotoxicity, being in the allowed range of medical application. The cytotoxicity tests on polyepoxy compound and glutaraldehyde were conducted too, and the cytotoxicity of polyepoxy compound was obviously lower than that of glutaraldehyde. Polyepoxy compound can be used as a more safe cross-link reagent for silk fibroin modification.

[Progress of silk fibroin in the cell scaffold of tissue engineering].

Recent researches about the application of silk fibroin in cell culture suggested that silk fibroin displayed high rate of cell attachment and growth in vitro culture of most kinds of cells, equivalent to collagen. So silk fibroin can be used for cell scaffold material of tissue engineering, and can be applied to several fields such as tissue engineering of skin, cartilage and blood vessel. The related researches and the prospect of the application of silk fibroin in cell scaffold of tissue engineering are reviewed in this paper.

Mineralization and biocompatibility of Antheraea pernyi (A. pernyi) silk sericin film for potential bone tissue engineering.

This study aimed to investigate the mineralization of Antheraea pernyi (A. pernyi) silk sericin. 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, and the corresponding results prove that A. pernyi sericin has Ca binding activity. Scanning electron microscope (SEM) observation shows that spherical crystals could be nucleated on the A. pernyi sericin film. These crystals were confirmed to be hydroxyapatite according to FT-IR and XRD spectra, indicating that A. pernyi sericin is capable of mineralization. In addition, cell adhesion and growth activity assay demonstrate that A. pernyi sericin shows excellent biocompatibility for the growth of MG-63 cells.

Preparation of a silk fibroin spongy wound dressing and its therapeutic efficiency in skin defects.

A novel silk fibroin spongy wound dressing (SFSD) incorporated with nano-Ag particles was prepared by coagulating with 1.25-5.0% (v/v) poly(ethylene glycol diglycidyl ether) (PGDE). The mechanical properties, moisture permeability and hygroscopicity of SFSD, and the nano-Ag release behavior from SFSD were evaluated. The results showed that the soft SFSD had satisfying tensile strength and flexibility, as well as excellent moisture permeability and absorption capability of wound exudates. The moisture permeability was 101 g/m(2) per h and the water absorption capacity of SFSD was 595.2% and 251.9% of its own weight in dry and wet states, respectively. The nano-Ag in the SFSD was released continuously at a relatively stable rate in PBS resulting in a remarkable antibacterial property. A rabbit model was used to dynamically observe the healing process of full-thickness skin defects. Full-thickness wounds were created on the dorsal side of rabbits, which were covered with SFSD and porcine acellular dermal matrix (PADM) for comparison. The mean healing time of the wounds covered with SFSD was 17.7 ± 2.4 days, significantly shorter than that with PADM. The histological analysis showed that the epidermal cell layer formed with SFSD was very similar to normal skin, suggesting that SFSD may provide a good component for the development of new wound dressings.

Fabrication and characterization of porous tubular silk fibroin scaffolds.

Silk fibroin (SF) has been one of promising resources of biotechnology and biomedical materials due to its unique properties. Here, different sizes of porous tubular scaffolds were fabricated from a SF aqueous solution with the addition of poly(ethylene glycol diglycidyl ether) (PGDE). The scaffolds were generally flexible and transparent at the wet state with a pore size of 81-128 mum and porosity of 90-96%, depending on the concentrations of SF and PGDE. The mechanical properties measurement showed that the tubular SF scaffolds had satisfying tensile and compression properties, especially the excellent deformation-recovery ability. FT-IR spectra indicated that the SF in the tubular scaffolds was in a beta-sheet structure, and no PGDE characteristic band was observed, suggesting that the PGDE could be removed from the scaffolds by soaking in deionized water. The cell compatibility of scaffolds was evaluated, and no obvious cytotoxicity to mouse L-929 fibroblasts was detected.

Preparation and characterization of nano-hydroxyapatite/silk fibroin porous scaffolds.

Novel tissue engineering scaffold materials of nano-hydroxyapatite (nHA)/silk fibroin (SF) biocomposite were prepared by freeze-drying. The needle-like nHA crystals of about 10 nm in diameter by 50-80 nm in length, which were uniformly distributed in the porous nHA/SF scaffolds, were prepared by a co-precipitation method with a size. The as-prepared nHA/SF scaffolds showed good homogeneity, interconnected pores and high porosity. XRD and FT-IR analysis suggested that the silk fibroin was in beta-sheet structure, which usually provides outstanding mechanical properties for silk materials. In this work, composite scaffolds containing as high as 70% (w/w) nHA were prepared, which had excellent compressive modulus and strength, higher than the scaffolds at low nHA content level and other porous biodegradable polymeric scaffolds often considered in bone-related tissue engineering reported previously. The cell compatibility of composite scaffolds was evaluated through cell viability by MTT assay. All these results indicated that these nHA/SF scaffold materials may be a promising biomaterial for bone tissue engineering.