Development of Small-Diameter Vascular Grafts Based on Silk Fibroin Fibers from Bombyx mori for Vascular Regeneration

In the field of surgical revascularization, the need for functional small-diameter (1.5–4.0 mm in diameter) 1vascular grafts is increasing. Several synthetic biomaterials have been tested for this purpose, but in many cases they cause thrombosis. In this study, we report the development of small-diameter vascular grafts made from silk fibroin fibers from the domestic silkworm Bombyx mori or recombinant silk fibroin fibers from a transgenic silkworm. The vascular grafts were prepared by braiding, flattening and winding the silk fibers twice onto a cylindrical polymer tube followed by coating with an aqueous silk fibroin solution. The grafts, which are 1.5 mm in inner diameter and 10 mm in length, were implanted into rat abdominal aorta. An excellent patency (ca. 85%, n= 27) at 12 months after grafting with wild-type silk fibers was obtained. Endothelial cells and smooth muscle cells migrated into the silk fibroin graft early after implantation, and became organized into an endothelium and a media-like smooth muscle layer.     

Mechanical Properties of Regenerated Bombyx mori Silk Fibers and Recombinant Silk Fibers Produced by Transgenic Silkworms

Regenerated silk fibroin fibers from the cocoons of silkworm, Bombyx mori, were prepared with hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or hexafluoroacetone-trihydrate (HFA), as dope solvents and methanol as coagulation solvent. The regenerated fiber prepared from the HFIP solution showed slightly larger tensile strength when the draw ratio is 1:3 than that of native silk fiber, but the strength of the regenerated fiber with draw ratio 1:3 from the HFA solution is much lower than that of native silk fiber. This difference in the tensile strength of the regenerated silk fibers between two dope solvents comes from the difference in the long-range orientation of the crystalline region rather than that of short-range structural environment such as the fraction of β-sheet structure. The increase in the biodegradation was observed for the regenerated silk fiber compared with native silk fiber. Preparations of regenerated silk fibroin fibers containing spider silk sequences were obtained by mixing silk fibroins and silk-like proteins with characteristic sequences from a spider, Naphila clavipes, to produce drag-line silk in E. coli in the fluoro solvents. A small increase in the tensile strength was obtained by adding 5% (w/w) of the silk-like protein to the silk fibroin. The production of silk fibroin fibers with these spider silk sequences was also performed with transgenic silkworms. Small increase in the tensile strength of the fibers was obtained without significant change in the elongation-at-break.     

Silk Fibroin/Sodium Carboxymethylcellulose Blended Films for Biotechnological Applications

The potential of silk protein is increased because of its importance as natural biopolymer for biotechnological and biomedical applications. The main disadvantage of silk fibroin films is their high brittleness. Thus, we studied blends of fibroin with other polymers to improve the film properties. Considering the possible applications of films in biomedical applications, we used a natural and biodegradable polymer as the second component. This study reports the fabrication and characterization of mulberry silk protein fibroin and sodium carboxymethylcellulose (NaCMC) blended films as potential substrates for in vitro cell culture. The blended films are investigated of their chemical interactions, morphologies, thermal, mechanical properties in addition to its swelling properties and biocompatibility. The addition of NaCMC improves the elasticity of fibroin films and its thermal properties. The change of morphology, swelling behavior and increase of surface roughness of the films were also observed in the blended films. The films become insoluble on alcohol treatment and are stable for longer duration in hydrolytic medium. The blended films are cytocompatible and supported adhesion and growth of mouse fibroblast cells. The results suggest that NaCMC blended silk fibroin films are found to be potential substratum for supporting cell adhesion and proliferation.     

Structural Characteristics and Properties of Silk Fibroin/Poly(lactic acid) Blend Films

This paper describes the preparation and characterization of blend films composed of regenerated silk fibroin (SF) and poly(lactic acid) (PLA). FT-IR and XRD of the SF/PLA blend films with different ratios indicated that the secondary structural transition of SF from Silk I to Silk II was induced upon blending with PLA. The effects of SF/PLA blend ratios on the mechanical and physical properties of the blend films were investigated. Compared to pure SF film, the mechanical and thermal properties of the blend films were improved, and surface hydrophilicity and swelling capacity decreased due to the secondary structural transition of SF to Silk II. Among the blend films with different ratios, the SF/PLA blend film with 7 wt% PLA content showed excellent mechanical properties. Meanwhile, the BSA adsorption amount on the blend film increased with the increase of PLA content. In vitro cell adhesion test showed that the blend film was a good matrix for the growth of L929 mouse fibroblast cells. Consequently, controlling the PLA content in the SF film can improve the mechanical and physical properties of the SF film and provide a promising opportunity to widen potential application of SF in the biomaterials field.     

Regenerated Silk Fibroin Nanofibrous Matrices Treated with 75% Ethanol Vapor for Tissue-Engineering Applications

As an excellent biocompatible and biodegradable protein polymer, silk fibroin (SF) has found wide applications, particularly serving as therapeutic agent for tissue-engineering applications, on which both post-spin treatment and sterilization processing are crucial to drug-loaded matrices. To find a safe, effective and appropriate post-spin treatment and sterilization approach for drug-loaded biomaterial matrices is one of the major problems in the field of tissue engineering at present. In this work, a simple, safe and effective approach skillfully integrating post-spin treatment with sterilization processing was developed to drug-loaded SF nanofibrous matrices. Electrospun SF nanofibrous matrices from its aqueous solution were post-treated with 75% ethanol vapor. ¹³C-NMR and WAXD analysis demonstrated that such post-spin treatment rendered the structure of SF nanofibrous matrices transform from the silk I form to the silk II form. Furthermore, biological assays suggested that as-treated SF nanofibrous matrices significantly promoted the development of murine connective tissue fibroblasts. Skillfully integrated with novel sterilization processing, 75% ethanol vapor treatment could be a potential approach to designing and fabricating diverse drug-loaded SF nanofibrous matrices serving as therapeutic agents for tissue-engineering applications in that it can effectively protect the drug from losing compared with traditional post-spin treatment and sterilization processing.     

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/m² 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.     

In Vitro and In Vivo Release of Basic Fibroblast Growth Factor Using a Silk Fibroin Scaffold as Delivery Carrier

Two different solvents were used to prepare two types of silk fibroin scaffolds via the salt-leaching technique, i.e., hexafluoroisopropanol (HFIP) and water. The in vitro release study suggests that the opposite charge between the silk fibroin and basic fibroblast growth factor (bFGF) at physiological pH rendered them to form a complex, and the difference in the solvents used to produce the silk fibroin scaffold did not affect the affinity of silk fibroin to bFGF. However, a higher degradation rate of the aqueous-derived silk fibroin scaffolds provided faster in vitro release kinetics of the bFGF, as compared to the HFIP-derived scaffolds. From the in vivo studies, the use of silk fibroin scaffolds as the carrier matrix enabled the control of the in vivo release of bFGF in a sustained fashion over two weeks, while the majority of the bFGF disappeared within one day after the injection of the bFGF in soluble form. In addition, the in vivo release of bFGF from the silk fibroin scaffolds was not affected by the mode of processing due to their similar degradation behavior in vivo.     

Characterization of Fibroin and PEG-Blended Fibroin Matrices for In Vitro Adhesion and Proliferation of Osteoblasts

Silk fibroin protein, isolated from cocoons of the domesticated mulberry silkworm, Bombyx mori, finds extensive application in biomaterial design. In this study, poly(ethylene glycol) (PEG) 4000 has been used for blending fibroin from both B. mori and Antheraea mylitta, the wild tropical non-mulberry silkworm. PEG-blended films have shown marked changes from the pure fibroin films with respect to thermal properties and mechanical properties. FT-IR spectroscopy confirmed incorporation of new functional groups like quinone oximes. Pure fibroin and PEG-blended fibroin films showed biocompatibility with the HOS osteosarcoma cell line. Von Kossa staining confirmed nodule formation due to mineralization and differentiation of osteoblasts on pure and blended matrices. On account of increased surface roughness, higher elongation percentage, higher thermostability and better activity of osteoblasts in terms of intracellular alkaline phosphatase production, PEG-blended A. mylitta fibroin film shows better potential than PEG-blended B. mori fibroin film for use as potential biomaterial.     

Evaluation of the properties of silk fibroin films from the non-mulberry silkworm Samia cynthia ricini for biomaterial design

Silk fibroin from a domesticated mulberry silkworm, Bombyx mori, is the most widely used in biomaterial design. We report for the first time the preparation of a relatively smooth (granule free) film of the nonmulberry Samia cynthia ricini fibroin for comparative evaluation of its cell-supporting properties against those of the B. mori fibroin film. The granule formation on the S. c. ricini fibroin film was successfully prevented by facilitating proper rearrangement of the protein molecules, as monitored by FT-IR, by dialysis through a stepwise decrease in the urea concentration in the dialysis media. The lower contact angle of the S. c. ricini fibroin film, compared to the B. mori fibroin film, corresponds well to its lower hydrophobic/hydrophilic amino-acid ratio and grand average of hydropathicity (GRAVY). L929 murine fibroblast cells on the granule-free S. c. ricini fibroin films exhibited greater proliferation and spreading rates than those on the B. mori fibroin films, possibly attributable to its higher content of hydrophilic and positively charged amino acids. It further suggests that fabrication, modification and/or engineering of S. c. ricini fibroin may provide a better biomaterial scaffold design than the more commonly used B. mori fibroin.     

Collaborative Cell-Resistant Properties of Polyelectrolyte Multilayer Films and Surface PEGylation on Reducing Cell Adhesion to Cytophilic Surfaces

Both poly(ethylene glycol) (PEG) grafting and layer-by-layer polyelectrolyte multilayer (PEM) deposition for surface modification of biomaterials have been shown to decrease cell adhesion. The aim of this study was to investigate the synergic efficacy of PEGylated PEM films on reducing cell adhesion. PEG grafted to poly(ethylene imine) (PEI) was deposited onto the top of PEI/PAA (poly(acrylic acid)) multilayer films which were deposited onto cytophilic substrates, including tissue culture polystyrene and collagen-based substrate. The efficacy of the PEGylated PEM films in blocking adhesion of L929 cells was investigated by varying the amount of conjugated PEG and the layer numbers of PEM films. We found that cell adhesion was reduced on the swollen PEM films and further decreased by deposition of PEI-g-PEG as the topmost layer. The ability in cell resistance was enhanced with increasing PEG contents of PEGylated PEM films. PEGylated PEM films were stable for long-term incubation in phosphate-buffered saline. We demonstrated that cell affinity of cytophilic surfaces could be depressed by deposition of PEGylated PEM films.     

Production and characterization of a silk-like hybrid protein, based on the polyalanine region of Samia cynthia ricini silk fibroin and a cell adhesive region derived from fibronectin

There are a variety of silkworms and silk fibroins produced by them. Silks have many inherent suitable properties for biomaterials. In this paper, a novel silk-like hybrid protein, [DGG(A)(12)GGAASTGRGDSPAAS](5), which consists of polyalanine region of silk fibroin from a wild silkworm, Samia cynthia ricini, and cell adhesive region including Arg-Gly-Asp (RGD) sequence, derived from fibronectin, was designed and produced. The genes encoding the hybrid protein were constructed and expressed in Escherichia coli. The main conformation of the polyalanine region, that is, either alpha-helix or beta-sheet, could be easily controlled by treatment with different acidic solvents, trifluoroacetic acid or formic acid, respectively. This structural change was monitored with 13C CP/MAS NMR. Higher cell adhesive and growth activities of the hybrid protein compared with those of collagen were obtained.     

Preparation and Characterization of Collagen from Soft-Shelled Turtle (Pelodiscus Sinensis) Skin for Biomaterial Applications

Collagen was isolated from the skin of soft-shelled turtle (Pelodiscus sinensis) by acid solubilization with pepsin. The yield of soft-shelled turtle collagen (STC) was 12.1% on a dry weight basis. The electrophoresis assay showed that STC consisted of a α ₁ α ₂ heterodimer similar to porcine collagen (PC). Amino-acid composition analysis showed that the hydroxyproline content of STC was 7.8%, which was lower than that of PC (9.5%). The denaturation temperature of STC was 36°C from optical rotation analysis. An accelerated fibrillogenesis of STC was observed in phosphate-buffered saline at 25°C. The resulting STC fibrillar gel had microfibrillar network with fibril diameter of ca. 124 nm, as revealed by observation with scanning electron microscopy. The compressive moduli of the STC gel and the PC gel were 3.2 ± 0.8 kPa and 3.6 ± 0.3 kPa, respectively. The potential of the STC gel for biomaterial applications was investigated by in vitro cell culture. Human dermal fibroblasts were three-dimensionally cultured in the STC gel and their growth was evaluated by DNA content measurement. Steady growth was observed in the STC gel for a 6-day culture period, although the growth rate was slower than in the PC gel. In conclusion, STC could be used as a novel collagen source for biomaterial applications.     

Silk Fibroin‐Based Films Enhance Rhodamine 6G Emission in the Solid State: A Chemical–Physical Analysis of their Interactions for the Design of Highly Emissive Biomaterials

Described herein is the preparation of dye‐doped films employing silk fibroin (SF) as a biomaterial, capable of preserving the optical properties of the monomeric dye in the solid state, a critical requisite for optical and biolaser applications. A comprehensive physical–chemical characterization is reported for SF films doped with Rhodamine 6G, an ideal candidate for photonics and optoelectronics. Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) and X‐Ray diffraction (XRD) provide information on SF secondary conformation in the presence of rhodamine. UV–vis absorption spectra and exciton CD inform on the structure of encapsulated rhodamine, while changes in dye photophysical properties illuminate the molecular mechanism of the involved host–guest interactions. SF host environment inhibits rhodamine dimer formation, indicating that SF is an optimum matrix to keep rhodamine essentially monomeric at concentrations as high as 7 mm in the film. The relevant optical properties of these films and the easiness of their preparation, make these systems optimal candidates for innovative photonic technologies.     

Metathesis Curing of Allylated Lignin and Different Plant Oils for the Preparation of Thermosetting Polymer Films with Tunable Mechanical Properties

Lignin and plant oils—two highly available renewable feedstocks—are cured via olefin metathesis to produce thermosetting polymer films with tunable mechanical properties. Therefore, an organosolv lignin, allylated in a recently described sustainable approach, is used. For the first time, unmodified plant oils are reacted with a lignin derivative. An increasing lignin content as well as an increasing amount of polyunsaturated fatty acids in the plant oil structure lead to higher Young's moduli and higher cross‐linking density. Stress–strain measurements reveal ductile behavior of the prepared films. Only a lignin content over 70% leads to brittle materials. To improve the overall sustainability of this approach, dimethyl carbonate is investigated as sustainable solvent for film formation, leading to materials with similar Young's moduli and tensile strength, but lower degree of cross‐linking if compared to the more volatile solvent dichloromethane.

     

Evaluation of adsorbents for the removal of metabolic wastes from blood

The adsorption properties of various adsorbents for metabolic wastes, such as urea, uric acid, creatinine and ammonium ions have been studied. Oxycellulose prepared by the periodic acid method having more aldehyde, adsorbs more than three times the amount of urea than the activated carbon supplied by Sarabhai Merck. Synthetic resins do not adsorb urea, uric acid and creatinine, but adsorb a significant amount of ammonium ions.     

电阻抗谱评价模拟失重对大鼠血液电特性的影响

在频率为104～108 Hz范围,采用Agilent 4294A阻抗分析仪测量大鼠血液交流阻抗,通过电阻抗谱、Bode图、Nyquist图和Nichols图的数据分析,观察60d模拟失重(SWL)对大鼠血液电阻抗频谱特性的影响。结果表明:(1)大鼠血液电阻抗降低,主要表现在复阻抗实部(Z′0和Z′∞)、电阻抗幅模值(|Z*|0和|Z*|∞)、阻抗驰豫幅度(ΔZ′、Δ|Z*|)和低频阻抗幅模值对数(Log|Z*|0)较对照(CON)组均降低;(2)大鼠血液电阻抗谱的特征频率和相位角增加,主要表现在特征频率(fC1、fC2)和相位角[θP(degree)、θP(radian)]较CON组增加。提示模拟失重引起血液电阻率降低,导电性能增加。     

Evaluation of the polymerase chain reaction for the detection of human papillomavirus from urine

The ability to detect the presence of human pap-illomavirus (HPV)-DNA sequences in urine was evaluated using polymerase chain reaction (PCR). DMA was purified and extracted from urine samples, and subjected to 40 cycles of amplification using the consensus primer pair MY11 and MY09. Coamplification using the β-globin primers, GH20 and PC04, was performed as an internal reaction control. Following assay optimization, urine samples from 22 women undergoing examination for cervical dysplasia were tested for the presence of HPV-DNA. PCR assay results were correlated with cytologic and histo-logic findings as well as Vira Type(tm) assay results. Overall, HPV was detected by PCR in 16 (76%) of the interpretable samples. HPV sequences were detected in 13 (87%) of the 15 specimens from women showing evidence of condylomata, dysplasia, or invasive carcinoma. HPV was detected in 3 (50%) of the women whose cytologic or his-tologic results were either negative or showed benign atypia. Although the sample size in this study is small, our results show that HPV can be detected by PCR in a majority of individuals showing evidence of HPV infection. The method described provides a means for the clinical laboratory to detect a broad range of HPV types from using a sample obtained by noninvasive techniques. The ability to easily obtain urine would allow for increased numbers of individuals to be tested, and thus, aid in our understanding of HPV. © 1995 Wiley-Liss, Inc.     

Tuning Block Polymer Structure,Properties, and Processability for the Design of Efficient Nanostructured Materials Systems

Block polymers (BPs) are ideal building blocks for nanoporous membranes, microelectronics templates, energy storage devices, and other technologies. For these systems, precise control over morphology, properties, and processability are essential to optimize performance. The Epps group has used a multidisciplinary approach to control BP nanostructure ordering and orientations, order‐disorder and glass transition temperatures, and mechanical and transport properties through the informed manipulation of BP effective segregation strengths and interfacial energetics. For example, alterations in macromolecular interactions between BP chains were achieved through the introduction of composition gradients between copolymer blocks or by adding dopants. In thin films, high‐throughput approaches were designed to modify and characterize substrate‐polymer interactions, and solvent annealing techniques were developed to facilitate nanoscale alignment and ordering. By manipulating BP interfacial energetics to gain exquisite tunability of nanostructures and materials' properties, the group is accelerating the development of next‐generation BPs that will be harnessed to fabricate inexpensive, efficient, and high‐performance devices.

     

Evaluation of biphasic culture system for mycobacterial isolation from the sputum of patients with pulmonary tuberculosis

Mycobacterial diseases continue to cause high morbidity and mortality. Isolation, identification and sensitivity testing form the backbone of laboratory investigations. M. tuberculosis isolation needs 6-8 weeks on conventional egg containing media. For rapid isolation various methods have been evaluated. We evaluated biphasic system (Middlebrook 7H11 agar slant+Middlebrook 9H broth) in comparison with Lowenstein-Jensen (LJ) medium. In smear positive cases biphasic system showed the recovery rate of 97.05% as against 79.41% on LJ on incubation for 21+/-4.44 and 28+/-3.76 days respectively. In smear negative and culture positive cases biphasic system and LJ showed isolation rates of 91.66% and 66.6% after 36+/-3.44 and 41+/- 4.09 days respectively. Biphasic system showed lower contamination rate (1.33%). Biphasic medium is superior to LJ medium in isolation of M. tuberculosis.