Regenerated Silk Fibroin Membrane Research Articles

Regenerated silk fibroin membranes as separators for transparent microbial fuel cells

In recent years novel applications of bioelectrochemical systems are exemplified by phototrophic biocathodes, biocompatible enzymatic fuel cells and biodegradable microbial fuel cells (MFCs). Herein, transparent silk fibroin membranes (SFM) with various fibroin content (2%, 4% and 8%) were synthesised and employed as separators in MFCs and compared with standard cation exchange membranes (CEM) as a control. The highest real-time power performance of thin-film SFM was reached by 2%-SFM separators: 25.7 ± 7.4 μW, which corresponds to 68% of the performance of the CEM separators (37.7 ± 3.1 μW). Similarly, 2%-SFM revealed the highest coulombic efficiency of 6.65 ± 1.90%, 74% of the CEM efficiency. Current for 2%-SFM reached 0.25 ± 0.03 mA (86% of CEM control). Decrease of power output was observed after 23 days for 8% and 4% and was a consequence of deterioration of SFMs, determined by physical, chemical and biological studies. This is the first time that economical and transparent silk fibroin polymers were successfully employed in MFCs.

Rational design of a stable, effective, and sustained dexamethasone delivery platform on a titanium implant: An innovative application of metal organic frameworks in bone implants

Endowing titanium implants with the capability of delivering dexamethasone in a sustained and controlled manner to promote osseointegration and osteogenesis has become an emerging field. However, most of the currently developed dexamethasone/titanium implants are far from satisfactory because of some inherent defects. In this work, we constructed a stable, effective, and sustained dexamethasone delivery platform on titanium disc by immobilizing dexamethasone@zeolitic imidazolate framework-8 nanoparticles into the micrometer-scale artificial etch pits on titanium substrate using methanol-induced regenerated silk fibroin membrane encapsulation. In addition, the titanium substrate was aminated and covalent interactions were established between the titanium substrate and the silk fibroin membrane through genipin crosslinking. Briefly, owing to the synergistic barrier effect of the zeolitic imidazolate framework-8 shell and the silk fibroin membrane, the silk fibroin-dexamethasone@zeolitic imidazolate framework-8-titanium could release dexamethasone in a controlled manner over 30days. Moreover, the pit walls together with the silk fibroin membrane protected the nanoparticles from detaching from the titanium substrate in the case of mechanical wear. The covalent interactions between the silk fibroin membrane and the titanium substrate prevented the silk fibroin membrane from self-peeling from the titanium substrate in a moist environment. In vitro cell culture indicated that the as-prepared titanium disc had good cytocompatibility with MC3T3-E1 cells. Furthermore, the cells cultured on the titanium disc demonstrated higher differentiation, calcium deposition, and expression of osteogenic genes than the cells cultured on the silk fibroin-zeolitic imidazolate framework-8-titanium and pristine titanium. In this work, dexamethasone was utilized as a drug model and could be replaced by other osteogenic drugs or growth factors. Thus, we believed that our design philosophy could inspire future research work on the development of Ti implants.

Glycerol-plasticised silk membranes made using formic acid are ductile, transparent and degradation-resistant

Regenerated silk fibroin membranes tend to be brittle when dry. The use of plasticisers such as glycerol improve membrane ductility, but, when combined with aqueous processing, can lead to a higher degradation rate than solvent-annealed membranes. This study investigated the use of formic acid as the solvent with glycerol to make deformable yet degradation-resistant silk membranes. Here we show that membranes cast using formic acid had low light scattering, with a diffuse transmittance of less than 5% over the visible wavelengths, significantly lower than the 20% transmittance of aqueous derived silk/glycerol membranes. They had 64% β-sheet content and lost just 30% of the initial silk weight over 6h when tested with an accelerated enzymatic degradation assay, in comparison the aqueous membranes completely degraded within this timeframe. The addition of glycerol also improved the maximum elongation of formic acid derived membranes from under 3% to over 100%. They also showed good cytocompatibility and supported the adhesion and migration of human tympanic membrane keratinocytes. Formic acid based, silk/glycerol membranes may be of great use in medical applications such as repair of tympanic membrane perforation or ocular applications where transparency and resistance to enzymatic degradation are important.

Construction of a functional silk‐based biomaterial complex with immortalized chondrocytes in vivo

To explore the feasibility of constructing a functional biomaterial complex with regenerated silk fibroin membrane and immortalized chondrocytes in vivo. Rat auricular chondrocytes (RACs) were transfected with the lentivirus vector pGC-FU-hTERT-3FLAG or pGC-FU-GFP-3FLAG, encoding the human telomerase reverse transcriptase (hTERT) or GFP gene. The effects of regenerated silk fibroin film on the adhesion, growth of immortalized chondrocytes and expression of collagen II in vitro were analyzed with immunofluorescent histochemistry. Immortalized RACs were transformed. Induction by nutrient medium promoted higher expression levels of collagen II in transformed chondrocytes. The regenerated silk fibroin film was not cytotoxic to immortalized chondrocytes and had no adverse influence on their adhesion. Collagen II expression was good in the immortalized chondrocytes in vivo. The construction of a silk-based biomaterial complex with immortalized chondrocytes may provide a feasible kind of functional biomaterial for the repair of cartilage defects in clinical applications.

Effect of regeneration of liquid silk fibroin on its structure and characterization

Silk derived from cocoons of the female silkworm (Bombyx mori) was made into three kinds of test material, degummed fiber, liquid fibroin and regenerated silk fibroin membrane (RSFM). The effect of the silk degumming and fiber dissolution system (SD–FDS) on the molecular structure and properties of silk fibroin were studied in detail. Degumming with Na2CO3 solution had the greatest impact on silk fibroin fiber, resulting in significant reduction of the thermal stability and crystallinity, and the fiber tensile property was only half of that degummed with urea buffer. The moderate degumming solution was urea buffer, followed by strongly alkaline electrolyzed water (SAEW). SDS-PAGE showed that the silk fibroin degummed with 8.0 M urea at 80 °C and dissolved in 9.3 M LiBr at 25 °C was similar to the natural silk fibroin in vivo, but boiling in Na2CO3 solution leads to serious breakage of the silk fibroin peptide chain. The degree of breakage of the peptide chain is positively correlated with the storage time of liquid silk fibroin. DSC showed the more nearly intact the silk fibroin peptide chain, the higher the thermal decomposition temperature. The regenerated condition of the silk protein is often overlooked during the research process, while our study showed that different silk degumming and fiber dissolution systems will have different influences. The results of this test help us gain a deeper understanding of the effect of silk degumming and fiber dissolution on the molecular structure and properties of silk fibroin and provide important experimental data for screening and using silk protein as advanced functional biomaterials, such as medical tissue engineering materials.

Cytocompatibility of regenerated silk fibroin film: a medical biomaterial applicable to wound healing

To explore the feasibility of using regenerated silk fibroin membrane to construct artificial skin substitutes for wound healing, it is necessary to evaluate its cytocompatibility. The effects of regenerated silk fibroin film on cytotoxicity, adhesion, cell cycle, and apoptosis of L929 cells, growth and vascular endothelial growth factor (VEGF) expression of ECV304 cells, and VEGF, angiopoietin-1 (Ang-1), platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2) expression of WI-38 cells were assessed by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, viable cell counting, flow cytometry (FCM), and enzyme-linked immunosorbant assay (ELISA). We showed that the regenerated silk fibroin film was not cytotoxic to L929 cells and had no adverse influence on their adhesion, cell cycle or apoptosis; it had no adverse influence on the growth and VEGF secretion of ECV304 cells and no effect on the secretion of VEGF, Ang-1, PDGF and FGF2 by WI-38 cells. The regenerated silk fibroin film should be an excellent biomaterial with good cytocompatibility, providing a framework for reparation after trauma in clinical applications.

Swelling and dissolution of silk fibroin (Bombyx mori) in N-methyl morpholine N-oxide

Bombyx mori silk fibers were dissolved in N-methyl morpholine N-oxide (MMNO), an organic cyclic amine oxide used for the solvent spinning of regenerated cellulosic fibers. The commercial MMNO monohydrate used in this study as a solvent for silk is a hygroscopic compound crystalline at room temperature, which becomes an active solvent after melting at 76°C. The degree of hydration of MMNO was checked by DSC measurements. The solvation power of MMNO towards silk fibroin drastically decreased at a water content ≥20–21% w/w. Dissolution of silk required both thermal and mechanical energy. The optimum temperature was 100°C. At lower temperatures dissolution proceeded very slowly. At higher temperatures, rapid depolymerization of silk fibroin occurred. The value of the Flory–Huggins interaction parameter χ for the MMNO–H2O–silk fibroin system was −8.5, suggesting that dissolution is a thermodynamically favored process. The extent of degradation of silk fibroin was assessed by measuring the intrinsic viscosity and determining the amino acid composition of silk after regeneration with an aqueous methanol solution, which was effective in removing the solvent and coagulating silk. Regenerated silk fibroin membranes were characterized by infrared spectroscopy, differential scanning calorimetry and scanning electron microscopy. The prevailing molecular conformation of silk fibroin chains was the β-sheet structure, as shown by the intense amide I-III bands at 1704, 1627, 1515, 1260, and 1230 cm−1. The value of the I1260/I1230 intensity ratio (crystallinity index) was 0.68, comparable to that of the fibers. The DSC thermogram was characteristic of a silk fibroin material with unoriented β-sheet crystalline structure, with an intense decomposition endotherm at 294°C. The SEM examination of fractured surfaces showed the presence of a dense microstructure with a very fine texture formed by densely packed roundish particles of about 100–200 nm diameter.

Immobilization of horseradish peroxidase with a regenerated silk fibroin membrane and its application to a tetrathiafulvalene-mediating H 2O 2 sensor

Horseradish peroxidase (HRP) was immobilized onto a membrane of the regenerated silk fibroin (RSF) from waste milk. The structure of the blend membrane of RSF and HRP was characterized by the use of IR spectra. A second generation of H 2O 2 sensor on the basis of the immobilized HRP was fabricated, in which tetrathiafulvalene acts as mediating electron transfer between the immobilized enzyme and a glassy carbon electrode. Dependencies of pH and temperature on the H 2O 2 biosensor were checked by utilizing cyclic voltammetry. The sensor exhibits high sensitivity, good reproducibility and storage stability.

Entrapment of both glucose oxidase and peroxidase in regenerated silk fibroin membraneCharacterization of the membrane structure and its application to an amperometric glucose sensor employing methylene green as an electron transfer mediato.

Two enzmyes, glucose oxidase and peroxidase, were for the first time simultaneously immobilized in regenerated silk fibroin membrane. The structure and morphology of the regenerated silk fibroin membrane containing both glucose oxidase and peroxidase were investigated with IR spectra and SEM. The bienzymes do not change the structures of the regenerated silk fibroin in the membrane, which has an islands-sea structure. For the first time, an amperometric methylene green mediating sensor for glucose based on co-immobilization of both glucose oxidase and peroxidase in regenerated silk fibroin was constructed. Cyclic voltammetry and amperometry were used to test the suitability of methylene green shuttling electrons between peroxidase and the glassy carbon electrode. The bienzyme-based system offers fast response and high sensitivity of the sensor to glucose. The effects of pH, temperature, and the concentration of the mediator on the response current were evaluated, and the dependence of the Michaelis-Menten constant K(m)(app) on the concentration of the mediator was investigated.

An Amperometric New Methylene Blue N-Mediating Sensor for Hydrogen Peroxide Based on Regenerated Silk Fibroin as an Immobilization Matrix for Peroxidase

A simple and effective procedure was described for the immobilization of peroxidase in regenerated silk fibroin membrane prepared from waste silk. The membranes of regenerated silk fibroin with or without peroxidase, before or after the ethanol treatment, were characterized by ir spectra. An amperometric H2O2sensor, based on the immobilized peroxidase in regenerated silk fibroin membrane, in the use of new methylene blue N as an electron transfer mediator, was fabricated. The characteristics of the sensor with respect to linearity, response time, effect of pH and temperature, stability, and reproducibility were investigated. Dependences of Michaelis–Menten constantKappMon the concentration of the mediator, and the applied potential were also studied and the results were presented. The sensor was highly sensitive to H2O2with a detection limit of 1.0 × 10−7Mand with response time of less than 40 s.

Feature of Entrapment of Glucose Oxidase in Regenerated Silk Fibroin Membranes and Fabrication of a 1,1′-Dimethylferrocene-Mediating Glucose Sensor

The structural characteristics of regenerated silk fibroin were determined IR spectra. Without ethanol treatment, the membrane has the structure of silk I. When treated with ethanol, the membrane possesses a mixed structure of silk I and silk II. The structures of the blend membrane were identified as a composite of the absorption bands characteristic of both molecules. SEM showed that the blend membrane has an island–sea structure and nonuniform distribution of glucose oxidase inside the membrane may exist. An amperometric glucose sensor was constructed by utilizing 1,1′-dimethylferrocene as electron transfer mediator. The effects of various experimental parameters such as pH, temperature, buffer concentration, applied potential, and scan rate are explored for optimum analytical performance. The glucose sensor responds rapidly to glucose, with steady-state current responses achieved in less than 50 s.

Characteristics of regenerated silk fibroin membrane in its application to the immobilization of glucose oxidase and preparation of a p-benzoquinone mediating sensor for glucose

The structures of the blend membrane of regenerated silk fibroin and glucose oxidase were investigated by means of FT-IR spectra, electronic absorption spectra and SEM. The structures of the membranes were partly transferred from silk I to silk II after the membrane had been treated with ethanol. It was found that the glucose oxidase in the membrane existed in molecular aggregates and the blend membrane had an islands sea structure. p-Benzoquinone in solution was employed to speed up the electron transfer between glucose oxidase in a regenerated silk fibroin membrane and a glassy carbon electrode. The effects of pH, temperature, concentration of p-benzoquinone and applied potential on the sensor were examined. The major advantage of water dispersed regenerated silk fibroin is its ability to immobilize the enzyme without any significant loss of enzymatic activity.

Fabrication and features of a Methylene Green-mediating sensor for hydrogen peroxide based on regenerated silk fibroin as immobilization matrix for peroxidase

Regenerated silk fibroin prepared from waste silk was employed as immobilization matrix for peroxidase and the structures of the blend membranes of regenerated silk fibroin and peroxidase were first investigated with IR and scanning electron microscopy. There was intermolecular interaction between peroxidase and regenerated silk fibroin in the immiscible state. Cyclic voltammetry and constant applied potential measurement showed that Methylene Green efficiently mediated electron transfer from oxidized horseradish peroxidase in regenerated silk fibroin membrane to a glassy carbon electrode. A sensor coupling immobilized peroxidase with Methylene Green responded rapidly to low H 2O 2 concentration and achieved 95% of the steady-state current in less than 25 s with a detection limit of 1.0 × 10 −7 M H 2O 2. The sensor was stable in continuous operation, indicating that peroxidase was entrapped in regenerated silk fibroin membrane and did not freely diffuse away from the sensor surface into solutions.

China - Two papers dealing with the use of a regenerated silk fibroin membrane in a biosensor: In J. CHEM. TECHNOL. BIOTECHNOL. (64/3 (269–276) 1995) Y. Liu, H. Liu, J. Qian, J. Deng & T. Yu of Fudan University report on ‘Immobilization of glucose oxidase in the regenerated silk fibroin membrane: Characterization of the membrane structure and its application to

China - Two papers dealing with the use of a regenerated silk fibroin membrane in a biosensor: In J. CHEM. TECHNOL. BIOTECHNOL. (64/3 (269–276) 1995) Y. Liu, H. Liu, J. Qian, J. Deng & T. Yu of Fudan University report on ‘Immobilization of glucose oxidase in the regenerated silk fibroin membrane: Characterization of the membrane structure and its application to

Feature of an amperometric ferrocyanide-mediating H 2O 2 sensor for organic-phase assay based on regenerated silk fibroin as immobilization matrix for peroxidase

Peroxidase was immobilized in the regenerated silk fibroin membrane prepared from waste silk and the structures of the blend membranes of regenerated silk fibroin and peroxidase were investigated with ir and scanning electron microscopy, indicating that the two kinds of macromolecules were immiscible and their intermolecular interactions existed in the blend membrane. Cyclic voltammetry and constant applied potential measurements tested the catalytic activity of the immobilized peroxidase in water-iso-propyl alcohol system by employing ferrocyanide as an electron transfer mediator between the enzyme and a glassy carbon electrode. Regenerated silk fibroin coating appeared very attractive for entrapping the enzyme onto the electrode since the immobilized peroxidase showed long-term stability in the organic media.

Characterization of regenerated silk fibroin membranes for immobilizing glucose oxidase and construction of a tetrathiafulvalene‐mediating glucose sensor

AbstractThe IR spectra, the electronic absorption bands, and scanning electron microscopy (SEM) of the blend membranes of regenerated silk fibroin and glucose oxidase were reported for the first time and the second generation of glucose sensor based on glucose oxidase immobilized in the regenerated silk fibroin membrane was first constructed. The IR absorption spectra of the pure regenerated silk fibroin membrane were ascribed to its structural characteristics by which the part transition from silk I to silk II was recognized in ethanol immersion. Those spectra of the blend membrane of regenerated silk fibroin and glucose oxidase were identified as a composite of the absorption bands characteristic of both macromolecules. The electronic absorption bands showed that the glucose oxidase in the membrane exists in aggregates. A sea islands' structure was observed by SEM. These findings suggest that the regenerated silk fibroin and glucose oxidase are incompatible and their molecular interactions are very weak. A tetrathiafulvalene‐mediating glucose sensor, employing immobilization of glucose oxidase by regenerated silk fibroin, was fabricated. The influences of temperature, applied potential, and pH on steady‐state electrocatalytic oxidation of glucose at the sensor were evaluated. The response of the sensor to glucose under N2 saturation reached 95% steady‐state current within 40 s. The sensor could be used repeatedly for 1.5 months without deterioration of the response. © 1995 John Wiley & Sons, Inc.

Immobilization of glucose oxidase in the regenerated silk fibroin membrane: Characterization of the membrane structure and its application to an amperometric glucose sensor employing ferrocene as electron shuttle

AbstractThe structural changes of the regenerated silk fibroin membranes for immobilizing glucose oxidase were characterized with FT‐IR spectra. The results of electronic absorption spectra and scanning electron microscopy illustrated that the glucose oxidase in the membranes existed in aggregates. The blend membranes exhibited an island‐like structure, attributable to the incompatible and weak intermolecular interactions between regenerated silk fibroin and glucose oxidase. The glucose sensor was constructed by combining glucose oxidase immobilized in regenerated silk fibroin membrane with an Eastman‐AQ‐ferrocene modified electrode. The advantages of the mediator include high stability and the ability to use a less positive potential for increased selectivity.

Characterization of regenerated silk fibroin membrane for immobilizing peroxidase and construction of an amperometric hydrogen peroxide sensor employing phenazine methosulphate as electron shuttle

Regenerated silk fibroin prepared from waste silk was used for the first time as the immobilization matrix of peroxidase. The structures of the blend membranes of regenerated silk fibroin and peroxidase were investigated using IR and scanning electron microscopy. It was found that the two macromolecules were immiscible despite their intermolecular interactions. An amperometric H 2O 2 sensor using phenazine methosulphate as the electron transfer agent between horseradish peroxidase in a regenerated silk fibroin membrane and glassy carbon electrode was fabricated. The sensor was highly sensitive to H 2O 2 with a detection limit of 1.0 × 10 −7 M H 2O 2 and a response time of less than 5 s. The effect of the applied potential and the mediator concentration on the Michaelis-Menten constant was calculated, and the influence of various experimental parameters such as pH, temperature, applied potential and the mediator concentration was investigated for optimum analytical performance.

Regenerated silk fibroin membrane as immobilization matrix for peroxidase and fabrication of a sensor for hydrogen peroxide utilizing methylene blue as electron shuttle

Regenerated silk fibroin prepared from waste silk was utilized to immobilize peroxidase. The structures of the composite membranes of regenerated silk fibroin and peroxidase were studied with IR and scanning electron microscopy. It was found that the two kinds of macromolecules are immiscible despite their intermolecular interactions. The major advantage in using water dispersed regenerated silk fibroin was its ability to dissolve the enzyme without any significant loss of enzymatic activity. An amperometric H 2O 2 biosensor based on methylene blue's mediating electron transfer between the immobilized horseradish peroxidase and a glassy carbon electrode was made. Electrocatalytic reduction of H 2O 2 at the electrode was evaluated with respect to solution pH, temperature, operating potential and concentration of methylene blue. The biosensor exhibited high sensitivity and a short response time of 30 s.

Structural changes of silk fibroin membranes induced by immersion in methanol aqueous solutions

AbstractStructural changes of native and regenerated silk fibroin membranes were induced by immersion in water‐methanol solutions and examined as a function of immersion time and methanol concentration. X‐ray diffractometry and infrared spectroscopy data showed that transition from random coil to β‐sheet structure occurred favorably when both native and regenerated silk fibroin membranes were immersed in water‐methanol solutions, regardless of the different immersion time. Only native silk membrane, treated for 2 min with pure methanol, maintained its original amorphous structure, as demonstrated by differential scanning calorimetric (DSC) curves. The degree of displacement, measured by thermomechanical analysis (TMA), was much greater for regenerated than for native silk fibroin membranes. SDS‐PAGE pattern showed that native silk fibroin has a molecular weight of 350, while the regenerated sample is formed by a large number of polypeptides in the range of 200‐50 KD. The amount of acidic and basic amino acids decreased slightly in regenerated silk fibroin. Physical properties of silk membranes treated with water‐methanol solutions are discussed in terms of membrane structure, treatment conditions, and chemical structure of starting material. © 1994 John Wiley & Sons, Inc.