Water-insoluble regenerated silk materials are normally achieved by increasing β-sheet content (silk II). In the present study, water-insoluble silk films were prepared by controlling very slow drying of B. mori silk solutions, resulting in the formation of stable films with dominating silk I instead of silk II structure. Wide angle x-ray scattering (WAXS) indicated that the silk films stabilized by slow drying were mainly composed of silk I rather than silk II, while water-and methanol-annealed silk films had a higher silk II content. The silk films prepared through slow drying had a globule-like structure in the core with nano-filaments. The core region was composed of silk I and silk II, and these regions are surrounded by hydrophilic nano-filaments containing random, turns, and α-helix secondary structures. The insoluble silk films prepared by slow drying had unique thermal, mechanical and degradative properties. DSC results revealed that silk I crystals had stable thermal properties up to 250°C, without crystallization above the Tg, but degraded in lower temperature than silk II structure. Compared with water-and methanol-annealed films, the films prepared through slow drying achieved better mechanical ductility and more rapid enzymatic degradation, reflective of the differences in secondary structure achieved via differences in post processing of the cast silk films. Importantly, the silk I structure, a key intermediate secondary structure for the formation of mechanically robust natural silk fibers, was successfully generated in the present approach of very slow drying, mimicking the natural process. The results also point to a new mode to generate new types of silk biomaterials, where mechanical properties can be enhanced, and degradation rates increased, yet water insolubility is maintained along with low beta sheet content.

We directly prepared insoluble silk films by blending with glycerol and avoiding the use of organic solvents. The ability to blend a plasticizer like glycerol with a hydrophobic protein like silk and achieve stable material systems above a critical threshold of glycerol is an important new finding with importance for green chemistry approaches to new and more flexible silk-based biomaterials. The aqueous solubility, biocompatibility, and well-documented use of glycerol as a plasticizer with other biopolymers prompted its inclusion in silk fibroin solutions to assess impact on silk film behavior. Processing was performed in water rather than organic solvents to enhance the potential biocompatibility of these biomaterials. The films exhibited modified morphologies that could be controlled on the basis of the blend composition and also exhibited altered mechanical properties, such as improved elongation at break, when compared with pure silk fibroin films. Mechanistically, glycerol appears to replace water in silk fibroin chain hydration, resulting in the initial stabilization of helical structures in the films, as opposed to random coil or beta-sheet structures. The use of glycerol in combination with silk fibroin in materials processing expands the functional features attainable with this fibrous protein, and in particular, in the formation of more flexible films with potential utility in a range of biomaterial and device applications.

Material systems are needed that promote stabilization of entrained molecules, such as enzymes or therapeutic proteins, without destroying their activity. We demonstrate that the unique structure of silk fibroin protein, when assembled into the solid state, establishes an environment that is conducive to the stabilization of entrained proteins. Enzymes (glucose oxidase, lipase and horseradish peroxidase) entrapped in these films over ten months retained significant activity, even when stored at 37°C, and in the case of glucose oxidase did not lose any activity. Further, the mode of processing of the silk protein into the films could be correlated to the stability of the enzymes. The relationship between processing and stability offers a large suite of conditions within which to optimize such stabilization processes. Overall, the techniques reported here result in materials that stabilize enzymes to a remarkable extent, without the need for cryoprotectants, emulsifiers, covalent immobilization or other treatments. Further, these systems are amenable to optical characterization, environmental distribution without refrigeration, are ingestible, and offer potential use in vivo, since silk materials are biocompatible and FDA approved, degradable with proteases and currently used in biomedical devices.

No abstract

No abstract

A silk fibroin gel system (e-gel), formed with weak electric fields has potential utility in medical materials and devices. The mechanism of silk e-gel formation was studied to gain additional insight into the process and control of the material properties. Silk fibroin nanoparticles with sizes of several ten nanometers, composed of metastable conformations, were involved in e-gel formation. Under electric fields, the nanoparticles rapidly assembled into larger nano- or microspheres with size ranges from tens nanometers to several microns. Repulsive forces from the negative surface charge of the acidic groups on the protein were screened by the local decrease in solution pH in the vicinity of the positive electrode. By controlling the formation and content of silk fibroin nanoparticles e-gel could be formed even from low concentration silk fibroin solutions (1%). When e-gel was reversed to the solution state, the aggregated nano- and microspheres dispersed into solution, a significant observation related to future applications for this process, such as for drug delivery.

Besides excellent biocompatibility and biodegradability, a useful tissue engineering scaffold should provide suitable macropores and nanofibrous structure, similar to extracellular matrix (ECM), to induce desired cellular activities and to guide tissue regeneration. In the present study, a mild process to prepare porous and nanofibrous silk based scaffolds from aqueous solution is described. Using collagen to control the self-assembly of silk, nanofibrous silk scaffolds were firstly achieved through lyophilization. Water annealing was used to generate insolubility in the silk based scaffolds, thereby avoiding the use of organic solvents. The nano-fibrils formed in the silk-collagen scaffolds had diameters of 20-100 nanometers, similar with native collagen in ECM. The silk-collagen scaffolds dissolved slowly in PBS solution, with about a 28% mass lost after 4 weeks. Following the dissolution or degradation, the nanofibrous structure inside the macropore walls emerged and interacted with cells directly. During in vitro cell culture, the nanofibrous silkcollagen scaffolds containing 7.4% collagen demonstrated significantly improved cellcompatibility when compared with salt-leached silk scaffolds and silk-collagen scaffolds containing 20% collagen that emerged less nano-fibrils. Therefore, this new process provides useful scaffolds for tissue engineering applications. Furthermore, the process involves all-aqueous, room temperature and pressure processing without the use of toxic chemicals or solvents, offering new green chemistry approaches, as well as options to load bioactive drugs or growth factors into process.

Silk fibroin solution was prepared by dissolving the silk fibroin in triad solvent CaCl2 · CH3CH2OH · H2O. In this article we tested and analyzed the state of frozen silk fibroin solution and fine structure of freeze dried porous silk fibroin materials. The results indicated that the glass transition temperature of frozen silk fibroin solution ranges from −34 to −20°C, and the initial melting temperature of ice in frozen solution is about −8.5°C. When porous silk fibroin materials are prepared by means of freeze drying, if freezing temperature is below −20°C, the structure of silk fibroin is mainly amorphous with a little silk II crystal structure, and if freezing temperature is above −20°C, quite a lot of silk I crystal structure forms. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 79: 2185–2191, 2001