Reversible hydrogel-solution system of silk with high beta-sheet content.

Silkworm silk has been widely used as a textile fiber, as biomaterials and in optically functional materials due to its extraordinary properties. The ß-sheet-rich natural nanofiber units of about 10-50 nm in diameter are often considered the origin of these properties, yet it remains unclear how silk self-assembles into these hierarchical structures. A new system composed of ß-sheet-rich silk nanofibers about 10-20 nm in diameter is reported here, where these nanofibers formed into "flowing hydrogels" at 0.5-2% solutions and could be transformed back into the solution state at lower concentrations, even with a high ß-sheet content. This is in contrast with other silk processed materials, where significant ß-sheet content negates reversibility between solution and solid states. These fibers are formed by regulating the self-assembly process of silk in aqueous solution, which changes the distribution of negative charges while still supporting ß-sheet formation in the structures. Mechanistically, there appears to be a shift toward negative charges along the outside of the silk nanofibers in our present study, resulting in a higher zeta potential (above -50 mV) than previous silk materials which tend to be below -30 mV. The higher negative charge on silk nanofibers resulted in electrostatic repulsion strong enough to negate further assembly of the nanofibers. Changing silk concentration changed the balance between hydrophobic interactions and electrostatic repulsion of ß-sheet-rich silk nanofibers, resulting in reversible hydrogel-solution transitions. Furthermore, the silk nanofibers could be disassembled into shorter fibers and even nanoparticles upon ultrasonic treatment following the transition from hydrogel to solution due to the increased dispersion of hydrophobic smaller particles, without the loss of ß-sheet content, and with retention of the ability to transition between hydrogel and solution states through reversion to longer nanofibers during self-assembly. These reversible solution-hydrogel transitions were tunable with ultrasonic intensity, time, or temperature.

Salt-leached silk scaffolds with tunable mechanical properties.

Substrate mechanical properties have remarkable influences on cell behavior and tissue regeneration. Although salt-leached silk scaffolds have been used in tissue engineering, applications in softer tissue regeneration can be encumbered with excessive stiffness. In the present study, silk-bound water interactions were regulated by controlling processing to allow the preparation of salt-leached porous scaffolds with tunable mechanical properties. Increasing silk-bound water interactions resulted in reduced silk II (ß-sheet crystal) formation during salt-leaching, which resulted in a modulus decrease in the scaffolds. The microstructures as well as degradation behavior were also changed, implying that this water control and salt-leaching approach can be used to achieve tunable mechanical properties. Considering the utility of silk in various fields of biomedicine, the results point to a new approach to generate silk scaffolds with controllable properties to better mimic soft tissues by combining scaffold preparation methods and silk self-assembly in aqueous solutions.

The biocompatibility of silk fibroin films containing sulfonated silk fibroin.

Sulfonation reaction may be an effective method for preparation of heparin-like materials. However, no sulfonated polymer based on protein backbone was used for improving the blood compatibility of biomaterials. In this study, the biocompatibility of new kind of composite materials films obtained by blending silk fibroin (SF) with sulfonated silk fibroin (SSF) was evaluated. The anticoagulant activity was characterized with prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT), which all increased remarkably, the clot times exceeded the measurement limit of clot detection instrument. Its platelet adhesion was also investigated as another parameter of blood compatibility. The cell compatibility of composite films was evaluated through cell morphologies on the films and cell viability by methyl thiazolyl tetrazolium (MTT) assay. Tensile strength and elongation at break of the composite films reached to 44.6 MPa and 50.3%, respectively. All these results indicated that SF/SSF composite film was a potential material for blood-contact materials and tissue engineering matrix.

Silk scaffolds with tunable mechanical capability for cell differentiation.

Bombyx mori silk fibroin is a promising biomaterial for tissue regeneration and is usually considered an "inert" material with respect to actively regulating cell differentiation due to few specific cell signaling peptide domains in the primary sequence and the generally stiffer mechanical properties due to crystalline content formed in processing. In the present study, silk fibroin porous 3D scaffolds with nanostructures and tunable stiffness were generated via a silk fibroin nanofiber-assisted lyophilization process. The silk fibroin nanofibers with high ß-sheet content were added into the silk fibroin solutions to modulate the self-assembly, and to directly induce water-insoluble scaffold formation after lyophilization. Unlike previously reported silk fibroin scaffold formation processes, these new scaffolds had lower overall ß-sheet content and softer mechanical properties for improved cell compatibility. The scaffold stiffness could be further tuned to match soft tissue mechanical properties, which resulted in different differentiation outcomes with rat bone marrow-derived mesenchymal stem cells toward myogenic and endothelial cells, respectively. Therefore, these silk fibroin scaffolds regulate cell differentiation outcomes due to their mechanical features.

Biomineralization of stable and monodisperse vaterite microspheres using silk nanoparticles.

The influence of silk fibroin (SF) on calcium carbonate (CaCO3) biomineralization has been investigated; however, the formation of small, uniform SF-regulated vaterite microspheres has not been reported. In this work, spherical CaCO3 was synthesized via coprecipitation in the presence of SF. SF nanostructures were first tuned by self-assembly at 60 °C to provide better control of the nucleation of CaCO3. Subsequently, monodisperse vaterite microspheres about 1.1 µm were generated by controlling aggregation and growth of CaCO3 under appropriate concentrations of SF and Ca ions. In contrast to unstable vaterite, the microspheres generated in the present study have sufficient stability in aqueous solution for at least 8 days without transformation into calcite, due to the electrostatic interactions between the Ca ions and the preassembled SF nanostructures. The microspheres as drug carriers of doxorubicin (DOX) were assessed and found to have good encapsulation efficiency, sustained drug release without burst release, and pH sensitivity. These new SF/CaCO3 hybrids may provide new options for various biomedical applications.

A novel solvent system for blending of polyurethane and heparin.

To improve the blood-compatibility of polyurethane, the co-solvent of tetrahydrofuran and water, a new solvent system for blending polyurethane and heparin, was proposed. After solvent casting, heparin was blended in a polyurethane film. The ATR-FTIR was used to analyze the surface chemical element and the contact angle was measured to investigate the hydrophilicity of the surface of the PUs. As the amount of heparin increased, the surface hydrophilicity was increased and all the clot times exceeded the measurement limit of the clot detection instrument when the heparin loaded on the polyurethane films was 3%, 5% and 7%. After the films were immersed in the phosphate buffered saline for 30 days, the activated partial thromboplastin time and thrombin time still exceeded the measurement limit of the clot detection instrument.

Hierarchical biomineralization of calcium carbonate regulated by silk microspheres.

As an analog of the main protein contained in nacre regenerated Bombyx mori silk fibroin has a significant influence on the morphology and polymorphic nature of CaCO3 in the biomineralization process. A number of studies have implied that the self-assembling aggregate structure of silk fibroin is a key factor in controlling CaCO3 aggregation. Further insight into this role is necessary with a particular need to prepare silk fibroin aggregates with homogeneous structures to serve as templates for the mineralization process. Here we have prepared homogeneous silk microspheres to serve as templates for CaCO3 mineralization in order to provide an experimental insight into silk-regulated crystallization processes. CaCO3 particles with different nano- and microstructures, and their polymorphs, were successfully formed by controlling the mineralization process. The key function of silk aggregation in controlling the morphology and polymorphic nature of CaCO3 particles was confirmed. A regulating effect of silk on the spatial features was also observed. A two-step process for silk fibroin-regulated biomineralization was found, with different levels of heterogeneous nucleation and aggregation. A full understanding of silk fibroin-regulated biomineralization mechanisms would help in understanding the function of organic polymers in natural biomineralization, and provide a way forward in the design and synthesis of new materials in which organic-inorganic interfaces are the keys to function, biological interfaces and many related material features.

Nanofibrous architecture of silk fibroin scaffolds prepared with a mild self-assembly process.

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 nm, 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 silk-collagen scaffolds containing 7.4% collagen demonstrated significantly improved cell compatibility 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.

Study on the preparation of collagen-modified silk fibroin films and their properties.

Blended films were prepared from a silk fibroin (SF) solution by adding a small amount of type I collagen (<5%). The mechanical properties of the wet films modified by collagen were improved obviously. The elongation at break reached 42%, and the smaller contact angles revealed that modified films had better hydrophilicity. 1% heparin was also added to modify the SF films to further improve the in vitro antithrombogenecity. The internal structure of the modified SF films was investigated with scanning electron microscopy, x-ray diffraction and Fourier transform infrared attenuated total reflection spectroscopy. The result indicates that the addition of a small amount of collagen and heparin did not change their conformation.

Blood compatibility of polyurethane immobilized with acrylic acid and plasma grafting sulfonic acid.

Sulfonic and carboxyl groups can effectively improve the blood compatibility of polyurethane. But it is difficult to obtain an optimum ratio of the two groups. In this article, polyurethane (PU) was dissolved with acrylic acid in a tetrahydrofuran solution and then spread on the glass plate to produce a film. At the same time, acrylic acid partly polymerized and immobilized with the PU films. The films (PU-AA) were exposed to sulfur dioxide plasma to graft sulfonic acid group on its surfaces. Through adjusting the quantity of acrylic acid and the plasma reaction condition, the antithrombin of polyurethane can be improved. The surface-modified PUs were characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscope (XPS) and a contact angle goniometer. The blood compatibility of the films was examined by using thrombin time (TT), activated partial thromboplastin time (APTT) and prothrombin time (PT). The TT and APTT were significantly prolonged for the surface-modified films of PU-AA by sulfur dioxide plasma and only APTT was elongated for PU-AA. The results suggest that sulfonic acid and acrylic acid have the different effect on the blood compatibility of surface-modified PUs.

The preparation of insoluble fibroin films induced by degummed fibroin or fibroin microspheres.

Blending degummed fibroin (DF) or insoluble fibroin microspheres with concentrated fibroin solution, the insoluble films were obtained through drying the solution at 40-45 degrees C. The conformation of silk fibroin films was analyzed by infrared spectrum and X-ray diffractometry. The results demonstrated that beta-sheet conformation increased rapidly when the degummed silk or insoluble microspheres blended with fibroin, while the pure SF membrane was mainly composed of alpha/random coil conformation when the other conditions kept same. This suggested that fibroin microspheres and degummed fibroin could induce the formation of beta-sheet crystal and the insoluble films, without methanol after-treatment, could be obtained at approximately room temperature. Although the fibroin films blending with DF had many protuberances, the films containing fibroin microspheres had the smooth surface and could be used effectively in biotechnological materials and biomedical application.