A study of the initial adhesive force of cells on silk fibroin-based materials using micropipette aspiration.

With the development of biomaterials, more attention is paid to the adhesion characteristics between cells and materials. It is necessary to study the adhesive force with a suitable method. Silk fibroin (SF) is widely investigated in biomedical application due to its novel biocompatibility and mechanical properties. In this article, the micropipette aspiration method and measurement pattern of uniform cells in round shape (UCR) was used to study the initial adhesive force of three types of cells on pure silk fibroin films (SFFs). We also compared the adhesive forces of modified SFFs with that of pure SFFs. The results of adhesive force in the initial adhesive stage were in concordance with the results of MTT assay and microscope observation, which were confirmed by the above three cell lines and four kinds of SFFs. The results indicated UCR was an efficient and quantitative measurement pattern in initial adhesion stage. This article also provides a useful method in identifying initial cell-materials interactions.

Structurally Well-Defined Au@Cu2- x S Core-Shell Nanocrystals for Improved Cancer Treatment Based on Enhanced Photothermal Efficiency.

Au@Cu2- x S core-shell nanocrystals (NCs) have been synthesized under large lattice mismatch with high crystallinity, controllable shape, and nonstoichiometric composition. Both experimental observations and simulations are used to verify the flexible dual-mode plasmon coupling. The enhanced photothermal effect is harnessed for diverse HeLa cancer cell ablation applications in the NIR-I window (750-900 nm) and the NIR-II window (1000-1400 nm).

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.

Heterovalent-Doping-Enabled Efficient Dopant Luminescence and Controllable Electronic Impurity Via a New Strategy of Preparing II-VI Nanocrystals.

Substitutional heterovalent doping represents an effective method to control the optical and electronic properties of nanocrystals (NCs). Highly monodisperse II-VI NCs with deep substitutional dopants are presented. The NCs exhibit stable, dominant, and strong dopant fluorescence, and control over n- and p-type electronic impurities is achieved. Large-scale, bottom-up superlattices of the NCs will speed up their application in electronic devices.

Phosphine-initiated cation exchange for precisely tailoring composition and properties of semiconductor nanostructures: old concept, new applications.

Phosphine-initiated cation exchange is a well-known inorganic chemistry reaction. In this work, different phosphines have been used to modulate the thermodynamic and kinetic parameters of the cation exchange reaction to synthesize complex semiconductor nanostructures. Besides preserving the original shape and size, phosphine-initiated cation exchange reactions show potential to precisely tune the crystallinity and composition of metal/semiconductor core-shell and doped nanocrystals. Furthermore, systematic studies on different phosphines and on the elementary reaction mechanisms have been performed.

Silk-regulated hierarchical hollow magnetite/carbon nanocomposite spheroids for lithium-ion battery anodes.

Hierarchical olive-like structured carbon-Fe3O4 nanocomposite particles composed of a hollow interior and a carbon coated surface are prepared by a facile, silk protein-assisted hydrothermal method. Silk nanofibers as templates and carbon precursors first regulate the formation of hollow Fe2O3 microspheres and then they are converted into carbon by a reduction process into Fe3O4. This process significantly simplifies the fabrication and carbon coating processes to form complex hollow structures. When tested as anode materials for lithium-ion batteries, these hollow carbon-coated particles exhibit high capacity (900 mAh g(-1)), excellent cycle stability (180 cycles) and rate performance due to their unique hierarchical hollow structure and carbon coating.

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.

Osteoinductive-nanoscaled silk/HA composite scaffolds for bone tissue engineering application.

Osteoinductive silk/hydroxyapatite (HA) composite scaffolds for bone regeneration were prepared by combining silk with HA/silk core-shell nanoparticles. The HA/silk nanoparticles were directly dispersed in silk solution to form uniform silk/HA blend and then composite scaffolds after a freeze-drying process. The HA/silk nanoparticles uniformly distributed in silk scaffolds at nanometer scale at varying HA content up to 40%, and substantially improved the compressive strength of the scaffolds produced. Rat bone mesenchymal stem cells (rBMSCs) were cultured in these scaffolds and cell proliferation was analyzed by confocal microscopy and DNA assay. Gene expression and biochemical assays were employed to study the influence of increasing HA/silk nanoparticles on in vitro osteogenic differentiation of rBMSCs. Increasing HA/silk nanoparticles inside silk scaffolds improved the growth and osteogenic capability of rBMSCs in the absence of osteogenic growth factors, and also significantly increased the calcium and collagen I deposition. In addition, compared to silk/HA composite scaffolds containing HA aggregates, the scaffolds loaded with HA/silk nanoparticles showed remarkably higher stiffness and better osteogenic property at same HA content, implying a preferable microenvironment for rBMSCs. These results suggest that the osteogenic property as well as mechanical property of silk/HA scaffolds could be further improved through fabricating their structure and topography at nanometer scale, providing more suitable systems for bone regeneration.

A mild process to design silk scaffolds with reduced ß-sheet structure and various topographies at the nanometer scale.

Three-dimensional (3-D) porous silk scaffolds with good biocompatibility and minimal immunogenicity show promise in a range of tissue regeneration applications. However, the challenge remains to effectively fabricate their microstructures and mechanical properties to satisfy the specific requirements of different tissues. In this study, silk scaffolds were fabricated to form an extracellular matrix (ECM) mimetic nanofibrous architecture using a mild process. A slowly increasing concentration process was applied to regulate silk self-assembly into nanofibers in aqueous solution. Then glycerol was blended with the nanofiber solution and induced silk crystallization in the lyophilization process, endowing freeze-dried scaffolds with water stability. The glycerol was leached from the scaffolds, leaving a similar porous structure at the micrometer scale but different topographies at the nanoscale. Compared to previous salt-leached and methanol-annealed scaffolds, the present scaffolds showed lower ß-sheet content, softer mechanical property and improved cell growth and differentiation behaviors, suggesting their promising future as platforms for controlling stem cell fate and soft tissue regeneration.

Silk Nanofiber Hydrogels with Tunable Modulus to Regulate Nerve Stem Cell Fate.

Reconstruction of damaged nerves remains a significant unmet challenge in clinical medicine. To foster improvements, the control of neural stem cell (NSC) behaviors, including migration, proliferation and differentiation are critical factors to consider. Topographical and mechanical stimulation based on the control of biomaterial features is a promising approach, which are usually studied separately. The synergy between topography and mechanical rigidity could offer new insights into the control of neural cell fate if they could be utilized concurrently in studies. To achieve this need, silk fibroin self-assembled nanofibers with a beta-sheet-enriched structure are formed into hydrogels. Stiffness is tuned using different annealing processes to enable mechanical control without impacting the nanofiber topography. Compared with nonannealed nanofibers, NSCs on methanol annealed nanofibers with stiffness similar to nerve tissues differentiate into neurons with the restraint of glial differentiation, without the influence of specific differentiation biochemical factors. These results demonstrate that combining topographic and mechanical cues provides the control of nerve cell behaviors, with potential for neurogenerative repair strategies.

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.

Nanoscale Control of Silks for Nanofibrous Scaffold Formation with Improved Porous Structure.

Silk-based porous scaffolds have been used extensively in tissue engineering because of their excellent biocompatibility, tunable biodegradability and robust mechanical properties. Although many silk-based scaffolds have been prepared through freeze-drying, a challenge remains to effectively control porous structures during this process. In the present study silk fibroin with different nanostructures were self-assembled in aqueous solution by repeated drying-dissolving process and then used to improve porous structure formation in lyophilization process. Viscosity, secondary structures and water interactions were also studied to exclude their influence on the formation and control of porous structures. Following nanofiber formation in aqueous solution, silk scaffolds with improved porous structure were directly formed after lyophilization and then stabilized with water or methanol annealing treatments. Compared to silk scaffolds derived from fresh solution, the nanofibrous scaffolds showed significantly better cell compatibility in vitro. Therefore, this nanoscale control of silk offers feasible way to regulate the matrix features including porous structure and nanostructure, which are important in regulating cell and tissue outcomes in tissue engineering and regeneration, and then achieve silk-based scaffolds with improved properties.

Biomineralization regulation by nano-sized features in silk fibroin proteins: synthesis of water-dispersible nano-hydroxyapatite.

In the present study, silk fibroin (SF) was used as a template to prepare nano-hydroxyapatite (nano-HA) via a biomineralization process. We observed that the content of SF affected both the morphology and water dispersibility of nano-HA particles. Scanning electron microscope (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), zetasizer, and Fourier transform infrared spectroscopy (FTIR) were used to examine nano-HA particle features including the surface morphology, aggregation performance, and crystallization. Rod-like nano-HA particles with desired water dispersibility were achieved when the ratio of SF/HA (calculated) was above 7:3. SEM, TEM, and zeta potential results revealed that nano-HA particles were enclosed by the SF which formed a negative charge layer preventing the aggregation of HA nanoparticles in aqueous solution. Moreover, the nano-HA particles were able to re-disperse in water without precipitation for two weeks at room temperature, 60°C, and 90°C. Our work suggested a facile and effective approach of designing water-dispersible nano-HA particles which may have wide potential application in tissue engineering especially bone regeneration.

Controlling structural symmetry of a hybrid nanostructure and its effect on efficient photocatalytic hydrogen evolution.

The existence of lattice strain between two different materials can be used to control the fine structural configuration in a hybrid colloidal nanostructure. Enabled by such, the relative position change of Au and CdX in Au-CdX from a symmetric to an asymmetric configuration is demonstrated, which can further lead to fine tuning of plasmon-exciton coupling and different hydrogen photocatalytic performance. These results provide new insight into plasmon enhanced photocatalytic mechanisms and provide potential catalysts for photoreduction reactions.

One-step synthesis of biocompatible magnetite/silk fibroin core-shell nanoparticles.

A one-step hydrothermal process with silk fibroin (SF) nanofibers as the template and coating was developed to synthesize core-shell magnetite/SF nanoparticles with limited controllable sizes. The Fe3O4 nanoparticles gradually aggregated into nanospheres with sizes increased from 120 to 500 nm by increasing the SF content in the reaction system. The magnetic properties and biocompatibility of Fe3O4/SF nanoparticles, as well as their functional ability with antibodies are also discussed to assess their possible applications in MRI and bio-separation. Compared to previous two-step processes, our one-step method provides a simpler and more cost-effective approach to prepare biocompatible core-shell magnetite nanoparticles.

Bilayered vascular grafts based on silk proteins.

A major block in the development of small diameter vascular grafts is achieving suitable blood vessel regeneration while minimizing the risk of thrombosis, intimal hyperplasia, suture retention, and mechanical failure. Silk-based tubular vessels for tissue engineering have been prepared by molding, dipping, electrospinning, or gel spinning, however, further studies are needed to improve the mechanical and blood compatibility properties. In the present study a bilayered vascular graft based on silk fibroin (SF) was developed. The graft was composed of an inner silk fiber-reinforced SF tube containing heparin and a highly porous SF external layer. Compared with previously fabricated SF tubes the fiber-reinforcement provided a comparable or higher mechanical strength, burst pressure, and suture retention strength, as well as mechanical compliance, to saphenous veins for vascular grafts. Heparin release was sustained for at least 1 month, affording blood compatibility to the grafts. The outer layer of the grafts prepared through lyophilization had a highly porous structure in which the macropore walls were composed of nanofibers similar to extracellular matrix, which offered an excellent environment for cell growth. In vitro studies showed good cytocompatibility and hemocompatibility.

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.

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.

Flexibility regeneration of silk fibroin in vitro.

Although natural silk fibers have excellent strength and flexibility, the regenerated silk materials generally become brittle in the dry state. How to reconstruct the flexibility for silk fibroin has bewildered scientists for many years. In the present study, the flexible regenerated silk fibroin films were achieved by simulating the natural forming and spinning process. Silk fibroin films composed of silk I structure were first prepared by slow drying process. Then, the silk fibroin films were stretched in the wet state, following the structural transition from silk I to silk II. The difference between the flexible film and different brittle regenerated films was investigated to reveal the critical factors in regulating the flexibility of regenerated silk materials. Compared with the methanol-treated silk films, although having similar silk II structure and water content, the flexible silk films contained more bound water rather than free water, implying the great influence of bound water on the flexibility. Then, further studies revealed that the distribution of bound water was also a critical factor in improving silk flexibility in the dry state, which could be regulated by the nanoassembly of silk fibroin. Importantly, the results further elucidate the relation between mechanical properties and silk fibroin structures, pointing to a new mode of generating new types of silk materials with enhanced mechanical properties in the dry state, which would facilitate the fabrication and application of regenerated silk fibroin materials in different fields.

Silk self-assembly mechanisms and control from thermodynamics to kinetics.

Silkworms and spiders generate fibers that exhibit high strength and extensibility. The underlying mechanisms involved in processing silk proteins into fiber form remain incompletely understood, resulting in the failure to fully recapitulate the remarkable properties of native fibers in vitro from regenerated silk solutions. In the present study, the extensibility and high strength of regenerated silks were achieved by mimicking the natural spinning process. Conformational transitions inside micelles, followed by aggregation of micelles and their stabilization as they relate to the metastable structure of silk are described. Subsequently, the mechanisms to control the formation of nanofibrous structures were elucidated. The results clarify that the self-assembly of silk in aqueous solution is a thermodynamically driven process where kinetics also play a key role. Four key factors, molecular mobility, charge, hydrophilic interactions, and concentration underlie the process. Adjusting these factors can balance nanostructure and conformational composition, and be used to achieve silk-based materials with properties comparable to native fibers. These mechanisms suggest new directions to design silk-based multifunctional materials.