Silk Films Research Articles

Morphological control and assembly of zinc oxide using a biotemplate

Zinc oxide is a wide band gap material that has significant applications in photovoltaics, piezoelectrics and optoelectronics. Traditionally, ZnO has been synthesized using high temperatures and harsh reaction conditions. Recently, benign reaction conditions have been used to synthesize ZnO using amine and citrate additives. In this study, peptide phage display was performed to identify a peptide, termed Z1, that binds to and directs the growth of ZnO hexagonal nanocrystals. By altering the concentration of Z1 peptide, the ZnO nanocrystal morphology can be tailored. Additionally, Z1 peptide was used to direct the growth of ZnO structures on free-standing silk films. The results presented here demonstrate the utility of peptides in controlling the structure and deposition of ZnO.

Processing methods to control silk fibroin film biomaterial features

Control of silk structural and morphological features is reported for fibroin protein films via all aqueous processing, with and without polyethylene oxide (PEO). Silk films with thicknesses from 500 nm to 50 μm were generated with controllable surface morphologies by employing soft-lithography surface patterning or by adjusting PEO concentrations. FTIR analysis indicated that water-annealing, used to cure or set the films, resulted in increased β-sheet and α-helix content within the films. Steam sterilization provided an additional control point by increasing β-sheet content, while reducing random coil and turn structures, yet retaining film transparency and material integrity. Increased PEO concentration used during processing resulted in decreased sizes of surface globule structures, while simultaneously increasing uniformity of these features. The above results indicate that both surface and bulk morphologies and structures can be controlled by adjusting PEO concentration. The combined tool set for controlling silk film geometry and structure provides a foundation for further study of novel silk film biomaterial systems. These silk film biomaterials have potential applicability for a variety of optical and regenerative medicine applications due to their optical clarity, impressive mechanical properties, slow degradability, and biocompatibility.

Radiologic and histologic characterization of silk fibroin as scaffold coating for rabbit tracheal defect repair

To explore the feasibility of silk fibroin as a biomaterial coating used in tracheal defect reconstruction. Silk fibroin was subcutaneously embedded in three rabbits and as an artificial implant material coating to reconstruct 12 rabbits' tracheal defects. The postoperative radiologic and histologic characterization was summarized. The thickness of the fibroblasts layer covering the porus and nonporus silk fibroin film was 240.4 +/- 9.9 and 302.3 +/- 10.5 microm, respectively, and there was no statistical difference (P > 0.05) within each group. There was no foreign-body granuloma or macrophagocyte infiltration around the silk film. The tracheal reconstruction study showed a normal mucous membrane with normal cilial growth on the artificial implant and no visible granulation tissue in the reconstructed tracheal cavity. Silk fibroin is a potential new biomaterial coating for tracheal defect reconstruction. The mechanism of silk fibroin appears to promote tracheal mucous membrane, which may be related to its molecular structure and biology.

Anisotropic growth of hydroxyapatite on the silk fibroin films

Bombyx mori silk fibroin is of practical interest for its excellent intrinsic properties utilizable in the biotechnological and biomedical fields. Here, the silk fibroin films were pretreated with different methods and then used as the template for the hydroxyapatite (HA) crystal growth. To study the effect of silk films’ surface structure on the protein biomineralization, the films were immersed into 1.5 times simulated body fluid (1.5 × SBF) to induce the HA deposition at 37 °C. The results showed that an anisotropic growth of HA crystals was observed on the different films as judging from XRD, TEM and HRTEM data. This was thought that the positions and density of carboxyl groups, C O and amino groups on the surface of SF films may be different, which play the key effect on HA crystal growth.

SILK FIBROIN FILMS CRYSTALLIZED BY MULTIWALLED CARBON NANOTUBES

Silk films prepared from regenerated silk fibroin are normally stabilized by β-sheet formation through the use of solvents (methanol, water etc.). Herein, we report a new method of preparing water-stable films without a β-sheet conformation from regenerated silk fibroin solutions by incorporating a small amount (0.2 wt%) of multiwalled carbon nanotubes (MWCNTs). To extend the biomaterial utility of silk proteins, forming water-stable silk-based materials with enhanced mechanical properties is essential. Scanning electron microscopy and transmission electron microscopy were used to observe the morphology of the MWCNT-incorporated silk films. The wide-angle X-ray diffraction provided clear evidence of the crystallization of the silk fibroin induced by MWCNT in the composite films without any additional annealing processing. The tensile modulus and strength of the composite films were improved by 108% and 51%, respectively, by the incorporation of 0.2 wt% of MWCNTs, as compared with those of the pure silk films. The method described in this study will provide an alternative means of crystallizing silk fibroin films without using an organic solvent or blending with any other polymers, which may be important in biomedical applications.

Bioactive Silk Protein Biomaterial Systems for Optical Devices

Silk-based biomaterial systems have been previously explored for a variety of medical and nonmedical materials needs. The unique biophysical features of silks provide options to generate highly tailored structures and morphologies with this unique family of fibrous proteins. To exploit these features, we have optimized the all aqueous processing of silk fibroin into novel surface nanopatterned protein materials. We have exploited control of this nanomorphology to optimize the optical features of these silk protein systems. We demonstrate control of surface morphology down to 125 nm, with fidelity over large length scales. This surface nanopatterning allows the silk protein to be formed into diffractive optics such as diffraction gratings, pattern generators, and lenses due to novel aqueous processing into optically clear materials via control of beta sheet crystallinity. Further, we incorporate biological components, such as hemoglobin and the enzyme peroxidase, during the process of forming the silk diffraction gratings. The ambient processing of the silk protein in water, in combination with these bioactive components, allows these entrained molecules to retain activity and provide added functions and selectivity to the optically active silk films. Thus, combinations of biochemical and optical readout is feasible and provides in a single, disposable/all degradable element with both spectral discrimination and biological function. These new surface nanopatterned, bioactive silk protein-based material systems offer a unique combination of features potentially useful for a range of biosensor needs, particularly when considered in concert with the remarkable mechanical properties of these proteins, their biocompatibility, and controllable biodegradation.

Characterization and optimization of RGD-containing silk blends to support osteoblastic differentiation

The effect of blending two silk proteins, regenerated Bombyx mori fibroin and synthetic spidroin containing RGD, on silk film material structure (β-sheet content) and properties (solubility), as well as on biological response (osteoblast adhesion, proliferation and differentiation) was investigated. Although the elasticity and strength of silks make them attractive candidates for bone, ligament, and cartilage tissue engineering applications, silk proteins generally lack bioactive peptides for enhancing cell functions. Thus, a synthetic spider silk, spidroin, containing two RGD cell adhesive sequences (RGD-spidroin) was engineered. RGD-spidroin was blended with different ratios of fibroin and spun coat into films on glass coverslips. β-Sheet formation, contact angle, surface topography and RGD surface presentation were characterized and correlated with cell behavior. We found that the amount of β-sheet formation was directly related to the RGD-spidroin content of the blends after annealing, with the pure RGD-spidroin demonstrating the highest amount of β-sheet content. The increased β-sheet content improved film stability under culture conditions. A new visualization technique demonstrated that the RGD presentation on the film surface was affected by both the RGD-spidroin content and annealing conditions. It was determined that 10 mass% RGD-spidroin was necessary to improve film stability and to achieve osteoblast attachment and differentiation.

Non-invasive optical characterization of biomaterial mineralization

Current approaches to study biomaterial mineralization are invasive and prevent dynamic characterization of this process within the same sample. Polarized light scattering spectroscopy (LSS) may offer a non-invasive alternative for assessing the levels of mineralization as well as some aspects of the organization of the mineral deposits. Specifically, we used LSS to characterize the formation of hydroxyapatite deposits on three types of silk films (water-annealed, methanol-treated and polyaspartic acid (PAA)-mixed) following 1, 3, 5 and 7 cycles of mineralization. We found that the total light scattering intensity provided a quantitative measure of the degree of mineralization as confirmed by thermal gravimetric analysis (TGA). The PAA-mixed silk films yielded the highest level of mineral deposition and the water-annealed ones the least, consistent with the β sheet content of the films prior to the onset of mineralization. The wavelength dependence of the singly backscattered light was consistent with a self-affine fractal morphology of the deposited films within scales in the range of 150–300nm; this was confirmed by Fourier analysis of scanning electron microscopy (SEM) images of the corresponding films. The deposits of minerals in the water-annealed films were predominantly flake-like, with positively correlated density fluctuations (Hurst parameter, H>0.5), whereas methanol-treated and PAA-mixed silk films resulted in densely-packed, bulk mineral deposits with negatively correlated density fluctuations (H<0.5). Therefore, LSS could serve as a valuable tool for understanding the role of biomaterial properties in mineral formation, and, ultimately, for optimizing biomaterial designs that yield mineral deposits with the desired organization.

Improving Cell-Adhesive Properties of Recombinant Bombyx mori Silk by Incorporation of Collagen or Fibronectin Derived Peptides Produced by Transgenic Silkworms

Silk of Bombyx mori can be used as various biomaterials. Especially, it is useful as a protein for coating the surface of cell culture plates since the silk possesses a biocompatibility to the cultured cells. However, the cell-adhesive ability is weaker than collagen or fibronectin, which are used for coating the plate more frequently (Yao et al. J. Biochem., 2004, 136, 643-649). To increase the biocompatibility of the silk, we constructed transgenic silkworms, inserting the modified fibroin light-chain genes for making recombinant silks that possessed partial collagen or fibronectin sequences, that is, [GERGDLGPQGIAGQRGVV(GER)3GAS]8GPPGPCCGGG or [TGRGDSPAS]8, respectively. Films were made from the recombinant silks, and the cell-adhesive activity for cultured mammalian cells was observed. The results showed that the two types of recombinant silk films possessed a much higher cell-adhesive activity as compared to the original unmodified silk. Especially, the recombinant silk with the sequence [TGRGDSPAS]8, produced by a transgenic Nd-sD mutant, gave a 6 times higher activity than the original unmodified silk.

Silk Fibroin Microfluidic Devices.

Silk Fibroin Microfluidic Devices.

Observation of Proliferation and Attachment of Three Different Cell Types on Nano-Fibrous Silk Film &amp; Scaffold

Attachment and viability of different cell types(fibrioblast, chondrocyte and osteoblast ) was observed on two forms of silk (mat &amp; Three-dimensional scaffolds). The osteoblasts behaviors cultured on silk mat were significantly higher than that found on 3-D silk fibroin scaffold (3-D SF scaffold). In the MTT assay, the cell viability of fibroblasts, chondrocyte and osteoblasts seeded on 2-D nanofiber mat was (2-D mat) significantly higher than that found on 3-D SF scaffold. Similar result could be seen from SEM observation and cell attachment study. However, alkaline phosphatase activity was significantly increased on 3-D SF scaffold than on2-D nanofiber

A new micro-tensile system for measuring the mechanical properties of low-dimensional materials—Fibers and films

A new micro-tensile system has been developed to evaluate the mechanical properties of low-dimensional materials, such as fibers and films. The proposed system uses a magnet-coil force actuator to apply a tensile load on the material and a digital image correlation (DIC) method to measure the corresponding displacement and strain of the material. The system combines a supporting frame, a hanging spring, a magnet-coil, a permanent magnet, two specimen clamps, an adjustable stage and an image acquisition unit. To verify the proposed technique, experiments were conducted on two polymeric materials, silkworm silk and polyethylene oxide film containing nano-composite SiO 2 (15%), to demonstrate the effectiveness of the system.

Nano-TiO2 induced secondary structural transition of silk fibroin studied by two-dimensional Fourier-transform infrared correlation spectroscopy and Raman spectroscopy

By sol–gel processing, regenerated nano-TiO2/SF (silk fibroin) composite films were synthesized. The experimental results revealed that the nano-TiO2 particles were well dispersed in the regenerated silk fibroin. Using FT-IR and Raman spectroscopy, the secondary structures of these composite films with concentrations of 0, 0.2, 0.4, 0.8 and 1.6 wt% were characterized. Concentration-perturbed two-dimensional (2D) correlation spectra were calculated for the spectra in the 1800–1600 cm−1 region. To investigate nano-TiO2 particles induced changes in the secondary structure and hydration, the slice spectra were calculated from the synchronous and asynchronous spectra, respectively. The transmittance IR and Raman spectra measurement indicated that the secondary structure of the pure silk film was mostly random coil and α-helix, while the composite films were β-sheet. With increasing nano-TiO2 content, the secondary structure of composite films was changed from typical Silk I to typical Silk II. However, it was found that the transition of the SF's secondary structures would be restrained by excessive nano-TiO2 (over 0.8%) introduced into the composite SF films. Through the FT-IR absorbance and 2D correlation spectra, it was demonstrated that the formation of nano-TiO2 particles could induce the partial transformation of SF conformation from Silk I to Silk II.

Effect of water on the thermal properties of silk fibroin

Silk fibroin films cast from water solution, and containing bound water, are quantitatively studied in this work. First, to obtain the solid and liquid heat capacities of the pure dry silk fibroin, cyclic heat treatment was used to monitor the process of removing the bound water. After water removal, the glass transition of pure non-crystalline silk was observed at 451 K (178 °C). The solid and liquid heat capacities of the pure silk fibroin were then measured using differential scanning calorimetry (DSC), temperature-modulated DSC (TMDSC), and quasi-isothermal TMDSC, and found to be: C p ( T) solid = 0.134 + 3.696 × 10 −3 T J/g K and C p ( T) liquid = 0.710 + 3.47 × 10 −3 T J/g K over the temperature region from 200 to 450 K. These heat capacities were used to construct the underlying baseline heat capacity for the combined silk–water system. When the combined silk–water system is studied, bound water is lost from the film during heating, and the loss of mass is quantified using thermogravimetric analysis (TGA). Bound water in the silk film acts as a plasticizer, and a lower glass transition of the silk–water system is observed. Comparison of the measured heat capacity of the silk–water system to the calculated total baselines was made in the vicinity of the water-induced glass transition. Results show that the total solid specific heat capacity is in good agreement with the calculated solid baseline in the low-temperature region below about 240 K. As temperature increases above the lower glass transition, all bound water eventually leaves the silk, and the free volume and the silk mobility are reduced. This allows the upper glass transition of the dried silk to be observed.

Patterned Silk Films Cast from Ionic Liquid Solubilized Fibroin as Scaffolds for Cell Growth

Silk is an attractive biomaterial for use in tissue engineering applications because of its slow degradation, excellent mechanical properties, and biocompatibility. In this report, we demonstrate a simple method to cast patterned films directly from silk fibroin dissolved in an ionic liquid. The films cast from the silk ionic liquid solution were found to support normal cell proliferation and differentiation. The versatility of the silk ionic liquid solutions and the ability to process large amounts of silk into materials with controlled surface topography directly from the dissolved silk ionic liquid solution could enhance the desirability of biomaterials such as silk for a variety of applications.

Regenerated spider silk as a new biomaterial for MEMS

Spider drag-line silk is introduced for the first time as a new biomaterial for Micro-Electro-Mechanical Systems (MEMS). The tasks accomplished in this paper were focused on mechanical characterization of regenerated spider silk under two conditions: (1) spin-coated thin film formed onto a silicon substrate; and (2) formation of a free-standing microbridge (800 x 800 x 40 microm3) obtained by a surface micromachining process. Micromechanical tests using a nano indentation machine showed the spider silk film having an elastic modulus of 7.3 GPa, a loss tangent of 0.044 and an UTS (Ultimate Tensile Strength) of 85.1 MPa.

Structural Analysis of Spider Silk Films

Due to their outstanding mechanical properties, spider silks have fascinated men for a long time. Silk is composed of proteins which can not only be processed into fibers, as found in nature, but also be cast to form films in vitro. Starting with a protein solution in hexafluoroisopropanol, we were able to cast films with different properties deviated from the two spider silk proteins employed. All as-cast films revealed an α-helixrich structure and were water soluble. However, the secondary structure of One-protein films (silk films cast from one spider silk protein) could be converted from an α-helical rich to a β-sheet rich secondary structure by post-cast treatment with methanol or potassium phosphate. The structural conversion was accompanied by a higher stability as seen by water-insolubility. Depending on the employed proteins, silk films were stable in protein denaturants such as urea and guanidinium hydrochloride. Strikingly, Two-protein films (silk films cast from a mixture of both spider silk proteins) showed properties derived from both proteins, indicating that the process of film casting based on silk proteins is closely related to film casting of traditional chemical polymers. Our results reveal novel possibilities to generate protein films for applications that demand stable biocompatible polymer films.

Determination of Poisson's Ratio of Thin low-k Films using Bidirectional Thermal Expansion Measurement

AbstractA recently developed bidirectional thermal expansion measurement (BTEM) method was applied to different types of low-k films to substantiate the reliability of the Poisson's ratio found with this technique and thereby to corroborate its practical utility. In this work, the Poisson's ratio was determined by obtaining the temperature gradient of the biaxial thermal stress from substrate curvature measurements, the temperature gradient of the whole thermal expansion strain along the film thickness from x-ray reflectivity (XRR) measurements, and reduced modulus of the film from nanoindentation measurements. For silicon oxide-based SiOC film having a thickness of 382.5 nm, the Poisson's ratio, Young's modulus and thermal extension coefficient (TEC) were determined to be Vf = 0.26, αf =21 ppm/K and Ef =9,7 GPa. These data are close to the levels of metals and polymers rather than the levels of fused silicon oxide, which is characterized by Vf = 0.17 and Er = 69.6 GPa. The alkyl component in the silicon oxide-based framework is thought to act as an agent in reducing the modulus and elevating the Poisson's ratio in SiOC low-k materials. In the case of an organic polymer SiLK film with a thickness of 501.5 nm, the Poisson's ratio, Young's modulus and TEC were determined to be Vf = 0.39, αf =74 ppm/K and Er =3.1 GPa, which are in the typical range of V= 0.34~0.47 with E =1.0~10 GPa for polymer materials. From the viewpoint of the relationship between the Poisson's ratio and Young's modulus as classified by different material types, the Poisson's ratios found for the silicon oxide-based SiOC and organic SiLK films are reasonable values, thereby confirming that BTEM is a reliable and effective method for evaluating the Poisson's ratio of thin films.

Nanoporous SiLK® Dielectric Films Prepared from Free‐Radical Graft Polymerization and Thermolysis

AbstractSummary: Thermally‐initiated free‐radical graft polymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) with the ozone‐pretreated SiLK® precursor was carried out. The chemical composition and structure of the graft copolymers were characterized by NMR and X‐ray photoelectron spectroscopy (XPS). Nanoporous low dielectric constant (κ) SiLK films were prepared by solution spin‐casting of the graft copolymers on the Si(100) substrates, followed by thermally‐induced cycloaddition and cross‐linking of the SiLK precursor and thermal decomposition of the labile PEGMA side chains. The nanoporous SiLK films so‐obtained had well‐preserved SiLK backbones and pore size in the range of 10–15 nm. The refractive index (RI) of resulting films has decreased to 1.47, from 1.62 for the pristine (non‐porous) SiLK dielectric film. A dielectric constant of about 2.2 was achieved from thermolysis of the SiLK graft copolymer containing about 15.2 wt.‐% of the PEGMA side chains.Preparation of the nanoporous SiLK dielectric film.magnified imagePreparation of the nanoporous SiLK dielectric film.

Fundamental Properties of Organic Low-k Dielectrics Usable in the Cu Damascene Process

The material parameters for organic low-k dielectrics usable in the damascene process were studied using two different types of polymers with similar low dielectric constants, namely, the PQ-600 thermoplastic polymer and the SiLK thermosetting polymer. The resistibility of these polymers in the damascene process was investigated through hard-mask (SiO2) deposition, etching and chemical mechanical polishing (CMP) processes using scanning probe microscopy (SPM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and a modified edge liftoff test (m-ELT). For the PQ-600 film, damage was observed in the deposition process and dissolution of the film occurred during chemical cleaning in the etching process. On the other hand, the SiLK film was combinable with the Cu damascene process and usable as an interlayer dielectric (ILD) in one-level Cu wiring. A high glass transition temperature (Tg) and chemical resistance resulting from the thermosetting structure are considered to be the essential properties required for the desired organic low-k dielectrics. In eddition, the electrical properies of the SiLK film were investigated using a one-level test element group (TEG) formed through a single Cu damascene process. The dielectric constant of the SiLK film extracted from the Cu damascene TEG compared with that of bulk SiO2 was reduced by 24%. The leakage current measured at 1 MV/cm between the adjoining Cu lines at the TEG pattern with a hard mask was 9.7×10-10 A/cm2, and dielectric breakdown occurred at 5.5 MV/cm.