Silk Films Research Articles

Cross-plane thermal transport in micrometer-thick spider silk films

This work reports on the first study of thermal transport capacity in the thickness direction (∼μm scale) for spider silk films. Fresh (minimally processed) and hexafluoroisopropanol (HFIP) films of Nephila clavipes and Latrodectus hesperus major ampullate silk are studied. Detailed Raman spectroscopy reveals that the fresh films have more crystalline secondary protein structures such as antiparallel β-sheets than the HFIP films for N. clavipes. For N. clavipes, the randomly distributed antiparallel β-sheets in fresh films have nearly no effect in improving thermal conductivity in comparison with HFIP films. For L. hesperus, the films mainly consist of α-helices and random coils while the fresh film has a higher concentration of α-helices. The higher concentration of α-helices in fresh films gives rise to a higher heat capacity than HFIP films, while the thermal conductivity shows little effect from the α-helices concentration. Thickened HFIP films are heated at different temperatures to study the effect of heat treatment on structure and thermal transport capacity. These experiments demonstrate that α-helices are formed by thermal treatment and that thermal effusivity increases with the appearance of α-helices in films.

Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay

Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications. To develop a novel affinity silk material, we generated a transgenic silkworm that spins silk protein containing the fibroin L-chain linked with the single-chain variable fragment (scFv) as a fusion protein. Previously, the scFv-conjugated “affinity” silk powder specifically immunoprecipitated its target protein, Wiskott-Aldrich syndrome protein. To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates. Enzyme-linked immunosorbent assay demonstrated specific detection of Wiskott-Aldrich syndrome protein, both as a recombinant protein and in its native form extracted from mouse macrophages. These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system.

Corneal stromal bioequivalents secreted on patterned silk substrates

Emulating corneal stromal tissue is believed to be the most challenging step in bioengineering an artificial human cornea because of the difficulty in reproducing its highly ordered microstructure, the key to the robust biomechanical properties and optical transparency of this tissue. We conducted a comparative study to assess the feasibility of human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on groove-patterned silk substrates. In serum-free keratocyte differentiation medium, hCSSCs successfully differentiated into keratocytes secreting multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 90–100 μm thick, containing abundant cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. In contrast, hCFs tended to differentiate into myofibroblasts that deposited less organized collagen in a pattern resembling that of corneal scar tissue. RGD surface coupling coupling was an essential factor in enhancing cell attachment, orientation, proliferation, differentiation and ECM deposition on the silk substratum. These results demonstrated that an approach of combining hCSSCs with an RGD surface-coupled patterned silk film offers a powerful tool to develop highly ordered collagen fibril-based constructs for corneal regeneration and corneal stromal tissue repair.

Quantitative characterization of mineralized silk film remodeling during long-term osteoblast–osteoclast co-culture

The goal of this study was to explore quantitative assessments of mineralized silk protein biomaterial films by co-cultures of human mesenchymal stem cell-derived osteoblasts and human acute monocytic leukemia cell line-derived osteoclasts during long-term culture (8–32 weeks). The remodeled films were quantitatively assessed using three different techniques during this extended cultivation to provide more comprehensive insight into the impact of co-cultures on surface remodeling. Scanning electron microscopy (SEM) with three dimensional surface reconstructions was used to quantitatively determine various surface morphological features and measures of roughness indicative of remodeling by the cells. Additionally, reconstructed surfaces were converted to depth images for Fourier analysis to quantify the potential fractal organization of biomineralization. The long-term remodeled films were also imaged using confocal reflectance microscopy and micro-computed tomography (micro-CT) to further quantify morphological changes. Films remodeled in co-culture demonstrated increased roughness parameters, fractal organization, and volume compared to films remodeled by osteoblasts alone. The combination of these techniques to quantify remodeling of mineralized protein films shows promise for quantifying processes related to mineralized surfaces.

Fabrication of transparent silk fibroin film for the regeneration of corneal endothelial cells; preliminary study

Dystrophies related to the corneal endothelium result in edema, loss of transparency, and finally, blindness. Currently, the only treatment of patients with these corneal endothelial dystrophies is transplantation. However, the limited availability of donor corneas is a main obstacle to transplanting healthy endothelia. In this study, we have chosen silk from the silkworm to make a substratum for corneal endothelial cells (CEnCs). This approach offers the possibility of overcoming the issue of limited supply of donor corneas. Two types of transparent silk films with different sericin contents have been prepared by boiling for 30 min and 1 h, respectively, as delivery vehicles for application on the corneal endothelium. The contents of sericin for high-sericin silk (HSS) and low-sericin silk (LSS) are 9.83% and 6.25%, respectively. Physical and chemical properties were analyzed by contact angle, transparency, tensile strength, FTIR, and DSC. Proliferation assay by MTT was conducted to confirm the viability of the CEnCs. These results demonstrate that the fabricated transparent silk films have sufficient physical and chemical properties as a possible carrier for CEnCs.

Film-based Implants for Supporting Neuron-Electrode Integrated Interfaces for The Brain.

Neural engineering provides promise for cell therapy by integrating the host brain with brain-machine-interface technologies in order to externally modulate functions. Long-term interfaces with the host brain remain a critical challenge due to insufficient graft cell survivability and loss of brain electrode sensitivity over time. Here, integrated neuron-electrode interfaces were developed on thin flexible and transparent silk films as brain implants. Mechanical properties and surface topography of silk films were optimized to promote cell survival and alignment of primary rat cortical cells. Compartmentalized cultures of living neural circuit and co-patterned electrode arrays were incorporated on the silk films with built-in wire connections. Electrical stimulation via electrodes embedded in the films activated surrounding neurons evoked calcium responses. In mice brains the silk film implants showed conformal contact capable of modulating host brain cells with minimal inflammatory response and stable indwelling for weeks. The approach of combining cell therapy and brain electrodes could provide sustained functional brain-machine interfaces with ex vivo control of neuron-electrode interface with spatial and temporal precision.

Low abdominal near-infrared spectroscopy values and elevated serum intestinal fatty acid-binding protein predict necrotizing enterocolitis in a premature piglet model

METHODS: Human neuroblastoma cells (KELLY) were injected into the left adrenal gland of Nude mice to create an orthotopic xenograft. When tumor volume reached 300mm3 (measured by ultrasound) 60-80% of the tumor was resected with electrocautery. A 7x11mm doxorubicin(200mg)-loaded sustained released silk film (DOX-film) was applied to the residual tumor cavity for six animals; silk films without doxorubicin (CONT-film) were applied in five animals. Subsequent tumor volume was followed for 28 days; tumor histology was analyzed with H&E, TUNEL assay, and Ki67. In vitro release profile of DOX-film was also measured. A repeated-measures generalized linear model was conducted with tumor volume as the outcome, DOX-film group and post-resection days as main effects, and a group-time interaction term.

Surgical resection combined with application of doxorubicin-loaded sustained release silk film as a treatment strategy in an orthotopic xenograft neuroblastoma mouse model

METHODS: Human neuroblastoma cells (KELLY) were injected into the left adrenal gland of Nude mice to create an orthotopic xenograft. When tumor volume reached 300mm3 (measured by ultrasound) 60-80% of the tumor was resected with electrocautery. A 7x11mm doxorubicin(200mg)-loaded sustained released silk film (DOX-film) was applied to the residual tumor cavity for six animals; silk films without doxorubicin (CONT-film) were applied in five animals. Subsequent tumor volume was followed for 28 days; tumor histology was analyzed with H&E, TUNEL assay, and Ki67. In vitro release profile of DOX-film was also measured. A repeated-measures generalized linear model was conducted with tumor volume as the outcome, DOX-film group and post-resection days as main effects, and a group-time interaction term.

Study on Silk Fibroin D-Mannose Blend Films

The preparation and properties of different proportion of D-mannose-fibroin membrane were studied in this paper. The change of crystal structure of silk fibroin (SF) in the process of becoming insoluble by adding D-mannose was studied. The blend silk fibroin films were casted by silk fibroin solutions mixing with D-mannose at a series of ratios at room temperature. The solubility, structure, and mechanical properties of the blend films were measured. The results show that with the content of D-mannose increasing, the solubility decreases. The blend films became insoluble when the mass ratios of D-mannose /SF are more than 1/10. The crystalline structure of SF is mostly changed into Silk I and there was almost no Silk II structure. Mechanical properties indicated that D-mannose could significantly improve the flexibility of silk films. With the good transmittance, D-mannose/SF blend membrane can be used in corneal tissue engineering.

Bladder tissue regeneration using acellular bi-layer silk scaffolds in a large animal model of augmentation cystoplasty

Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a porcine model of augmentation cystoplasty. Two bi-layer matrix configurations were fabricated by solvent-casting/salt leaching either alone (Group 1) or in combination with silk film casting (Group 2) to yield porous foams buttressed by heterogeneous surface pore occlusions or homogenous silk films, respectively. Bladder augmentation was performed with each scaffold group (6 × 6 cm2) in juvenile Yorkshire swine for 3 m of implantation. Augmented animals exhibited high rates of survival (Group 1: 5/6, 83%; Group 2: 4/4, 100%) and voluntary voiding over the course of the study period. Urodynamic evaluations demonstrated mean increases in bladder capacity over pre-operative levels (Group 1: 277%; Group 2: 153%) which exceeded nonsurgical control gains (144%) encountered due to animal growth. Similarly, elevations in bladder compliance were substantially higher in augmented animals from baseline (Group 1: 357%; Group 2: 147%) in comparison to controls (41%). Gross tissue evaluations revealed that both matrix configurations supported extensive de novo tissue formation throughout the entire original implantation site which exhibited ultimate tensile strength similar to nonsurgical counterparts. Histological and immunohistochemical analyses showed that both implant groups promoted comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent cytokeratin, uroplakin, and p63 protein expression in both matrix groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Ex vivo organ bath studies demonstrated that regenerated tissues supported by both silk matrices displayed contractile responses to carbachol, α,β-methylene-ATP, KCl, and electrical field stimulation similar to controls. Our data detail the ability of acellular silk scaffolds to support regeneration of innervated, vascularized smooth muscle and urothelial tissues within 3 m with structural, mechanical, and functional properties comparable to native tissue in a porcine model of bladder repair.

Neural responses to electrical stimulation on patterned silk films

Peripheral nerve injury is a critical issue for patients with trauma. Following injury, incomplete axon regeneration or misguided axon innervation into tissue will result in loss of sensory and motor functions. The objective of this study was to examine axon outgrowth and axon alignment in response to surface patterning and electrical stimulation. To accomplish our objective, metal electrodes with dimensions of 1.5 mm × 4 cm, were sputter coated onto micropatterned silk protein films, with surface grooves 3.5 μm wide × 500 nm deep. P19 neurons were seeded on the patterned electronic silk films and stimulated at 120 mV, 1 kHz, for 45 min each day for 7 days. Responses were compared with neurons on flat electronic silk films, patterned silk films without stimulation, and flat silk films without stimulation. Significant alignment was found on the patterned film groups compared with the flat film groups. Axon outgrowth was greater (p < 0.05) on electronic films on days 5 and 7 compared with the unstimulated groups. In conclusion, electrical stimulation, at 120 mV, 1 kHz, for 45 min daily, in addition to surface patterning, of 3.5 μm wide × 500 nm deep grooves, offered control of nerve axon outgrowth and alignment.

Structure and Mechanical Properties of Spider Silk Films at the Air–Water Interface

The kinetics of adsorption of solubilized spider major ampullate (MA) silk fibers at the air-water interface and the molecular structure and mechanical properties of the interfacial films formed have been studied using various physical techniques. The data show that Nephila clavipes MA proteins progressively adsorb at the interface and ultimately form a highly cohesive thin film. In situ infrared spectroscopy shows that as soon as they reach the interface the proteins predominantly form β sheets. The protein secondary structure does not change significantly as the film grows, and the amount of β sheet is the same as that of the natural fiber. This suggests that the final β-sheet content is mainly dictated by the primary structure and not by the underlying formation process. The measure of the shear elastic constant at low strain reveals a very strong, viscous, cohesive assembly. The β sheets seem to form cross-links dispersed within an intermolecular network, thus probably playing a major role in the film strength. More importantly, the molecular weight seems to be a crucial factor because interfacial films made from the natural proteins are ~7 times stronger and ~3 times more viscous than those obtained previously with shorter recombinant proteins. Brewster angle microscopy at the air-water interface and transmission electron microscopy of transferred films have revealed a homogeneous organization on the micrometer scale. The images suggest that the structural assembly at the air-water interface leads to the formation of macroscopically solid and highly cohesive networks. Overall, the results suggest that natural spider silk proteins, although sharing similarities with recombinant proteins, have the particular ability to self-assemble into ordered materials with exceptional mechanical properties.

Accelerated In Vitro Degradation of Optically Clear Lowβ-Sheet Silk Films by Enzyme-Mediated Pretreatment

To design patterned, transparent silk films with fast degradation rates for the purpose of tissue engineering corneal stroma. β-sheet (crystalline) content of silk films was decreased significantly by using a short water annealing time. Additionally, a protocol combining short water annealing time with enzymatic pretreatment of silk films with protease XIV was developed. Low β-sheet content (17%-18%) and enzymatic pretreatment provided film stability in aqueous environments and accelerated degradation of the silk films in the presence of human corneal fibroblasts in vitro. The results demonstrate a direct relationship between reduced β-sheet content and enzymatic pretreatment, and overall degradation rate of the protein films. The novel protocol developed here provides new approaches to modulate the regeneration rate of silk biomaterials for corneal tissue regeneration needs. Patterned silk protein films possess desirable characteristics for corneal tissue engineering, including optical transparency, biocompatibility, cell alignment, and tunable mechanical properties, but current fabrication protocols do not provide adequate degradation rates to match the regeneration properties of the human cornea. This novel processing protocol makes silk films more suitable for the construction of human corneal stroma tissue and a promising way to tune silk film degradation properties to match corneal tissue regeneration.

Accelerated In Vitro Degradation of Optically Clear Low–β Sheet Silk Films by Enzyme-Mediated Pretreatment

Results: Low b-sheet content (17%–18%) and enzymatic pretreatment provided film stability in aqueous environments and accelerated degradation of the silk films in the presence of human corneal fibroblasts in vitro. The results demonstrate a direct relationship between reduced b-sheet content and enzymatic pretreatment, and overall degradation rate of the protein films. Conclusions: The novel protocol developed here provides new approaches to modulate the regeneration rate of silk biomaterials for corneal tissue regeneration needs. Translational Relevance: Patterned silk protein films possess desirable characteristics for corneal tissue engineering, including optical transparency, biocompatibility, cell alignment, and tunable mechanical properties, but current fabrication protocols do not provide adequate degradation rates to match the regeneration properties of the human cornea. This novel processing protocol makes silk films more suitable for the construction of human corneal stroma tissue and a promising way to tune silk film degradation properties to match corneal tissue regeneration.

The performance of silk scaffolds in a rat model of augmentation cystoplasty

The diverse processing plasticity of silk-based biomaterials offers a versatile platform for understanding the impact of structural and mechanical matrix properties on bladder regenerative processes. Three distinct groups of 3-D matrices were fabricated from aqueous solutions of Bombyx mori silk fibroin either by a gel spinning technique (GS1 and GS2 groups) or a solvent-casting/salt-leaching method in combination with silk film casting (FF group). SEM analyses revealed that GS1 matrices consisted of smooth, compact multi-laminates of parallel-oriented silk fibers while GS2 scaffolds were composed of porous (pore size range, 5–50 μm) lamellar-like sheets buttressed by a dense outer layer. Bi-layer FF scaffolds were comprised of porous foams (pore size, ∼400 μm) fused on their external face with a homogenous, nonporous silk film. Silk groups and small intestinal submucosa (SIS) matrices were evaluated in a rat model of augmentation cystoplasty for 10 weeks of implantation and compared to cystotomy controls. Gross tissue evaluations revealed the presence of intra-luminal stones in all experimental groups. The incidence and size of urinary calculi was the highest in animals implanted with gel spun silk matrices and SIS with frequencies ≥57% and stone diameters of 3–4 mm. In contrast, rats augmented with FF scaffolds displayed substantially lower rates (20%) and stone size (2 mm), similar to the levels observed in controls (13%, 2 mm). Histological (hematoxylin and eosin, Masson's trichrome) and immunohistochemical (IHC) analyses showed comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites supported by all matrix groups similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent uroplakin and p63 protein expression in all experimental groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by Fox3-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. In comparison to other biomaterial groups, cystometric analyses at 10 weeks post-op revealed that animals implanted with the FF matrix configuration displayed superior urodynamic characteristics including compliance, functional capacity, as well as spontaneous non voiding contractions consistent with control levels. Our data demonstrate that variations in scaffold processing techniques can influence the in vivo functional performance of silk matrices in bladder reconstructive procedures.

Protein‐Protein Nanoimprinting of Silk Fibroin Films

Protein-protein imprinting of silk fibroin is introduced as a rapid, high-throughput method for the fabrication of nanoscale structures in silk films, through the application of heat and pressure. Imprinting on conformal surfaces is demonstrated with minor adjustments to the system, at resolutions comparable to other currently available nonplanar nanoimprint lithography techniques.

Nanoscale investigations of synthetic spider silk fibers modified by physical and chemical processes

Spider silk has biocompatibility and biodegradability properties and is known for the mechanical, physical and chemical properties that make it a promising building block in the development of novel biofibers. Its unique properties partially result from the repetitive polypeptide sequences that compose the silk proteins. The strength is related to the polyalanine motifs organized into β-sheet structures, and the elasticity is attributed to glycine-rich regions, β turns and 310 helix structures. Some alcohols were shown to induce β-sheet formation in spidroins and spider silk films, while water increases the overall structure ordering of silkworm fibroins. Furthermore, fiber stretching induces β-sheet formation in synthetic spider fibers. However, there is a lack of information relating the physical and mechanical behaviors that might contribute to improving the microstructure and performance of synthetic fibers. In this work, we reported the surface nanostructure and the nanomechanical behavior of synthetic spider fibers, which were composed of modified recombinant proteins to combine strength and extensibility motifs. Our present study evaluated synthetic fibers qualitatively and quantitatively, and indicated that atomic force microscopy (AFM) and scanning electron microscopy (SEM) were complementary tools to describe particular details of the surface structure and the mechanical features of synthetic spider fibers. Therefore, AFM and SEM would support the development of spinning systems and the characterization of novel biomaterials. Synthetic spider fibers have particular structural and mechanical features that can be improved through extrusion treatments. The surface irregularity, elasticity and strength of fibers are likely to be dependent on their molecular organization and an alcohol bath appears to initiate the transition of random coil structures to β-sheets, resulting in smoother, stronger and more elastic fibers. Atomic force microscopy/force spectroscopy complemented by scanning electron microscopy was able to qualitatively describe synthetic fibers and quantify their roughness and mechanical properties, representing a significant step toward spider silk characterization, and for its application as a biomaterial.

Fabrication and characterization of biomaterial film from gland silk of muga and eri silkworms

This study discusses the possibilities of liquid silk (Silk gland silk) of Muga and Eri silk, the indigenous non mulberry silkworms of North Eastern region of India, as potential biomaterials. Silk protein fibroin of Bombyx mori, commonly known as mulberry silkworm, has been extensively studied as a versatile biomaterial. As properties of different silk-based biomaterials vary significantly, it is important to characterize the non mulberry silkworms also in this aspect. Fibroin was extracted from the posterior silk gland of full grown fifth instars larvae, and 2D film was fabricated using standard methods. The films were characterized using SEM, Dynamic contact angle test, FTIR, XRD, DSC, and TGA and compared with respective silk fibers. SEM images of films reveal presence of some globules and filamentous structure. Films of both the silkworms were found to be amorphous with random coil conformation, hydrophobic in nature, and resistant to organic solvents. Non mulberry silk films had higher thermal resistance than mulberry silk. Fibers were thermally more stable than the films. This study provides insight into the new arena of research in application of liquid silk of non mulberry silkworms as biomaterials.

Effect of silk protein processing on drug delivery from silk films.

Sericin removal from the core fibroin protein of silkworm silk is a critical first step in the use of silk for biomaterial-related applications, but degumming can affect silk biomaterial properties, including molecular weight, viscosity, diffusivity and degradation behavior. Increasing the degumming time (10, 30, 60, and 90 min) decreases the average molecular weight of silk protein in solution, silk solution viscosity, and silk film glass-transition temperature, and increases the rate of degradation of a silk film by protease. Model compounds spanning a range of physical-chemical properties generally show an inverse relationship between degumming time and release rate through a varied degumming time silk coating. Degumming provides a useful control point to manipulate silk's material properties.

Controllable cell adhesion, growth and orientation on layered silk protein films.

Due to their mechanical stability, biocompatibility and biodegradability, silks are promising materials for various biomedical applications including tissue engineering. Since the shape and the organisation of cells in and on scaffolds both affect their function, we tested patterned silk scaffolds made of three different silk proteins concerning their influence on cell adhesion, growth and orientation. Two different cell lines, BALB/3T3 fibroblasts and C2C12 myoblasts, showed controllable cell adhesion as well as orientation dependent on the silk proteins used and patterns made. Surprisingly, the presence of the integrin binding motif RGD did not influence cell adhesion and orientation on structured silk films, although it did so significantly on flat films.