Silk Films Research Articles

Varying surface hydrophobicities of coatings made of recombinant spider silk proteins

The engineered spider silk protein eADF4(C16) reveals similarities to amphiphilic block copolymers. Drop cast of protein solutions on different hydrophobic as well as hydrophilic templates out of different starting solvents (hexafluoroisopropanol, formic acid and aqueous buffers) generated silk films varying in structure and surface properties. Here, the underlying secondary structure of the proteins, the mechanical integrity at increased temperatures, homogeneity and surface topography of silk films, as well as the wettability were investigated in detail. Interestingly, the used templates had impact on microphase separation of the silk molecules as seen by the content of β-sheet structures; as well as on silk film surface hydrophobicities.

Preparation of Eri silk fibroin and gelatin blend film loaded chlorhexidine using as model for hydrophilic drug release

The objective of this research was to prepare Eri silk fibroin solution for preparing silk film loaded chlorhexidine drug as model for hydrophilic drug release. The Eri silk cocoons were boiled in 0.5%NaCO3 solution at 90℃, and then left in air dried at room temperature. The fibroin was dissolved in 9M (Ca(NO3)2) with ethanol (2 by mole) and heated at 70℃. The silk fibroin (SF) solution was then dialyzed to exclude salt in phosphate buffer. The SF and gelatin (G) solutions were mixed for preparation of films in both with and without chlorhexidine. The films were observed their morphology under scanning electron microscope. The results found that all of films were rough of their surfaces, homogeneous texture without phase separation. The native SF film composed of pores throughout the film area but did not observe in native G film. The results also showed that the SF and G can be good interacted to form hydrogen bonds. These were indicated from FTIR spectra and thermal analysis. The chlorhexidine drug has not affect on the changes of film properties. However, the releasing pattern of chlorhexidine from each film was varied. The highest rate of drug releasing was found in the native SF film while the native G film was the lowest. It might be suggested that the drug releasing rate was depended on polarity of each polymer components.

A Statistical Algorithm for Quantifying Cellular Alignment Distributions

Statistical techniques for the analysis of cellular alignment data are currently limited due to heuristic and qualitative approaches. For example, generally a cut-off degree limit, commonly 20 degrees, is arbitrarily defined within which cells are considered ‘aligned.’ The efficacy of patterned biomaterials in guiding the alignment of cells, such as neurons, is often critical in correlating materials design with biological outcomes. We developed a statistical algorithm for processing cellular alignment data, which implicitly determines an ‘angle of alignment.’ This was accomplished in the following manner: 1) Neuronally-differentiated PC-12 cells were seeded on flat and micropatterned silk fibroin films. Cells ‘aligning’ with an underlying, anisotropic scaffold display uniformly distributed angles up to a cut-off point, determined by how effective the biomaterial is in aligning cells. 2) We then determined where the data sets diverged from the uniform distribution by measuring the spacing between the collected, increasingly ordered angles, modeling this spacing as a time series. 3) This time series was then analyzed for uniformity using a normalized cumulative periodogram (NCP)-criterion. Following this protocol, we were able to define cellular alignments for the patterned silk films, of dimensions 300 patterns/mm, with 8° and 17° pitch angles. Their corresponding alignment indices, as determined through the NCP-criterion were 19° and 8°, respectively, which suggests that steeper pitch angles yield superior alignment. This proposed algorithm offers a novel way to implicitly define cellular alignment, with respect to various patterned biomaterial formats. This method may also offer an alternative, quantitative way to assess cellular alignment, which could offer improved predictive measures related to biological outcomes. Furthermore, the current algorithm is used for evaluating biomaterials in 2D; however, the NCP-criterion may be modified to accommodate 3D data, which will be important for understanding and quantifying regenerative outcomes in vivo.

Impact of processing parameters on the haemocompatibility of Bombyx mori silk films

Silk has traditionally been used for surgical sutures due to its lasting strength and durability; however, the use of purified silk proteins as a scaffold material for vascular tissue engineering goes beyond traditional use and requires application-orientated biocompatibility testing. For this study, a library of Bombyx mori silk films was generated and exposed to various solvents and treatment conditions to reflect current silk processing techniques. The films, along with clinically relevant reference materials, were exposed to human whole blood to determine silk blood compatibility. All substrates showed an initial inflammatory response comparable to polylactide-co-glycolide (PLGA), and a low to moderate haemostasis response similar to polytetrafluoroethylene (PTFE) substrates. In particular, samples that were water annealed at 25 °C for 6 h demonstrated the best blood compatibility based on haemostasis parameters (e.g. platelet decay, thrombin-antithrombin complex, platelet factor 4, granulocytes-platelet conjugates) and inflammatory parameters (e.g. C3b, C5a, CD11b, surface-associated leukocytes). Multiple factors such as treatment temperature and solvent influenced the biological response, though no single physical parameter such as β-sheet content, isoelectric point or contact angle accurately predicted blood compatibility. These findings, when combined with prior in vivo data on silk, support a viable future for silk-based vascular grafts.

High-performance bioanode based on the composite of CNTs-immobilized mediator and silk film-immobilized glucose oxidase for glucose/O2 biofuel cells

A high-performance bioanode based on the composite of carbon nanotubes (CNTs)-immobilized mediator and silk film (SF)-immobilized glucose oxidase (GOD) was developed for glucose/O2 biofuel cell (BFC). Ferrocenecarboxaldehyde (Fc) was used as the mediator and covalently immobilized on the ethylenediamine (EDA)-functionalized CNTs (CNTs-EDA). GOD was cross-linked on the SF with glutaraldehyde (GA) as the cross-linking agent. The resulting electrode (CNTs-Fc/SF-GOD/glassy carbon (GC) electrode) exhibited good catalytic activity towards glucose oxidation and excellent stability. For the assembled glucose/O2 BFC with the CNTs-Fc/SF-GOD/GC electrode as the bioanode and a commercial E-TEK Pt/C modified GC electrode as the cathode, the open circuit potential is 0.48V and the maximum power density of 50.70μWcm−2 can be achieved at 0.15V.

Rapid nano impact printing of silk biopolymer thin films

In this paper, nano impact printing of silk biopolymer films is described. An indenter is rapidly accelerated and transfers the nanopattern from a silicon master into the silk film during an impact event that occurs in less than 1 ms. Contact stresses of greater than 100 MPa can be achieved during the short impact period with low power and inexpensive hardware. Ring shaped features with a diameter of 2 µm and a ring width of 100–200 nm were successfully transferred into untreated silk films using this method at room temperature. Mechanical modeling was carried out to determine the contact stress distribution, and demonstrates that imprinting can occur for contact stresses of less than 2 MPa. Thermal characterization at the impact location shows that raising the temperature to 70 °C has only a limited effect on pattern transfer. Contact stresses of greater than approximately 100 MPa result in excessive deformation of the film and poor pattern transfer.

Biocompatibility of physico-crosslinked regenerated silk fibroin film as tissue engineered cornea

Background Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterial had been characterized to meet these functional requirements. Objective This study was to investigate the feasibility of physico-crosslink regenerated silk fibroin film as tissue engineered corneal scaffold. Methods Human corneal epithelial cells(CECs) links were cultured by regular method and CECs in logarithmic phase were than incubated on physico-crosslink regenerated silk fibroin film membrane. The shape of cultured human CECs was observed after 24,48 and 72 hours under the inverted microscope and scanning electron microscope( SEM ) ,and the CECs were cultured on culture plates as controls. The growth state of CECs on regenerated silk fibroin film was observed daily for 7 days by MTT, and cell cycle analysis and the presence of apoptosis of human CECs were examined by flow cytometry after incubation on regenerated silk fibroin film. Regenerated silk fibroin filmCECs (4 mm×3 mm) were implanted into the corneal stroma of the right eyes of New Zealand white rabbits. At the end of 4 and 8 weeks after implantation, the appearance of the ocular surface was examined using slit lamp and corneal neovascular area was measured. Corneal histopathological examination was carried out to assess the degradation of graft materials and immunohistochemistry was performed to detect the expression of CD34 in the corneal tissue after operation. Results The morphology and structure of CECs were identical using the two cultured Methods when observed under the inverted microscope and SEM after 24,48 and 72 hours. No significant difference was found in the A490 value 1,2,3,4,5,6 or 7 days after incubation on regenerated silk membrane and in culture plates ( Fmethod =0. 641 ,P＞0.05 ). The apoptosis rates of CECs on regenerated silk membrane or culture plates were 1.8％ and 2.0％ and the amount of cells in G2/G1 phase was 1. 956 and 1. 945, respectively. Histopathological examination showed that the regenerated silk membrane material degraded and was replaced by regular collagen tissue 2 months after implantation,and the presence of neovascular area and inflammatory cells were less prominent in 2 months than 1 month post-implantation. The expression level of CD34 in corneal tissue was evidently lower 1 and 2 months after operation than the Ad-VEGF165-induced positive control group (P＜0. 05), and no significant differences were seen when compared with normal CECs(P＞0.05). Conclusions Physico- crosslink regenerated silk fibroin film is an excellent biomaterial for tissue engineered corneal scaffold with good biocompatibility. Key words: Silk fibroin film/ physico-crosslinked regenerated; Cornea; Tissue engineering; Biocompatibility; Corneal epithelial cell; Corneal transplantation

Effect of β-sheet crystalline content on mass transfer in silk films

The material properties of silk are favorable for drug delivery due to the ability to control material structure and morphology under ambient, aqueous processing conditions. Mass transport of compounds with varying physical–chemical characteristics was studied in silk fibroin films with control of β-sheet crystalline content. Two compounds, vitamin B12 and fluorescein isothiocynate (FITC) labeled lysozyme were studied in a diffusion apparatus to determine transport through silk films. The films exhibited size exclusion phenomenon with permeability coefficients with contrasting trends with increases in β-sheet crystallinity. The size exclusion phenomenon observed with the two model compounds was characterized by contrasting trends in permeability coefficients of the films as a function of β-sheet crystallinity. The diffusivity of the compounds was examined in the context of free volume theory. Apart from the β-sheet crystallinity, size of the compound and its interactions with silk influenced mass transfer. Diffusivity of vitamin B12 was modeled to define a power law relationship with β-sheet crystallinity. The results of the study demonstrate that diffusion of therapeutic agents though silk fibroin films can be directed to match a desired rate by modulating secondary structure of the silk proteins.

실크/PLGA 필름에서 실크 함량이 망막색소 상피세포의 부착 및 증식 거동에 미치는 영향

망막 조직공학을 위한 생체 재료는 기계적 안전성, 생체적합성, 낮은 분해속도 등을 포함하여 in vivo에서 잠재적인 유용성을 위한 몇 가지 중요한 특징이 입증되어야 한다. 실크 필름 생체재료는 이러한 기능적인 요구에 맞게 디자인되었다. 0, 10, 20, 40, 및 80 wt%의 실크가 함유된 천연/합성물질과 하이브리드화된 silk/PLGA 필름을 용매 증발법으로 제조하였다. 1, 2, 및 3일 후에 부착된 세포 수를 확인하기 위해 MTT 분석을 하였고 SEM을 통해 필름에 부착된 세포 모폴로지를 확인하였다. 또한, mRNA 발현정도를 알아보기 위해 retinal pigment epithelium(RPE) 세포의 프라이머인 RPE65를 사용하여 RT-PCR을 실시하였고 RPE 세포의 특정 단백질인 cytokeratin의 발현을 확인하고 세포의 증식을 비교하기 위해 면역화학염색을 실시하였다. 본 실험을 통해 실크/PLGA 필름에서 20∼40 wt%실크를 함유한 경우에 RPE 세포의 부착과 증식에 가장 좋은 영향을 미치는 것을 확인하였다.

Inżynierowany jedwab pajęczy: inteligentny biomateriał przyszłości. Część II

The development and progress in engineered spider silk manufacturing has enabled its practical application. Recombinant spider silk can assemble in several morphological forms such as films, hydrogels, fibers, scaffolds, microcapsules, and micro- and nanospheres. The in vitro fiber formation takes place by mimicking the natural spinning process in the spider spinning gland: in the presence of phosphate ions and dragging forces. Films are obtained by evaporation of solvent from the silk solution, while the result of evaporation of the solvent in the presence of porogens is a silk scaffold. Hydrogels are formed by spontaneous polymerization of silk particles in solutions at low pH. The silk film assembled at the interface of two immiscible phases forms microcapsules. The smallest of the described forms--silk spheres--are obtained by salting out the silk protein solution after addition of the phosphate ions. Common properties of the silk biomaterials are biocompatibility and biodegradability, which make them suitable for a number of applications in medicine and pharmacy. Moreover, the strategy of hybrid proteins which provides the desired function to biomaterial will further expand their potential use.

Genetically Engineered Chimeric Silk–Silver Binding Proteins

AbstractComposite or hybrid materials are commonly found in Nature, formed through the concentration and subsequent nucleation of ions upon organic templates that are most often protein based. Examples include the deposition of calcium containing salts in bone, teeth and the inner ear and iron oxide structures in magnetotactic bacteria. Biological organisms use a limited number of metal ions, the principal ones being calcium and iron, with lesser amounts of strontium, and barium. The ability to utilize other ions to generate composites offers the possibility of new material properties. New materials incorporating silver would be useful in the context of antimicrobial functions. Therefore, in the present study, a new route to such functionalized biomaterials is reported. Genetically engineered fusion proteins are created by the incorporation of nucleotides corresponding to short silver binding peptides identified by a combinatorial biopanning process into the consensus sequence of silk from the spider, Nephila clavipes. The resulting chimeric silk–silver binding proteins nucleated Ag ions from a solution of silver nitrate while the silk protein provided a stable template material which could be processed into films, fibers, and three‐dimensional scaffolds. The silk films inhibited microbial growth of both Gram‐positive and Gram‐negative microrganisms on agar plates and in liquid culture, thus highlighting the potential of these chimeric material systems as antimicrobial biomedical coatings.

Clay enriched silk biomaterials for bone formation

The formation of silk protein/clay composite biomaterials for bone tissue formation is described. Silk fibroin serves as an organic scaffolding material offering mechanical stability suitable for bone-specific uses. Clay montmorillonite (Cloisite® Na+) and sodium silicate are sources of osteoinductive silica-rich inorganic species, analogous to bioactive bioglass-like bone repair biomaterial systems. Different clay particle–silk composite biomaterial films were compared with silk films doped with sodium silicate as controls for the support of human bone marrow derived mesenchymal stem cells in osteogenic culture. The cells adhered to and proliferated on the silk/clay composites over 2weeks. Quantitative real time polymerase chain reaction analysis revealed increased transcript levels for alkaline phosphatase, bone sialoprotein, and collagen type 1 osteogenic markers in the cells cultured on the silk/clay films in comparison with the controls. Early evidence of bone formation based on collagen deposition at the cell–biomaterial interface was also found, with more collagen observed for the silk films with higher contents of clay particles. The data suggest that silk/clay composite systems may be useful for further study for bone regenerative needs.

Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing

We present a simple and effective method to obtain refined control of the molecular structure of silk biomaterials through physical temperature-controlled water vapor annealing (TCWVA). The silk materials can be prepared with control of crystallinity, from a low content using conditions at 4 °C (α helix dominated silk I structure), to highest content of ∼60% crystallinity at 100 °C (β-sheet dominated silk II structure). This new physical approach covers the range of structures previously reported to govern crystallization during the fabrication of silk materials, yet offers a simpler, green chemistry, approach with tight control of reproducibility. The transition kinetics, thermal, mechanical, and biodegradation properties of the silk films prepared at different temperatures were investigated and compared by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), uniaxial tensile studies, and enzymatic degradation studies. The results revealed that this new physical processing method accurately controls structure, in turn providing control of mechanical properties, thermal stability, enzyme degradation rate, and human mesenchymal stem cell interactions. The mechanistic basis for the control is through the temperature-controlled regulation of water vapor to control crystallization. Control of silk structure via TCWVA represents a significant improvement in the fabrication of silk-based biomaterials, where control of structure-property relationships is key to regulating material properties. This new approach to control crystallization also provides an entirely new green approach, avoiding common methods that use organic solvents (methanol, ethanol) or organic acids. The method described here for silk proteins would also be universal for many other structural proteins (and likely other biopolymers), where water controls chain interactions related to material properties.

Mechanisms of Controlled Release from Silk Fibroin Films

The controlled release of fluorescein-iso-thio-cyanate (FITC)-labeled dextrans from methanol-treated and untreated silk fibroin films was modeled to characterize the release kinetics and mechanisms. Silk films were prepared with FITC-dextrans of various molecular weights (4, 10, 20, 40 kDa). Methanol treatment was used to promote crystallinity. The release data were assessed with two different models, an empirical exponential equation commonly fit to release data and a mechanism-based semiempirical model derived from Fickian diffusion through a porous film. The FITC-dextran release kinetics were evaluated as a function of molecular weight and compared between the untreated- and methanol-treated films. For the empirical model, the estimated values of the model parameters decreased with the molecular weight of the analyte and showed no significant difference between untreated- and methanol-treated films. For the diffusion-based model, the estimated diffusion coefficient was smaller for the methanol-treated films than for the untreated films. Also, the diffusion coefficient was observed to decrease linearly with increasing molecular weight of the analyte. The percent of FITC-dextran loading entrapped and not released was less for the methanol-treated films than for untreated films and linearly increased with molecular weight. A linear regression was fit to the relationship between molecular weight and the percent of entrapped FITC-dextran particles. Using these defined linear relationships, we present an updated version of the diffusion model for simulating release of FITC-dextran of varied molecular weights from methanol-treated and untreated silk films.

Structural Origins of Silk Piezoelectricity.

Uniaxially oriented, piezoelectric silk films were prepared by a two-step method that involved: (1) air drying aqueous, regenerated silk fibroin solutions into films, and (2) drawing the silk films to a desired draw ratio. The utility of two different drawing techniques, zone drawing and water immersion drawing were investigated for processing the silk for piezoelectric studies. Silk films zone drawn to a ratio of λ= 2.7 displayed relatively high dynamic shear piezoelectric coefficients of d(14) = -1.5 pC/N, corresponding to over two orders of magnitude increase in d(14) due to film drawing. A strong correlation was observed between the increase in the silk II, β-sheet content with increasing draw ratio measured by FTIR spectroscopy (C(β)∝ e(2.5) (λ)), the concomitant increasing degree of orientation of β-sheet crystals detected via WAXD (FWHM = 0.22° for λ= 2.7), and the improvement in silk piezoelectricity (d(14)∝ e(2.4) (λ)). Water immersion drawing led to a predominantly silk I structure with a low degree of orientation (FWHM = 75°) and a much weaker piezoelectric response compared to zone drawing. Similarly, increasing the β-sheet crystallinity without inducing crystal alignment, e.g. by methanol treatment, did not result in a significant enhancement of silk piezoelectricity. Overall, a combination of a high degree of silk II, β-sheet crystallinity and crystalline orientation are prerequisites for a strong piezoelectric effect in silk. Further understanding of the structural origins of silk piezoelectricity will provide important options for future biotechnological and biomedical applications of this protein.

Safety evaluation of silk protein film (A novel wound healing agent) in terms of acute dermal toxicity, acute dermal irritation and skin sensitization

Acute dermal toxicity study was conducted in rats. The parameters studied were body weight, serum biochemistry and gross pathology. The animals were also observed for clinical signs and mortality after the application of test film. The dermal irritation potential of silk protein film was examined using Draize test. In the initial test, three test patches were applied sequentially for 3 min, 1 and 4 hours, respectively, and skin reaction was graded. The irritant or negative response was confirmed using two additional animals, each with one patch, for an exposure period of 4 hours. The responses were scored at 1, 24, 48 and 72 hours after the patch removal. Skin sensitization study was conducted according to Buehler test in guinea pigs, in which on day 0, 7 and 14, the animals were exposed to test material for 6 hours (Induction phase) and on day 28, the animals were exposed for a period of 24 hours (Challenge phase). The skin was observed and recorded at 24 and 48 hours after the patch removal. In acute dermal toxicity study, the rats dermally treated with silk film did not show any abnormal clinical signs and the body weight, biochemical parameters and gross pathological observations were not significantly different from the control group. In acute dermal irritation study, the treated rabbits showed no signs of erythema, edema and eschar, and the scoring was given as “0” for all time points of observations according to Draize scoring system. In skin sensitization study, there were no skin reactions 24 and 48 hours after the removal of challenge patch, which was scored “0” based on Magnusson/Kligman grading scale.

Impact of initial solvent on thermal stability and mechanical properties of recombinant spider silk films

Spider silk fibers are one of the most remarkable biopolymers displaying a unique combination of mechanical properties, biocompatibility and biodegradability. The recombinant production of spider silk proteins now allows the processing of silk proteins into novel materials with the aim of future applications. Here, we analyzed films made of a recombinantly produced, engineered spider silk protein with a sequence derived from the dragline silk protein ADF4 of the European garden spider Araneus diadematus. An influence of different initial solvents (aqueous buffer, hexafluoro-2-propanol and formic acid) on certain film properties was identified in as cast as well as methanol post-treated films: while no significant effects on the films' thermal stability were observed, a significant influence on their mechanical properties could be shown. Interestingly, solvent-induced effects were sustained after methanol post-treatment and could be correlated with the presence and arrangement of secondary structure elements. Insights into molecular orientation of individual structural elements within the films upon applied load were revealed by combined polarized IR spectroscopy and mechanical measurements.

Quantifying Osteogenic Cell Degradation of Silk Biomaterials

The degradation of silk protein films by human mesenchymal stem cells (hMSCs), osteoblasts and osteoclasts, cells involved in osteogenic functions in normal and diseased bone, was assessed in vitro. The involvement of specific matrix metalloproteinases (MMPs) and integrin signaling in the degradation process was determined. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to quantitatively compare degradation by the different cell types using surface patterned silk films. Osteoblasts and osteoclasts demonstrated significant degradation of the silk films in vitro in comparison to the hMSCs and the film controls without cells. The osteoclasts degraded the silk films the most and also generated the highest level of MMPs 1 and 2. The osteoblasts upregulated integrins α5 and β1, while the osteoclasts upregulated integrins α2 and β1. There was significant contrast in responses on the silk matrices between osteogenic cells versus undifferentiated hMSCs to illustrate in vitro the role of cell type on matrix remodeling. These are important issues in matching biomaterial matrix features and studies in vitro to remodeling in vivo, in both normal and disease tissue systems. Cell populations and niche factors impact tissue regeneration, wound healing, physiological state, and the ability to better understand the role of different cell types is critical to overall regenerative outcomes.

Nanoscale Control of Silica Particle Formation via Silk−Silica Fusion Proteins for Bone Regeneration

The biomimetic design of silk/silica fusion proteins was carried out, combining the self assembling domains of spider dragline silk (Nephila clavipes) and silaffin derived R5 peptide of Cylindrotheca fusiformis that is responsible for silica mineralization. Genetic engineering was used to generate the protein-based biomaterials incorporating the physical properties of both components. With genetic control over the nanodomain sizes and chemistry, as well as modification of synthetic conditions for silica formation, controlled mineralized silk films with different silica morphologies and distributions were successfully generated; generating 3D porous networks, clustered silica nanoparticles (SNPs), or single SNPs. Silk serves as the organic scaffolding to control the material stability and multiprocessing makes silk/silica biomaterials suitable for different tissue regenerative applications. The influence of these new silk-silica composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on the silk/silica films. The presence of the silica in the silk films influenced osteogenic gene expression, with the upregulation of alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col 1) markers. Evidence for early bone formation as calcium deposits was observed on silk films with silica. These results indicate the potential utility of these new silk/silica systems towards bone regeneration.

Rapid Nanoimprinting of Doped Silk Films for Enhanced Fluorescent Emission

Rapid Nanoimprinting of Doped Silk Films for Enhanced Fluorescent Emission