Electrospun Silk Fibroin Research Articles

Electrospun silk fibroin–poly (lactic-co-glycolic acid) membrane for nerve tissue engineering

Silk fibroin–poly(lactic-co-glycolic acid) fibrous membranes were electrospun by varying the weight ratios for silk fibroin to poly(lactic-co-glycolic acid). The hydrophilicity, mechanical property, and biodegradability of the fibrous in vitro were evaluated. Contact angle test demonstrated that the hydrophilicity of poly(lactic-co-glycolic acid) fibrous membrane could be improved by introducing silk fibroin ingredient. Mechanical test showed that the strain–elongation performances of silk fibroin–poly(lactic-co-glycolic acid) fibrous can be controlled by changing the silk fibroin percentage. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay test showed that the silk fibroin–poly(lactic-co-glycolic acid) 2:8 fibrous enhanced the nerve cell proliferation compared to poly(lactic-co-glycolic acid) fibrous. Silk fibroin–poly(lactic-co-glycolic acid) fibrous membrane has been made into the nerve guidance conduit by the reeling and the sewing processing. The poly(lactic-co-glycolic acid) nerve guidance conduit and silk fibroin–poly(lactic-co-glycolic acid) nerve guidance conduit were implanted into a 10-mm sciatic nerve defect part of mice for nerve regeneration and the nerve regenerated at 12 weeks. Nerve regeneration test showed that the regenerated nerve in the silk fibroin–poly(lactic-co-glycolic acid) nerve guidance conduit group was more organized and mature than that in the poly(lactic-co-glycolic acid) nerve guidance conduit group. The results suggest that the silk fibroin–poly(lactic-co-glycolic acid) (2:8) nerve guidance conduits have potential applications in nerve regeneration.

Effect of Korean Bombyx mori variety on electro-spinning performance of regenerated silk fibroin

The electro-spinning of silk has attracted the attention of researchers in biomedical fields because of 1) the unique properties of silk, including its biocompatibility, and 2) the simplicity of electro-spinning for nanoweb fabrication. Although there are hundreds of varieties of Bombyx mori, the effect of silkworm variety has not been investigated in the study of silk electro-spinning. As an effort to develop electro-spun silk with diverse performances, the effect of Korean silkworm variety on the electro-spinning of silk fibroin (SF) was examined in this study. The regenerated SF solutions showed different viscosities depending on the silkworm variety, which resulted in differences in the maximum electro-spinning rates and diameters of electro-spun SF fibers. However, the crystallization of SF during electro-spinning and the water content of the electro-spun SF webs remained the same between different silkworm varieties.

Tissue performance of bladder following stretched electrospun silk fibroin matrix and bladder acellular matrix implantation in a rabbit model.

The goal of this study was to investigate the tissue performance of bladder following stretched electrospun silk fibroin matrix (SESFM) implantation compared with bladder acellular matrix (BAM). We compared SESFM with BAM based on porosity and pore size. Scaffolds were separately transplanted into opposite walls of the bladder of 30 rabbits after stripping the bladder mucosa and smooth muscle (1.5 × 2.0 cm(2)). Gross anatomical observation, histological analysis and muscle contractility studies were performed at 2, 4, and 8 weeks post-op. SESFM has higher porosity and larger pore size compared with BAM (p < 0.05). At 2 weeks, the presence of vesical calculus was evident in 7/10 rabbits. Histological analysis showed that SESFM and BAM promoted similar degree of urothelium regeneration (p > 0.05). However, SESFM promoted a higher degree of smooth muscle and vessel regeneration compared to BAM (p < 0.05). In addition, muscle strips supported by SESFM displayed higher contractile responses to carbachol, KCl, and phenylephrine compared with BAM. At 8 weeks, both matrices elicited similar mild acute and chronic inflammatory reactions. Our results demonstrated that SESFM has greater ability to promote bladder tissue regeneration with structural and functional properties compared to BAM, and with similar biocompatibility.

Membrane-reinforced three-dimensional electrospun silk fibroin scaffolds for bone tissue engineering

Electrospun silk fibroin (SF) scaffolds have drawn much attention because of their resemblance to natural tissue architecture such as extracellular matrix, and the biocompatibility of SF as a candidate material to replace collagen. However, electrospun scaffolds lack the physical integrity of bone tissue scaffolds, which require resistance to mechanical loadings. In this work, we propose membrane-reinforced electrospun SF scaffolds by a serial process of electrospinning and freeze-drying of SF solutions in two different solvents: formic acid and water, respectively. After wet electrospinning followed by replacement of methanol with water, SF nanofibers dispersed in water were mixed with aqueous SF solution. Freeze-drying of the mixed solution resulted in 3D membrane-connected SF nanofibrous scaffolds (SF scaffolds) with a thickness of a few centimeters. We demonstrated that the SF concentration of aqueous SF solution controlled the degree of membrane reinforcement between nanofibers. It was also shown that both increase in degree of membrane reinforcement and inclusion of hydroxyapatite (HAP) nanoparticles resulted in higher resistance to compressive loadings of the SF scaffolds. Culture of human osteoblasts on collagen, SF, and SF-HAP scaffolds showed that both SF and SF-HAP scaffolds had biocompatibility and cell proliferation superior to that of the collagen scaffolds. SF-HAP scaffolds with and without BMP-2 were used for in vivo studies for 4 and 8 weeks, and they showed enhanced bone tissue formation in rat calvarial defect models.

Tissue performance of bladder following stretched electrospun silk fibroin matrix and bladder acellular matrix implantation in a rabbit model

Tissue performance of bladder following stretched electrospun silk fibroin matrix and bladder acellular matrix implantation in a rabbit model

Optimization of nanofibrous silk fibroin scaffold as a delivery system for bone marrow adherent cells: in vitro and in vivo studies.

Electrospun silk fibroin nanofibrous scaffolds (ESFNSs) were successfully prepared by electrospinning of various Bombyx mori silk fibroin concentrations (10, 12, and 14% in formic acid). After characterizing the purified silk fibroin, the morphology, porosity, fibers' diameter, and uniformity of the prepared scaffolds were examined in detail. In addition, biological responses such as effects on bone marrow cell viability, cytotoxicity, and cell adhesion were evaluated in vitro. Biocompatibility and bioactivity properties of the ESFNSs were evaluated in vitro and in vivo by cell culturing and subcutaneous implantation in rat models for 7 and 28 days, respectively. According to the obtained results, no beaded fibers were seen in any of the prepared scaffolds, whereas ESFNS-10% provided more uniformity and porosity with nanoscaled fibers (90 ± 0.021 nm). Furthermore, the scaffolds also showed good cell adhesion and spreading (68.7 ± 11.8 and 7.6 ± 3.3 total length and width, respectively) with no detectable effect on cell viability and cytotoxicity. The in vivo biocompatibility evaluation indicated that the scaffolds did not stimulate detectable cellular inflammatory response (lymphocytes) and increased the total cell number (cellularity) in the implantation area. Furthermore, the results suggest the potential use of the prepared ESFNS-10% bone marrow cell constructs in direct implantation for tissue engineering applications.

Development of novel electrospun nanofibrous scaffold from P. Ricini And A. Mylitta silk fibroin blend with improved surface and biological properties.

Biomaterials that stimulate cell attachment and proliferation without any surface modification (e.g. RGD coating) provide potent and cost effective scaffold for regenerative medicine. This study assessed the physico-chemical properties and cell supportive potential of a silk fibroin blend scaffold derived from eri (Philosamia ricini) and tasar (Antheraea mylitta) silk (ET) respectively by electrospinning process. The scanning electron microscopy and transmission electron microscopy study found that the fiber diameters are in 200 to 800nm range with flat morphology. The porosity of ET scaffold is found to be 79±5% with majority of pore diameter between 2.5 to 5nm. Similarly, Bombyx mori (BM) silk fibroin and gelatin nanofibrous scaffolds were prepared and taken as control. The ultimate tensile strength of the ET and BM scaffold are found to be 1.83±0.13MPa and 1.47±0.10MPa respectively. The measured contact angle (a measure of hydrophilicity) for ET (54.7°±1.8°) is found to be lower than BM (62°±2.3°). The ability to deposit apatite over ET is comparable to that of BM nanofibers. All the scaffolds were seeded with cord blood derived mesenchymal stem cells (hMSCs) and cultured for 14days in vitro. The immunofluorescence study reveals enhanced cell attachment with higher metabolic activity for MSCs grown over ET than BM and gelatin. The ET scaffold also demonstrated expression of higher amount cell adhesion molecules (CD29/CD44) and higher proliferation rate than BM and gelatin as confirmed by MTT assay, DNA content estimation assay, flow cytometry study and SEM study. Overall, it may be concluded that ET scaffold may have potential in developing bone tissue grafts for clinical applications in the future.

Experimental study of electrospun silk fibroin matrices seeded with urothelial cells for urethral reconstruction

Objective To investigate the feasibility of urethral reconstruction by using stretched electrospun silk fibroin matrices.Methods Stretched electrospun silk fibroin matrix was prepared,and the structure of the material was assessed by electron microscopy.Canine urothelial cells were isolated,expanded and seeded onto the material for 1 week to obtain a tissue-engineered graft.The tissue-engineered graft was assessed using HE staining and electron microscopy scanning.A dorsal urethral mucosa defect was created in 9 female beagle dogs.In the experimental group,tissue-engineered mucosa was used to repair urethral mucosa defects in 6 dogs.No substitute was used in the 3 dogs of the control group.Retrograde urethrography was performed at 1,2 and 6 months after grafting.The urethral grafts were analyzed grossly and histologically.Results Electron microscopy scanning revealed that the material had a 3 dimensional porous structure.Urothelial cells grew on the material and showed good biocompatibility with the stretched silk fibroin matrices.Canines implanted with tissue-engineered mucosa voided without difficulty.Retrograde urethrography revealed no signs of stricture,and histological staining showed gradual epithelial cell development and stratified epithelial layers at 1,2 and 6 months.The canines in the control group showed difficulty in voiding.Retrograde urethrography showed urethral stricture,and histological staining showed that no or only one layer of epithelial cells developed.A severe inflammatory reaction was also observed in the control group.Conclusion Stretched electrospun silk fibroin matrices have good biocompatibility with urothelial cells,and could be a potential material for urethral reconstruction. Key words: Urethra; Tissue engineering; Silk fibroin; Biomaterials; Urothelial cells

Preparation of electrospun silk fibroin nanofibers from solutions containing native silk fibrils

ABSTRACTElectrospinning solutions containing native silk fibrils with varied diameter and length were firstly achieved by dissolving silk in CaCl2/Formic acid solvents. The structure of nanofibrils significantly improved the spinnability of electrospinning solution. The diameter of electrospun silk fibroin (SF) nanofibers increased from 40 nm to 1.8 μm, which could be achieved through increasing the solution concentration from 2 to 10%, implying a good size control over a wide range in this process. The structure of SF nanofibers transferred from random coil to beta‐sheet, before and after ethanol treatment, respectively. The mechanical properties of the SF nanofibers were improved significantly with stress and strain at break of 11.15 MPa and 7.66% in dry state, and 3.32 MPa and 174.0% in wet state. The strategy for preparing SF nanofibers with improved mechanical properties and fiber diameter control over a wide range provides benefits for the application of this material. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci.2015,132, 41236.

Development of artificial dermis using 3D electrospun silk fibroin nanofiber matrix.

Several auto- or alloplastic dermal substitutes have been used for full thickness skin defects. However, there is no ideal dermal substitute that is biocompatible and offers adequate mechanical properties and sufficient thickness and volume. Silk fibroin can be considered a possible alternative material for the construction of dermal substitutes. Nanofibers fabricated by electrospinning silk fibroin were considered an excellent candidate due to their desirable biocompatibility. However, it is generally difficult for cells to infiltrate the electrospun silk fibroin due to its small pore size. To overcome this challenge, a key parameter is the control of the pore size of silk fibroin electrospun such that cells can easily infiltrate into the nanofibers and proliferate internally. In this work, a novel approach is introduced by dropping two different size NaCl crystals above the rotating collector, which become incorporated into the nanofibers. The structures and properties of the, resulting electrospun silk fibroin were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and its wound healing effect for a full thickness skin defect was compared with Matriderm. Although the dermal regenerative effect of the 3D-SF (3D electrospun silk fibroin nanofiber matrix) was similar to that of Matriderm, the 3D-SF was almost completely degraded and did not induce wound contracture like Matriderm. We suggest that 3D-SF can be applied to the treatment of full thickness skin defects.

Organotypic culture to assess cell adhesion, growth and alignment of different organs on silk fibroin.

Glass sheets covered with aligned electrospun silk fibroin (Bombyx mori) were compared to tissue culture-treated Thermanox® coverslips, using an organotypic culture method. Different chick embryo organ behaviours were analysed in terms of circularity, cell growth and cell adhesion after being cultivated in contact with these two materials. The circularity (cell layer shape corresponding to the trend of the biomaterials to induce a specific directionality) depends on the organ used when in contact with silk fibroin. This biomaterial induced higher cell adhesion (kidney) or lower cell adhesion (spine) compared to Thermanox. Cell growth, represented by the cell layer area (mm2 ), was also drastically reduced (gonad) or increased (blood vessel) on the silk fibroin. Organotypic culture is a rapid, cost effective and relatively simple method to evaluate different parameters, allowing prescreening of morphology and cytocompatibility to select the appropriate applications for new biomaterials. In the present study we compared the morphology of different organotypic cultures on orientated silk and Thermanox as growth supports to rapidly evaluate the benefit of a silk-based biomaterial for tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

The role of physiological mechanical cues on mesenchymal stem cell differentiation in an airway tract-like dense collagen–silk fibroin construct

Airway tracts serve as a conduit of transport in the respiratory system. Architecturally, these are composed of cartilage rings that offer flexibility and prevent collapse during normal breathing. To this end, the successful regeneration of an airway tract requires the presence of differentiated chondrocytes and airway smooth muscle cells. This study investigated the role of physiological dynamic mechanical stimulation, in vitro, on the differentiation of mesenchymal stem cells (MSCs), three-dimensionally seeded within a tubular dense collagen matrix construct-reinforced with rings of electrospun silk fibroin mat (TDC–SFC). In particular, the role of either shear stress supplied by laminar fluid flow or cyclic shear stress in combination with circumferential strain, provided by pulsatile flow, on the chondrogenic differentiation, and contractile lineage of MSCs, and their effects on TDC–SFC morphology and mechanical properties were analysed. Chondrogenic differentiation of MSCs was observed in the presence of chondrogenic supplements under both static and laminar flow cultures. In contrast, physiological pulsatile flow resulted in preferential cellular orientation within TDC–SFC, as dictated by dynamic circumferential strain, and induced MSC contractile phenotype expression. In addition, pulsatile flow decreased MSC-mediated collagen matrix remodelling and increased construct circumferential strength. Therefore, TDC–SFC demonstrated the central role of a matrix in the delivery of mechanical stimuli over chemical factors, by providing an in vitro niche to control MSC differentiation, alignment and its capacity to remodel the matrix.

Relationship between rheology and electro-spinning performance of regenerated silk fibroin prepared using different degumming methods

Electro-spun silk fibroin (SF) has been studied for biomedical applications because of its good biocompatibility, cyto-compatibility, and simple fabrication method. SF is obtained by a degumming process and the degumming method can affect the degree of molecular degradation of SF during the degumming process. In the present study, the effect of the degumming method on the rheology and electro-spinning performance of a silk solution was examined. In addition, the relationship between the rheology and electrospinnability was investigated. Regardless of the degumming method, all silk formic acid solutions exhibited almost Newtonian fluid behavior. The order of the viscosity of the silk solution was as follows: HTHP method > acid method > soap/soda method. An analysis of the correlation between the viscosity and electrospun morphology showed that the viscosity played a key role in determining the electro-spun morphology, and the critical viscosity for good fiber formation without beads in electro-spinning exists between 0.13 and 0.20 Pa·s. The viscosity also determines the maximum electro-spinning rate of the SF formic acid solution. The morphology and diameter of the electro-spun fiber were almost unaffected by the electro-spinning rate of the SF solution.

Mechanically-reinforced electrospun composite silk fibroin nanofibers containing hydroxyapatite nanoparticles

Electrospun silk fibroin (SF) scaffolds provide large surface area, high porosity, and interconnection for cell adhesion and proliferation and they may replace collagen for many tissue engineering applications. Despite such advantages, electrospun SF scaffolds are still limited as bone tissue replacement due to their low mechanical strengths. While enhancement of mechanical strengths by incorporating inorganic ceramics into polymers has been demonstrated, electrospinning of a mixture of SF and inorganic ceramics such as hydroxyapatite is challenging and less studied due to the aggregation of ceramic particles within SF. In this study, we aimed to enhance the mechanical properties of electrospun SF scaffolds by uniformly dispersing hydroxyapatite (HAp) nanoparticles within SF nanofibers. HAp nanoaprticles were modified by γ-glycidoxypropyltrimethoxysilane (GPTMS) for uniform dispersion and enhanced interfacial bonding between HAp and SF fibers. Optimal conditions for electrospinning of SF and GPTMS-modified HAp nanoparticles were identified to achieve beadless nanofibers without any aggregation of HAp nanoparticles. The MTT and SEM analysis of the osteoblasts-cultured scaffolds confirmed the biocompatibility of the composite scaffolds. The mechanical properties of the composite scaffolds were analyzed by tensile tests for the scaffolds with varying contents of HAp within SF fibers. The mechanical testing showed the peak strengths at the HAp content of 20wt.%. The increase of HAp content up to 20wt.% increased the mechanical properties of the composite scaffolds, while further increase above 20wt.% disrupted the polymer chain networks within SF nanofibers and weakened the mechanical strengths.

Effect of Sodium Carbonate Concentrations on the Formation and Mechanism of Regenerated Silk Fibroin Nanofibers by Electrospinning

Degumming is the first process for the preparation of all silk‐based products. In this paper, effect of sodium carbonate concentrations for silk degumming on the formation of electrospun silk fibroin nanofibers was investigated and the reason for the silk electrospinning process was explained for the first time by differences from the microstructure of regenerated silk fibroin. With increasing the sodium carbonate concentration, microstructure both in the aqueous solutions and in the electrospinning solutions transformed from nanofibrils to nanoparticles, leading to obvious changes on rheological property; electrospinning solutions with nanofibrils behaved like the native silk dope and owned remarkably higher viscosity than the solutions with nanoparticles showing very low viscosity. More interestingly, nanofibrils favored the formation of silk nanofibers with ease, and even nanofibers could be electrospun at concentration 2%. However, nanoparticles were completely unable to generate nanofibers at high spinning concentration 8%. Importance of sodium carbonate concentrations is heavily emphasized for impacting the microstructure types and further influencing the electrospinning performance of regenerated silk. Hence, sodium carbonate concentrations provide a controllable choice for the preparation of silk‐based electrospun biomaterials with desired properties.

Electrospun silk fibroin fiber diameter influences in vitro dermal fibroblast behavior and promotes healing of ex vivo wound models

Replicating the nanostructured components of extracellular matrix is a target for dermal tissue engineering and regenerative medicine. Electrospinning Bombyx mori silk fibroin (BMSF) allows the production of nano- to microscale fibrous scaffolds. For BMSF electrospun scaffolds to be successful, understanding and optimizing the cellular response to material morphology is essential. Primary human dermal fibroblast response to nine variants of BMSF scaffolds composed of nano- to microscale fibers ranging from ~250 to ~1200 nm was assessed in vitro with regard to cell proliferation, viability, cellular morphology, and gene expression. BMSF support of epithelial migration was then assessed through utilization of a novel ex vivo human skin wound healing model. Scaffolds composed of the smallest diameter fibers, ~250 -300 nm, supported cell proliferation significantly more than fibers with diameters approximately 1 μm (p < 0.001). Cell morphology was observed to depart from a stellate morphology with numerous cell -fiber interactions to an elongated, fiber-aligned morphology with interaction predominately with single fibers. The expressions of extracellular matrix genes, collagen types I and III (p < 0.001), and proliferation markers, proliferating cell nuclear antigen (p < 0.001), increased with decreasing fiber diameter. The re-epithelialization of ex vivo wound models was significantly improved with the addition of BMSF electrospun scaffolds, with migratory keratinocytes incorporated into scaffolds. BMSF scaffolds with nanofibrous architectures enhanced proliferation in comparison to microfibrous scaffolds and provided an effective template for migratory keratinocytes during re-epithelialization. The results may aid in the development of effective BMSF electrospun scaffolds for wound healing applications

Silk dissolution and regeneration at the nanofibril scale.

Native silk features strong, extensible and tough properties which originate from its hierarchical nanofibril structure. In the present study, native silk nanofibrils were obtained by dissolving degummed silk in salt-formic acid (FA), such as CaCl2-FA. The CaCl2-FA dissolved silk by breaking hydrogen bonds in the crystalline region while preserving the nanofibril structures. The dissolution behavior of silk from fibre to nanofibril was determined by Na2CO3 concentration during the degumming process, and was regulated by CaCl2 concentration during the dissolution process. The resulting solution containing silk fibroin (SF) nanofibrils could be easily processed into films with nanofibrous structures and high strength. In addition, high-quality electrospun SF nanofibres could also be generated easily from this solution. The novel dissolution and regeneration behavior of silk at the nanofibril scale provides new insights into methods for the preparation of high-quality silk materials for application in high-tech areas.

Facile and highly efficient approach for the fabrication of multifunctional silk nanofibers containing hydroxyapatite and silver nanoparticles

In this study, a good combination consisting of electrospun silk fibroin nanofibers incorporated with high-purity hydroxyapatite (HAp) nanoparticles (NPs) and silver NPs is introduced as antimicrobial for tissue engineering applications. The variable pressure field emission scanning electron microscope results confirmed randomly placed nanofibers are produced with highly dispersed HAp and silver NPs in nanofibers after electrospinning. The X-ray diffraction results demonstrated crystalline features of each of the three components used for electrospinning. Moreover, the TEM-EDS analysis confirmed the presence and chemical nature of each component over individual silk nanofiber. The FT-IR analyses was used confirm the different vibration modes caused due to functional groups present in silk fibroin, Hap, and silver NPs. The obtained nanofibers were checked for antimicrobial activity by using two model organisms Escherichia coli and Staphylococcus aureus. Subsequently, the antimicrobial tests have indicated that prepared nanofibers do possess good bactericidal activity. The ability of N,N-dimethylformamide and silk fibroin used to reduce silver nitrate into silver metal was evaluated using MTT assay. The nanofibers were grown in presence of NIH 3T3 fibroblasts, which revealed toxic behavior to fibroblasts at higher concentrations of silver nitrate used in this study. Furthermore, cell attachment studies on nanofibers for 3 and 12 days of incubation time were minutely observed and correlated with the results of MTT assay. The reported results confirmed the high amounts of silver nitrate can lead to toxic effects on viability of fibroblasts and had bad effect in cell attachment.

Electrospun silk fibroin composite nanofibrous mats loaded with vitamin A and E

Electrospun silk fibroin composite nanofibrous mats loaded with vitamin A and E

Bubble electrospun silk fibroin for tissue engineering scaffolds

Bubble electrospun silk fibroin for tissue engineering scaffolds