Electrospun Silk Fibroin Scaffolds Research Articles

Electrospun Silk Fibroin Scaffolds for Tissue Regeneration: Chemical, Structural, and Toxicological Implications of the Formic Acid-Silk Fibroin Interaction.

The dissolution of Bombyx mori silk fibroin (SF) films in formic acid (FA) for the preparation of electrospinning dopes is widely exploited to produce electrospun SF scaffolds. The SILKBridge® nerve conduit is an example of medical device having in its wall structure an electrospun component produced from an FA spinning dope. Though highly volatile, residual FA remains trapped into the bulk of the SF nanofibers. The purpose of this work is to investigate the type and strength of the interaction between FA and SF in electrospun mats, to quantify its amount and to evaluate its possible toxicological impact on human health. The presence of residual FA in SF mats was detected by FTIR and Raman spectroscopy (new carbonyl peak at about 1,725 cm−1) and by solid state NMR, which revealed a new carbonyl signal at about 164.3 ppm, attributed to FA by isotopic 13C substitution. Changes occurred also in the spectral ranges of hydroxylated amino acids (Ser and Thr), demonstrating that FA interacted with SF by forming formyl esters. The total amount of FA was determined by HS-GC/MS analysis and accounted for 247 ± 20 μmol/g. The greatest part was present as formyl ester, a small part (about 3%) as free FA. Approximately 17% of the 1,500 μmol/g of hydroxy amino acids (Ser and Thr) theoretically available were involved in the formation of formyl esters. Treatment with alkali (Na2CO3) succeeded to remove the greatest part of FA, but not all. Alkali-treated electrospun SF mats underwent morphological, physical, and mechanical changes. The average diameter of the fibers increased from about 440 nm to about 480 nm, the mat shrunk, became stiffer (the modulus increased from about 5.5 MPa to about 7 MPa), and lost elasticity (the strain decreased from about 1 mm/mm to about 0.8 mm/mm). Biocompatibility studies with human adult dermal fibroblasts did not show significant difference in cell proliferation (313 ± 18 and 309 ± 23 cells/mm2 for untreated and alkali-treated SF mat, respectively) and metabolic activity. An in-depth evaluation of the possible toxicological impact of residual FA was made using the SILKBridge® nerve conduit as case study, following the provisions of the ISO 10993-1 standard. The Potential Patient Daily Intake, calculated from the total amount of FA determined by HS-GC/MS, was 2.4 mg/day and the Tolerable Exposure level was set to 35.4 mg/day. This allowed to obtain a value of the Margin of Safety of 15, indicating that the amount of FA left on SF mats after electrospinning does not raise concerns for human health.

Chondrogenic Differentiation of Mesenchymal Stem Cells from Rat Bone Marrow on the Elastic Modulus of Electrospun Silk Fibroin Scaffolds

Chondrogenic Differentiation of Mesenchymal Stem Cells from Rat Bone Marrow on the Elastic Modulus of Electrospun Silk Fibroin Scaffolds

Reduced graphene oxide/titanium dioxide hybrid nanofiller-reinforced electrospun silk fibroin scaffolds for tissue engineering

Reduced graphene oxide/titanium dioxide (RGO@TiO2) hybrid nanoparticle was synthesized and used as a filler to study the synergistic effects on electrospun regenerated silk fibroin (RSF) mats. The incorporation of the hybrid filler increased the average diameter of RSF fibers, but decreased the content of β-sheet conformation. The breaking strength of RSF/RGO@TiO2 composite mat increased by 220%. Moreover, the composite mat supported the growth and expansion of cells and had a good antibacterial property. This study may provide a new development perspective of multifunctional nanocomposite based on RGO@TiO2 hybrid filler.

Characterization of Electrospun Silk Fibroin Scaffolds for Bone Tissue Engineering: A Review

Silk Fibroin (SF) is a natural polymer obtained from the Bombyx mori silkworm. It has been used in bone tissue engineering thanks to its favorable biocompatibility, adhesion, low biodegradability, and tensile strength properties. Electrospinning is a technique to develop nanofibers. It uses high voltages to convert polymer solutions into porous nanostructured scaffolds with a good ratio between superficial area and volume. In this paper, we examine the effect of the electrospinning parameters on fiber morphology once the spun fibers have been treated. In addition, we present different physicochemical characterizations of electrospun SF scaffolds such as their morphology (via Scanning Electron Microscopic—SEM—), crystalline structure (via Fourier Transform Infrared—FTIR—spectroscopy and X-Ray Diffraction—XRD—), thermal characteristics (via Differential Scanning Calorimetry—DSC—and Thermogravimetric Analysis—TGA—), and mechanical properties (tensile strength). Finally, we discuss the potential applications and impacts of electrospun SF in bone tissue engineering and future research trends.

Differential Effects of Directional Cyclic Stretching on the Functionalities of Engineered Cardiac Tissues.

Cardiac tissue engineering aims to regenerate functional cardiac tissues through recapitulating the native microenvironmental cues, where cardiomyocytes are highly oriented and rhythmically contract along their long-axis direction in the native myocardium. In addition, the different oriented layers of the left ventricle lead to a dynamic biaxial stretching on cardiomyocytes, which makes the mechanical microenvironment in the myocardium much more complex. Thus, it is important to investigate the effect of dynamic mechanical stimulation along different directions to preoriented cardiomyocytes on the functionalities of engineered cardiac tissues. In this study, we regenerate oriented cardiac tissues using electrospun silk fibroin scaffolds, followed by applying the dynamic mechanical stimulation parallel or perpendicular to the cell orientation. The design and utilization of nanofibrous scaffolds combining the aligned topography and random substrate ensure the realization of dynamic mechanical stimulation with a homogeneous strain along both directions. The results reveal that the parallel mechanical stretching promotes the alignment of cardiomyocytes and the formation of sarcomeres and gap junctions, while the perpendicular stimulation interferes with the cell alignment and gap junction formation. This study would provide guidance for promoting the functionalities of regenerated cardiac tissues through directional dynamic mechanical stimulation.

Surface Modification of Multiple Bioactive Peptides to Improve Endothelialization of Vascular Grafts.

Endothelialization is an effective approach to prevent thrombus formation and enhance vascular graft survival. Surface modification of biomolecules has been proved to be effective in regulating endothelial cell behaviors. In this study, several peptides including YIGSR, RGD, and REDV sequences are covalently immobilized on the surface of electrospun silk fibroin scaffolds and the effects of combined application of these peptides on cell behaviors are studied. The results show that, compared with the scaffolds modified with single peptides, the scaffolds modified with dual peptides (YIGSR+RGD) could significantly enhance the proliferation of human umbilical vein endothelial cells (HUVECs). However, the combination of REDV+RGD or YIGSR+REDV does not promote the adhesion or proliferation of HUVECs. Notably, YIGSR-modified scaffolds improved HUVEC migration significantly in comparison to REDV- or RGD-modified groups. Moreover, its combination with either of these two peptides also presents excellent effect on cell migration. Thus, all the data suggest that the combined application of peptides might be a promising method to enhance the endothelialization of small-diameter vascular grafts.

Comparison of silk fibroin electrospun scaffolds with poloxamer and honey additives for burn wound applications

A primary aim in wound-healing research is to construct an inexpensive, biodegradable dermal regeneration template with heightened moisture retention and permeability properties. The presence of moisture is important for optimal burn wound healing as it creates an environment for re-epithelialization and minimizes the risk of infections. Permeability can be achieved through a process known as electrospinning. This scaffold fabrication technique creates a mat of randomly oriented nanofibers that can readily mimic native extracellular matrix. Novel electrospun silk fibroin scaffolds were fabricated with poloxamer 407 (P407) and Manuka honey additives for a burn wound dermal regeneration template application. Enhanced human dermal fibroblast adhesion and cell infiltration were observed with the inclusion of P407, and scaffolds incorporated with Manuka honey demonstrated increased water uptake and a higher cell density within the scaffolds at the end of a 28-day period. Overall, this study established that both the silk fibroin/P407 and silk fibroin/Manuka honey scaffolds have the potential to be successful dermal regeneration templates, with P407 increasing surface wettability and Manuka honey modulating moisture retention.

Electrospun silk fibroin scaffolds coated with reduced graphene promote neurite outgrowth of PC-12 cells under electrical stimulation

Novel approaches to neural research require biocompatible materials capable to act as electrode structures or scaffolds for tissue engineering in order to stimulate or restore the functionality of damaged tissues. This work offers promising results that indicate the potential use of electrospun silk fibroin (SF) scaffolds coated with reduced graphene oxide (rGO) in this sense. The coated material becomes conductor and electroactive. A complete characterisation of SF/rGO scaffolds is provided in terms of electrochemistry, mechanical behaviour and chemical conformation of fibroin. The excellent biocompatibility of this novel material is proved with cultures of PC-12 cells. The coating with rGO improved the adhesion of cells in comparison with cells growing onto the surface of pure SF scaffolds. Also, the use of SF/rGO scaffolds combined with electrical stimulation promoted the differentiation into neural phenotypes reaching comparable or even superior levels to those obtained by means of the traditional treatment with neural growth factor (NGF).

Three-dimensional electrospun silk-fibroin nanofiber for skin tissue engineering

Tissue-engineered skin substitutes may offer an effective therapeutic option for the treatment of patients with skin damages. In this study, a novel three-dimensional (3D) scaffold composed of electrospun silk fibroin (SF) nanofiber was fabricated using electrospinning with the addition of NaCl crystals. It has well known that the electrospun SF nanofibers were excellent scaffold for tissue. However, it is generally difficult for cells to infiltrate the electrospun silk fibroin due to its small pore size. To resolve this problem, we dropped the NaCl crystals above the rotating collector, which become incorporated into the nanofibers. Three methods (freeze-drying, salt-leaching, and electrospinning with NaCl) for fabrication of SF scaffolds were compared to the difference of their characteristics using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), mechanical strength, porosity, swelling abilities, and cell proliferation. Additionally, using air-liquid culture system, keratinocytes were co-cultured with fibroblasts in each type of SF scaffolds to construct an artificial bilayer skin in vitro. In our experimental results, histologic findings in only electrospun SF scaffolds showed more proliferation of fibroblasts in deep layer and more differentiation of keratinocytes in superficial layer. The present study suggests that 3D electrospun SF scaffolds might be a suitable for skin tissue engineering.

Fabrication of electrospun silk fibroin scaffolds coated with graphene oxide and reduced graphene for applications in biomedicine

Silk fibroin and graphene are both promising biomaterials described in the bibliography. Hybrid scaffolds combining their properties could be attractive for tissue engineering applications. In this work, a new methodology to produce electrospun fibroin scaffolds coated with graphene materials is provided. The mechanical, electrical and electrochemical properties of the materials attained were characterised. The fibre diameters were measured (from 3.9 to 5.2μm). The samples coated with reduced graphene were electronic conductors and electroactive in liquid electrolytes, showing maximum oxidation and reduction (around −0.4V peak). The chronoamperometric responses showed a reduction shoulder, pointing to the entrance of balancing cations from the solution by nucleation–relaxation: the reaction induced structural changes in the graphene. In order to check the biocompatibility of the materials, they were seeded with L929 fibroblasts. The excellent biocompatibility of silk fibroin meshes was maintained after coating with graphene, being the proliferation results equal in all the treatments 7days after the seeding (Tukey, p>0.05).The conductive and electroactive properties of meshes coated with reduced graphene allow the potential application of local electric fields or local ionic currents to cell cultures, biological interfaces or animal models without host response.

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.

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.

Creation of macropores in electrospun silk fibroin scaffolds using sacrificial PEO‐microparticles to enhance cellular infiltration

Electrospun scaffolds are widely used in tissue engineering; however, a common problem is the poor cell infiltration because of the small pore size and tightly packed structure of these fibrous scaffolds. To address this issue, a novel technique was developed to fabricate electrospun silk fibroin (SF) scaffolds with rather macropores and high porosity using electrospraying-generated PEO microparticles as porogen. The morphology and pore size of MPES scaffolds were evaluated by scanning electron microscopy. It was revealed that MPES scaffold had a relatively loose structure with an increase of mean pore size (i.e., approx. 30 μm of MPES vs. approx. 5 μm of traditional electrospun scaffolds (TES) and porosity (i.e., 95% vs. 84% of TES). Culture of mouse 3T3 fibroblast in TES and MPES scaffold revealed that both scaffolds could support cell attachment, spread and proliferation. Yet, cell inflitration in vitro under the static culture condition only occurred in the MPES scaffold. Subcutaneous implantation of scaffolds in rats further confirmed that the tissue ingrowth was more efficient in the MPES scaffold compared to TES scaffold. Thus, the use of PEO microparticles as porogen was a feasible and effective method for creating macroporous electrospun SF scaffold, which provided an alternative to address the limitation of cell infiltration associated with electrospun fibrous scaffold.

Fabrication of conductive electrospun silk fibroin scaffolds by coating with polypyrrole for biomedical applications

Scaffolds constituted by micro and nanofibers of silk fibroin were obtained by electrospinning. Fibers of fibroin meshes were coated with polypyrrole (pPy) by chemical polymerization; chemical linkages between polymers were observed by SEM and IR spectroscopy. Mechanical resistance of the meshes was improved by polypyrrole coating. Furthermore, coated meshes present a high electroactivity allowing anion storage and delivery during oxidation/reduction reactions in aqueous solutions. Uncoated and pPy coated materials support the adherence and proliferation of adult human mesenchymal stem cells (ahMSCs) or human fibroblasts (hFb). The bioactivity of fibroin mesh overcomes that of the polypyrrole coated meshes.

In vitro and in vivo degradation behavior of aqueous-derived electrospun silk fibroin scaffolds

We investigated the in vitro degradation behavior of the electrospun silk fibroin (SF) scaffolds by protease XIV. Phosphate-buffered saline (PBS) without enzyme was used as a control. About 65% of the electrospun SF scaffolds were degraded within 24 d in protease XIV, while almost no scaffolds were degraded in PBS. A great deal of fragments was visible in protease XIV solution. SEM indicated surface erosion of the scaffolds increased during protease degradation with increasing exposure time. FTIR and XRD indicated the crystalline structure of the scaffolds decreased after protease degradation for 24 d, and a degradation mechanism was proposed. Furthermore, the results of the in vivo degradation by implantation in rats showed that the scaffolds were completely degraded in vivo after implantation for 8 weeks and well tolerated by the host animals. The insights gained in this study can serve as a guide to match desired degradation behavior with specific applications for the electrospun SF scaffolds, such as tissue engineering and drug delivery.

Electrospun Silk Fibroin Scaffolds with Macropores for Bone Regeneration: An In Vitro and In Vivo Study

We developed three-dimensional electrospun silk fibroin (ESF) scaffolds with controllable pore size. The purpose of this study was to evaluate ESF scaffolds with pores (P-ESF) for bone regeneration via in vitro and in vivo studies, with a comparison to a commercially available porous three-dimensional polylactic acid (PLA) scaffold. P-ESF supported significantly higher proliferation and alkaline phosphatase activity of osteoblasts than PLA in vitro (p < 0.05). Moreover, higher expression levels of activated adhesion-related proteins, including focal adhesion kinase, were observed in the P-ESF than in PLA, as confirmed by western blot analyses. Microcomputed tomography revealed that 78.30% of the original bone volume was attained in the P-ESF implantation group at 7 weeks after critical bone defect formation in rat calvaria. Comparatively, the PLA implantation group showed only 49.31%. Histological evaluation also showed new bone tissue formation upon P-ESF implantation. Taken together, the P-ESF scaffold may be a good bone substitute for bone regeneration.

Guidance of Olfactory Ensheathing Cell Growth and Migration on Electrospun Silk Fibroin Scaffolds

Transplantation of olfactory ensheathing cells (OECs) is a potential treatment for spinal cord injury (SCI). However, this process lacks extracellular matrix guiding cell growth, tissue morphogenesis, and remodeling. In order to solve this problem, we fabricated silk fibroin scaffolds (SFS) with different fiber diameters by electrospinning. The behaviors of OECs on 300 and 1800 nm SFS were studied by analyzing cell morphological feature, distribution, and proliferation. The results showed the 300 nm SFS with good potential to guide OECs growth. Subsequently, the properties of 300 nm SFS were further investigated along with PLL. With 300 nm SFS, the preservation of cell phenotype was confirmed by the presence of cell-specific markers, including nerve growth factor receptor p75 and glial fibrillary acidic protein. And the migration behaviors of OECs were also observed by Leica AF6000. In addition, migration tracks, turning behavior, migration distances, migration speeds, and forward migration indices were calculated. Furthermore, the expression of neurotrophic factors was assayed at transcription and protein levels using RT-PCR and ELISA. All these results indicated the diameter of the fiber played an important role in guiding cell adhesion, growth, and migration in vitro and the 300 nm SFS could be suitable to construct tissue-engineered scaffolds for SCI repair.

Electrospun scaffolds from silk fibroin and their cellular compatibility

Electrospinning offers an attractive opportunity for producing silk fibroin (SF) nano/micro fibrous scaffolds with potential for tissue regeneration and repair. Electrospun scaffolds of silk fibroin were fabricated as a biomimetic scaffold for tissue engineering. The morphology of the electrospun scaffolds was investigated with SEM and AFM. The SEM images indicated that electrospun SF fibers were ribbon-shaped and the average width increased with increasing SF concentrations. The AFM images revealed that, after treated with methanol, there was a groove on the surface of fiber, which is conducive to cell attachment. The structure of electrospun SF fibers was characterized by NMR, WAXD, and DSC. The results displayed that SF in electrospun fibers was present in a random coil conformation, SF conformation transformed from random coil to beta-sheet when treated with methanol. Cell attachment and proliferation studies with pig iliac endothelial cells (PIECs) demonstrated that electrospun SF scaffolds significantly promoted cell attachment and proliferation in comparison with cast SF films. These results suggest electrospun SF scaffolds may be potential candidates for cardiovascular tissue engineering.

Dynamic culture conditions to generate silk-based tissue-engineered vascular grafts

Tissue engineering is an alternative approach for the preparation of small-diameter (<6 mm) vascular grafts due to the potential to control thrombosis, anastomotic cellular hyperplasia and matrix production. This control also requires the maintenance of graft patency in vivo, appropriate mechanical properties and the formation of a functional endothelium. As a first step in generating such tissue-engineered vascular grafts (TEVGs), our objective was to develop a tissue-engineered construct that mimicked the structure of blood vessels using tubular electrospun silk fibroin scaffolds (ESFSs) with suitable mechanical properties. Human coronary artery smooth muscle cells (HCASMCs) and human aortic endothelial cells (HAECs) were sequentially seeded onto the luminal surface of the tubular scaffolds and cultivated under physiological pulsatile flow. The results demonstrated that TEVGs under dynamic flow conditions had better outcome than static culture controls in terms of cell proliferation and alignment, ECM production and cell phenotype based on transcript and protein level assessments. The metabolic activity of HCASMCs present in the TEGs indicated the advantage of dynamic flow over static culture in effective nutrient and oxygen distribution to the cells. A matrigel coating as a basement membrane mimic for ECM significantly improved endothelium coverage and retention under physiological shear forces. The results demonstrate the successful integration of vascular cells into silk electrospun tubular scaffolds as a step toward the development of a TEVG similar to native vessels in terms of vascular cell outcomes and mechanical properties.

In vitro evaluation of electrospun silk fibroin scaffolds for vascular cell growth

Human aortic endothelial (HAEC) and human coronary artery smooth muscle cell (HCASMC) responses on electrospun silk fibroin scaffolds were studied to evaluate potential for vascular tissue engineering. Cell proliferation studies supported the utility of this biomaterial matrix by both HAECs and HCASMCs. Alignment and elongation of HCASMCs on random non-woven nanofibrous silk scaffolds was observed within 5 days after seeding based on SEM and confocal microscopy. Short cord-like structures formed from HAECs on the scaffolds by day 4, and a complex interconnecting network of capillary tubes with identifiable lumens was demonstrated by day 7. The preservation of cell phenotype on the silk fibroin scaffolds was confirmed by the presence of cell-specific markers, including CD146, VE-cadherin, PECAM-1 and vWF for HAECs, and SM-MHC2 and SM-actin for HCASMCs at both protein and transcription levels using immunocytochemistry and real-time RT-PCR, respectively. Formation of ECM was also demonstrated for the HCASMCs, based on the quantification of collagen type I expression at protein and transcription levels. The results indicate a favorable interaction between vascular cells and electrospun silk fibroin scaffolds. When these results are factored into the useful mechanical properties and slow degradability of this protein biomaterial matrix, potential utility in tissue-engineered blood vessels can be envisioned.