Preparation and characterization of blended <Emphasis Type="Italic">Bombyx mori</Emphasis> silk fibroin scaffolds

The aim of this study was to compare physical, mechanical and biological properties of 3-dimensional scaffolds prepared from Bombyx mori silk fibroin (SF), fibroin blended with collagen (SF/C), and fibroin blended with gelatin (SF/G) using a freeze-drying technique. The prepared scaffolds were sponge-like structure that exhibited homogeneous porosity with highly interconnected pores. Average pore size of these scaffolds ranged from 65–147 μm. All biodegradable scaffolds were capable of water absorption of 90 %. The degradation behavior of these scaffolds could be controlled by varying the amount of blended polymer. The SF/C and SF/G scaffolds showed higher compressive modulus than that of SF scaffolds which could be attributed to the thicker pore wall observed in the blended constructs. The less crystalline SF structure was observed in SF/G scaffolds as compared to SF/C scaffolds. Thus, the highest compressive modulus was observed on SF/C matrix. To investigate the feasibility of the scaffolds for cartilage tissue engineering application, rat articular chondrocytes were seeded onto the scaffolds. The MTT assay demonstrated that blending collagen or gelatin into SF sponge facilitated cell attachment and proliferation better than SF scaffolds. The blended SF scaffolds possessed superior physical, mechanical and biological properties in comparison to SF scaffolds and showed high potential for application in cartilage tissue engineering.     

Mechanism of silk fibroin scaffolds with oriented multichannels and its cytocompatibility

As a biomaterial, besides excellent biocompatibility and biodegradability, suitable macropores and pores structure should be provided to guide cell extension and migration. In present study, the silk fibroin (SF) scaffold with uniaxial channels was prepared by directional temperature field freezing technique. The average pore diameter, pore density and porosity of the scaffold with oriented channels are ～128.7 µm, ～158 mm^?2 and ～91.4 %, respectively. By controlling of the temperature gradient direction, the oriented multichannels of the scaffolds were formed in longitudinal easily. In process of the scaffolds fabrication, the directional growth of ice crystal could shear and draft to the silk fibroin molecule segments, which resulted in the new crystal nucleus formation in new zone and increase of β-sheet components in the scaffolds. In vitro, L929 cells were seeded on the scaffolds with oriented channels to evaluate the effect on cell behavior. Cell viability, adhesion and morphology were determined by methyl thiazolyl tetrazolium, confocal microscope and scanning electron microscope. The results showed that the cells anchored on the oriented channels, spread along the direction of the channels and hold a higher viability on the scaffolds with oriented channels. These new oriented multichannel scaffold could guide the adhesion and proliferation of L929 cells, which hold a potential in tissue engineering.     

Growth of primary hippocampal neurons on multichannel silk fibroin scaffold

Particular attention has been given to axonal outgrowth of neurons to understand how topographical surface cues influence attachment and subsequent directional migration and growth. In present study, the silk fibroin (SF) scaffold with uniaxial channels was prepared by directional freeze-drying processes. The average pore diameter, the porosity, and pore density of the scaffold are 120 µm, 88 %, and 203 mm^?2, respectively. Further, hippocampal neurons were seeded onto the scaffold and the hippocampal neurons morphology was investigated. Cell-cell networks and cell-matrix interactions had been established by newly formed axons and the diversity of neurons was much higher after culturing 7 days. The neurons expressed β-III-tubulin and nerve filament, while glial fibrillary acidic protein immunofluorescence was barely above background. These results indicated that the SF scaffolds with uniaxial multichannels could be guided axons of neurons spread along the channels. SF scaffolds with oriented pores have a potential for nerve tissue regeneration.     

Chondrogenic differentiation of rat mesenchymal stem cells on silk fibroin/chondroitin sulfate/hyaluronic acid ternary scaffolds

Cartilage repair is a challenge in bone tissue reconstruction. In this study, silk fibroin (SF), chondroitin sulfate (CS) and hyaluronic acid (HA) were employed to fabricate scaffolds for tissue engineered cartilage by freeze drying technique. The secondary pores were formed in the main pores of SF/CS/HA scaffold which improved the pore connectivity and equilibrium swelling of the scaffold. Furthermore, rat bone marrow mesenchymal stem cells were seeded on the scaffolds to evaluate the cell adhesion and proliferation. Results of hematoxylin/eosin staining and cell counting kit-8 assay showed that the cells migration and differentiation of SF/CS/HA (80/15/5) scaffold were better than that of SF/CS/HA scaffolds with different ratios after 7 days culture. Moreover, immunohistochemistry and scanning electron microscope demonstrated that large amounts of collagen II and proteoglycans of the cells were expressed in the SF/CS/HA 3D scaffold, while the expression of collagen I was barely visible by immunohistochemistry. Abound of extracellular matrix was formed to morphologically round and distributed uniformly throughout the scaffolds. The 3D ternary scaffold could promote the cells chondrogenic differentiation without using any inductive agent and offer potential for cartilage tissue regeneration.     

Silk fibroin/hyaluronic acid porous scaffold for dermal wound healing

The use of silk protein as a biomaterial has been studied for decades. In this study, silk fibroin (SF)/hyaluronic acid (HA) blend scaffolds were prepared by freeze-drying technique. The structure and properties of the blend scaffolds were examined and analyzed. The results demonstrated that the secondary structures of the SF/HA scaffolds were mainly amorphous and β-sheet structures. The pore radius and porosity of the scaffolds decreased with a decrease in the freezing temperature decrease and an increase in the HA ratio. The pore radius and porosity were regulated from 32.22 μm to 290.76 μm and from 74.1 % to 91.15 %, respectively. In vitro, the SF/HA scaffolds could support the fibroblast cell adhesion and proliferation and showed good cytocompatibility. In vivo, the SF/HA scaffolds were implanted into the dorsum of Sprague Dawley rats to evaluate their bioactivity for dermal tissue reconstruction. The vascular-like structures appeared more rapidly in SF/HA scaffolds than that in the PVA group, and a new dermal layer was formed, as determined by histological analysis. The SF/HA porous scaffolds have promise as a dermal substitute.     

Electrospinning of <Emphasis Type="Italic">Bombyx mori</Emphasis> silk fibroin nanofiber mats reinforced by cellulose nanowhiskers

Cellulose nanowhisker (CNW) reinforced electrospun Bombyx mori silk fibroin (SF) nanofibers were fabricated. The morphology, structure, and mechanical properties of nanofibers were investigated by FE-SEM, TEM, FTIR, and tensile testing. It was found that the nanofiber size decreased obviously from 250 nm in the unreinforced mat to 77–160 nm in the CNW reinforced mats depending on the CNW content due to the increased conductivity of spinning dope. In the reinforced mats, the CNWs were embedded in the SF matrix separated from each other, and aligned along the fiber axis. There was a positive correlation between the CNW content and the tensile strength and Young’s modulus of reinforced mats. However the strain at break dropped gradually with the increase of CNW. When the CNW content was 2 w/w%, the tensile strength and Young’s modulus of reinforced SF nanofiber mats were about 2 times higher than those of unreinforced mat.     

Application of electrospun silk fibroin nanofibers as an immobilization support of enzyme

Silk fibroin (SF) nanofibers were prepared by electrospinning and their application as an enzyme immobilization support was attempted. By varying the concentration of SF dope solution the diameter of SF nanofiber was controlled. The SF nanofiber web had high capacity of enzyme loading, which reached to 5.6 wt%. The activity of immobilizedα-chymotrypsin (CT) on SF nanofiber was 8 times higher than that on silk fiber and it increased as the fiber diameter decreased. Sample SF8 (ca. 205 nm fiber diameter) has excellent stability at 25°C by retaining more than 90 % of initial activity after 24 hours, while sample SF11 (ca. 320 nm fiber diameter) shows higher stability in ethanol, retaining more than 45% of initial activity. The formation of multipoint attachment between enzyme and support might increase the stability of enzyme. From these results, it is expected that the electrospun SF nanofibers can be used as an excellent support for enzyme immobilization.     

Fabrication of silk fibroin/eggshell nanofiber membranes for facemasks

We report our study on fabrication of soluble eggshell membrane (SESM) and silk fibroin (SF) nanofibers composite (SF/SESM) for facemasks by electrospinning. Biocompatibility of the SF and SESM, determined from hydrophilicity results, is exploited in SF/SESM nanocomposite for facemask application. The SF/SESM nanocomposites were prepared in different ratios of SF and SESM. The samples were characterized by scanning electron microscopy (SEM), FTIR and water droplet adsorption tests conducted via water contact angle (WCA) and water droplet diffusion. The results revealed that addition of SESM has insignificant effect on the electrospinnability of SF nanofibers in the studied ratios. The SEM results depicted regular morphology of the nanofibers except increase in nanofiber diameter with addition of SESM. The FTIR results confirmed respective peaks of SF and SESM in SF/SESM nanocomposite. WCA of the nanofibers decreased with addition of SESM such that for SF/SESM30, 30 % SESM, it reduced to 0 ° from 101 ° for pure SF nanofibers. The research results demonstrate SF/SESM30 nanocomposite as optimum ratio of SF and SESM for facemasks and other biomedical applications.     

EDC/NHS crosslinked electrospun regenerated tussah silk fibroin nanofiber mats

The tussah silk fibroin (TSF) nanofibers with 611 nm diameters were prepared by electrospinning with the solvent hexafluoroisopropanol (HFIP). And then, the TSF nanofibers were crosslinked by 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide/N-Hydroxysuccinimide (EDC/NHS) crosslinking agent. The morphology and microstructure of the crosslinked TSF nanofibers were characterized by scanning electron microscopy (SEM), Fourier transforms infrared analysis (FTIR), X-ray diffraction, Instron electronic strength tester, and cell culture. After treatment with EDC/NHS crosslinking agent, the TSF nanofibers swelled and its average diameter increased from 611 to 841 nm. FTIR and X-ray diffraction results demonstrated that random coil, ??-helix, and ??-sheet co-existed in the TSF nanofiber mats, but the content of ??-sheet increased from 25.26 to 45.34 %, and the random coil content decreased from 32.47 to 24.94 %. Compared with the electrospun pure TSF nanofiber mats, the crosslinked TSF nanofiber mats exhibited a lower breaking tenacity and initial modulus, which were 5.51 MPa and 9.86 MPa, respectively. At the same time, the extension at break of the crosslinked TSF nanofiber achieved 109.38 %. In cell culture evaluation, the crosslinked TSF nanofibers were found to support cell adhesion and spreading fibroblast L373 and bone marrow mesenchymal stem cells (BMSCs), which had potential utility in a range of tissue engineering.     

Enhanced mechanical properties of silk fibroin-based composite plates for fractured bone healing

We successfully fabricated bacterial cellulose/silk fibroin (BC/SF) composite plates having similar strength to that of human cortical bone (12.8–17.7 GPa). The mechanical properties of the BC/SF composite plates were investigated at various BC nanofiber contents. The BC nanofibers acted as good reinforcements for the stress transfer produced by the interactions between the BC nanofibers and the SF matrix, as confirmed by the molecular deformation of the BC nanofibers. The BC/SF composite plates have a promising potential as a replacement material for existing metal bone plate.     

Preparation and characterization of Ceftazidime loaded electrospun silk fibroin/gelatin mat for wound dressing

Fabrication of Ceftazidime (CTZ) loaded silk fibroin/gelatin (SF/GT) nanofibers (NFs) without the loss of structure and bioactivity of CTZ was demonstrated by electrospinning method. The structure, morphology and mechanical properties of the electrospun SF/GT nanofibrous mats were characterized using FT-IR, SEM and DSC. The drug release profile of different electrospun fibers was analyzed using spectrophotometric method, and also diffusion method was applied to assess the antibacterial effect of NFs. Cell viability was evaluated by MTT assay. The results show that the average diameter of drug loaded NFs at the optimum polymer to drug feeding ratio (10:1) was 276.55±35.8 nm, while increasing the feeding ratio to 1:1 increases the average diameter to 825.02±70.3 nm. FT-IR of drug loaded NFs was revealed that CTZ was successfully encapsulated into NFs while viability study approved cytocompatibility of SF/GT NFs. CTZ was released from NFs during 6 h, and formation of inhibition zone in diffusion test demonstrated the antibacterial effect of drug loaded NFs. Altogether, the CTZ loaded SF/GT NFs can improve the drug effectiveness particularly in the prevention of post-surgical adhesions and infections for wound dressing.     

Highly porous three-dimensional poly(lactide-co-glycolide) (PLGA) microfibrous scaffold prepared by electrospinning method: A comparison study with other PLGA type scaffolds on its biological evaluation

In this study, a three-dimensional (3D) poly(lactide-co-glycolide) (PLGA) microfibrous scaffold with high porosity (ca. 90 % porosity) was developed for evaluating its performance in tissue engineering application. A dope solution of PLGA/polyethylene oxide (PEO) blend was electrospun into a methanol coagulation bath for fabricating highly porous 3D PLGA scaffold and a salt leaching method was used for making interconnected pores of 100?C200 ??m size inside the scaffold. The morphological structure, pore size and porosity of the microfibrous scaffold were determined, and compared with twodimensional (2D) mat-type and 3D sponge-type of PLGA scaffold. Also, swelling ratio, water uptake and compressive strength were compared in order to elucidate the structure-property relationships of different types of the scaffolds, especially in a wet condition. As a result of scanning electron microscopy (SEM) observation, normal human dermal fibroblasts (nHDF) were migrated, attached, and proliferated well inside the 3D scaffold. MTT assay confirmed that the highly porous 3D PLGA microfibrous scaffold had superior cell adhesion and proliferation abilities due to fibrous structure of large specific surface area, and interconnected pore structure. Therefore, this high performance 3D PLGA scaffold can have a high potentiality for application in tissue engineering in comparison with conventional PLGA scaffolds.     

Three-dimensional porous collagen/chitosan complex sponge for tissue engineering

A three-dimensional, porous collagen/chitosan complex sponge was prepared to closely simulate basic extracellular matrix (ECM) constitutes, collagen and glycosaminoglycan. The complex sponge was prepared by a lyophilization method and had the regular network with highly porous structure, suitable for cell adhesion and growth. The pores were well interconnected, and their distribution was fairly homogeneous. The complex sponge was crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to increase its biological stability and enhance its mechanical properties. The crosslinking medium had a great effect on the inner structure of the sponge. The homogeneous, porous structure of the sponge was remarkably collapsed in an aqueous crosslinking medium. However, the morphology of the sponge remained almost intact in a water/ethanol mixture crosslinking milieu. Mechanical properties of the collagen/chitosan sponge were significantly enhanced by EDC-mediated crosslinking. The potential of the sponge as a scaffold for tissue engineering was investigated using a Chinese hamster ovary cell (CHO-K1) line.     

Polyanizidine and Polycaprolactone Nanofibers for Designing the Conductive Scaffolds

The main objective of this work was chemically bioactivation of the conducting polyanizidine (PANIZ) by incorporating a polyester such as polycaprolactone (PCL). Modified PANIZ nanocomposites were synthesized through ring opening and chemical oxidation polymerizations. A four-point probe was applied to measure the conductivity of newly synthesized star-like block copolymer (S-PCL-PANIZ) nanocomposite, which was about 0.44 S cm^-1. Conductive biodegradable nanofibers were prepared by electrospinning with 25 and 75 % (wt/wt) S-PCL-PANIZ to PCL. The contact angle of each prepared nanofiber was 87±3°, supporting their usefulness for cell culture. The cultured mouse osteoblast MG63 cells demonstrated normal morphology and significantly higher adhesion and spreading on the nanofiber. The bioactivated PANIZ based nanocomposite may be fruitful in tissue engineering to fabricate conducting biodegradable scaffolds with improved cell adhesion properties for various cell cultures.     

Metal ion adsorbability of electrospun wool keratose/silk fibroin blend nanofiber mats

In this study, electrospun wool keratose (WK)/silk fibroin (SF) blend nanofiber was prepared and evaluated as a heavy metal ion adsorbent which can be used in water purification field. The WK, which was a soluble fraction of oxidized wool keratin fiber, was blended with SF in formic acid. The electrospinnability was greatly improved with an increase of SF content. The structure and properties of WK/SF blend nanofibers were investigated by SEM, FTIR, DMTA and tensile test. Among various WK/SF blend ratios, 50/50 blend nanofiber showed an excellent mechanical property. It might be due to some physical interaction between SF and WK molecules although FTIR result did not show any evidence of molecular miscibility. As a result of metal ion adsorption test, WK/SF blend nanofiber mats exhibited high Cu²⁺ adsorption capacity compared with ordinary wool sliver at pH 8.5. It might be due to large specific surface area of nanofiber mat as well as numerous functional groups of WK. Consequently, the WK/SF blend nanofiber mats can be a promising candidate as metal ion adsorption filter.     

Montelukast incorporated poly(methyl vinyl ether-co-maleic acid)/poly(lactic-co-glycolic acid) electrospun nanofibers for wound dressing

The aim of the present study was to prepare nanofibers loaded with montelukast, a cysteinyl leukotrienes (CysLTs) inhibitor, with anti-inflammatory properties effective on wound healing. Polymeric nanofibers containing montelukast were spun by electrospinning method using different ratios of the blend of two biodegradable polymers of poly(methyl vinyl etherco-maleic acid) (PMVEMA) and poly(lactic-co-glycolic acid) (PLGA) at the total polymer concentration of 37 %, the distance of the needle to rotating screen of 19 cm, the voltage of 12 Kv and the rate of injection of 0.2 ml/h. The ratio of two polymers in the blend and the concentration of montelukast were optimized based on the diameter of the nanofibers, drug loading percent and release efficiency by a full factorial design. The morphology, diameter and diameter distribution of the nanofibers were studied by scanning electron microscopy (SEM). Drug loading percent in the nanofibers was determined by extracting the loaded drug from a specific surface of the nanofibers which was subsequently analyzed spectrophotometrically. The drug release rate from the nanofibers was studied in phosphate buffer solution (pH 7.4) containing 0.5 % Tween 20 at predetermined time intervals until 10 days. The cytotoxicity of the designed nanofibers was evaluated on mouse fibroblast cells using trypan blue method, their platelet adherence property was quantified by measuring the lactate dehydrogenase (LDH) activity and confirmed by SEM micrographs. The optimized ratio of PLGA/PMVEMA was 3:1 with the total concentration of polymers as 37 % loaded with 30 % of montelukast produced nanofibers with a diameter of 157.6 nm, drug loading percent of 43.7 % and release efficiency of 75 % after 10 days. The cell viability was similar in nanofibers and the negative control group. The platelets adhesion to the nanofibers was more than the negative control group (p<0.05).     

Preliminary Evaluation of 3D Printed Chitosan/Pectin Constructs for Biomedical Applications

In the present study, chitosan (CS) and pectin (PEC) were utilized for the preparation of 3D printable inks through pneumatic extrusion for biomedical applications. CS is a polysaccharide with beneficial properties; however, its printing behavior is not satisfying, rendering the addition of a thickening agent necessary, i.e., PEC. The influence of PEC in the prepared inks was assessed through rheological measurements, altering the viscosity of the inks to be suitable for 3D printing. 3D printing conditions were optimized and the effect of different drying procedures, along with the presence or absence of a gelating agent on the CS-PEC printed scaffolds were assessed. The mean pore size along with the average filament diameter were measured through SEM micrographs. Interactions among the characteristic groups of the two polymers were evident through FTIR spectra. Swelling and hydrolysis measurements confirmed the influence of gelation and drying procedure on the subsequent behavior of the scaffolds. Ascribed to the beneficial pore size and swelling behavior, fibroblasts were able to survive upon exposure to the ungelated scaffolds.     

The Effect of Melanocyte Stimulating Hormone and Hydroxyapatite on Osteogenesis in Pulp Stem Cells of Human Teeth Transferred into Polyester Scaffolds

Presently, tissue engineering is employed in the restoration and repair of tissue defects. Degradable scaffolds, stem cells and stimulating factors are employed in this method. In this study, the effect of melanocyte-stimulating hormone (MSH) and/or hydroxyapatite (HA) on proliferation, osteoblast differentiation, and mineralization of human dental pulp stem cells (hDPSCs) seeded on PLLA-PCL nanofibrous scaffolds was evaluated. For this aim, PLLA-PCL-HA nanofibrous scaffolds were fabricated using electrospinning method. FE-SEM images exhibited that all nanofibers had bead-free morphologies with average diameters ranging from 150–205 nm. Human DPSCs seeded into PLLA-PCL nanofibers were treated with MSH. Cell viability, proliferation, morphology, osteogenic potential, and the expression of tissue-specific genes were assessed by means of MTT assay, FE-SEM, alizarin red S staining, and RT-PCR analysis. hDPSCs exhibited improved adhesion and proliferation capacity on the PLLA-PCL-HA nanofibers treated with MSH compared to other groups (p<0.05). Additionally, PLLA-PCL-HA nanofibers treated with MSH exhibited significantly higher mineralization and alkaline phosphatase activity than other groups. RT-PCR results confirmed that PLLA-PCL-HA nanofibers enriched with MSH could significantly unregulated the gene expression of BMP2, osteocalcin, RUNX2 and DSPP that correlated to osteogenic differentiation (p<0.05). Based on results, incorporation of HA nanoparticles in PLLA-PCL nanofibers and addition of MSH in media exhibited synergistic effects on the adhesion, proliferation, and osteogenesis differentiation of hDPSCs, and therefore assumed to be a favorable scaffold for bone tissue engineering applications.     

Effect of sericin blending on molecular orientation of regenerated silk fiber

The use of regenerated silk fiber is limited due to its inferior mechanical properties in spite of high potential in a wide variety of applications. Many studies have been conducted in order to improve the mechanical properties of the regenerated silk materials, but no one has so far suggested an obvious solution. Meanwhile, some reports showed evidence that structural development of silk protein can be manipulated by physical interactions between silk fibroin (SF) and silk sericin (SS) during the regeneration process, especially in recrystallization process of SF. Such a hypothesis suggests a promising clue to enhance the mechanical properties of silk-based materials. Therefore, in this study, we tried to elucidate how SS can promote developing the molecular chain orientation of SF, resulting in an improvement of mechanical properties of regenerated silk fiber during spinning process. The tensile properties of the regenerated silk fiber were significantly improved compared to those of pure SF fiber when a proper amount of SS was blend with SF; both tenacity and breaking elongation increased by approximately 30 % and 70 % at three fold draw ratio, respectively. Quantitative analysis of X-ray diffraction and Herman’s orientation coefficient confirmed that such an improvement of tensile property was mainly caused by an increase of molecular orientation induced by sericin during the drawing process.     

Prediction of diameter in blended nanofibers of polycaprolactone-gelatin using ANN and RSM

Fabrication of nanofibers with a defined diameter is a primary purpose of the electrospinning process. The diameter of nanofiber is directly related to its individual features, such as mechanical property and porosity. The motivation to conduct the current study was to explore the diameter of hybrid nanofibers of polycaprolactone-gelatin (PCL-GT) as one of the most attractive scaffolds employed in various research fields, such as tissue engineering and industrial fields. We have developed two predictive models describing the electrospinning process of PCL-GT using response surface methodology (RSM) and artificial neural network (ANN). The effect of 4 variables on diameter was analyzed, including total polymer concentration, ratio of PCL to Gel, voltage, and tip-to-collector distance. The individual and interactive effects of the mentioned factors were analyzed using RSM. The total polymer concentration had the most significant individual effect on the diameter of PCL-Gel nanofiber, whereas the other three factors showed less strong individual effects, although, the interactive effects of these factors were more remarkable. It was demonstrated that both models, especially the ANN model, could accurately predict the diameter of PCL-GT nanofiber (regression coefficient > 0.92, mean absolute percentage error < 5.7). The represented predictive models could facilitate construction of electrospun nanofibers from PCL-Gel with wellcontrolled diameter required for any intended purpose.