Earticle

3D-printed biodegradable cellulose nanocrystal (CNC)-reinforced chitosan/silk fibroin scaffolds were synthesized as an advanced material for regenerative medicine application. The developed bio-inks were characterized using Fourier transform infrared spectroscopy and X-ray diffraction analysis. The composite scaffolds exhibited better swelling potential and better degradation potential than the pure polymer scaffolds. The cytotoxicity of the developed scaffolds was measured using the WST-1 assay in the presence of human bone marrow-derived mesenchymal stem cells (hMSCs). Improved cell viability and mineral deposition were observed with composite scaffolds vis-à-vis pure polymer scaffolds, showing their improved biocompatibility and mineralization potential. Upregulation of osteogenic associated gene markers was observed in the composite scaffold-treated media compared to the control, indicating enhanced osteogenic efficiency. The osteogenic induction process occurs via the mitogen-activated protein kinase pathway. Higher M1 macrophages polarization occurred in the scaffolds treated media after 24 h of incubation, which assisted the angiogenesis in bone tissue regeneration during the initial stage. Moreover, shifting from M1 to M2 macrophages polarization was observed in the scaffolds treated media after 3 days of incubation, suggesting its tissue regeneration potential. Compared to the control group, enhanced bone regeneration was observed in the calvaria defect rat model with the printed scaffolds, indicating their superior osteogenic efficiency. These results demonstrate that the printed scaffolds are promising materials for vascularized bone-healing applications and provide practical approaches for biomaterial development.

Objective: The purpose of this study was to determine the feasibility of an artificial esophagus using a silk fibroin (SF) scaffold. Method: The artificial esophagus was a three-layered, hybrid-type prosthesis composed of an outer and inner layer of poly(ε-caprolactone) (PCL) with a middle layer of SF. After depositing the inner layer of the PCL scaffold by electrospinning, the lyophilized middle SF layer was created. The outer layer of PCL was produced following the same procedure used to make the inner PCL layer. Result: In the cross-sectional image of the PCL-SF scaffold, we can clearly observe the nanofiber structure and the porous structure of the middle silk layer. The average stress values for the PCL-silk scaffold were 2.3460.12 MPa (dry) and 2.2660.23 MPa (wet). The CCK-8 assay results showed that prepared scaffolds modified with SF are generously nontoxic to the cells and that the presence of silk favored the growth of fibroblasts; therefore, this material is compatible with biomedical applications. Conclusion: Our study proposes a more practical experimental model for studying a three-layered PCL-SF scaffold in the esophagus.

In this study, fibroin ingestion is effective to improve concentration and performance is to determine whether. For this purpose, the fibroin ingestion period for archery athletes, measured changes in concentration and performance. Intake and how to eat one day and two times a week and 500mg intake. Concentration and memory before starting the diet, either, were tested 3 weeks after ingestion. Concentration and memory before starting the diet, either, for 3 weeks after ingestion test was done one month before the pibeuroinseopchwi intake and 1 month after the game and the scores were compared with the National Sports Festival. The statistical analysis of difference and group and 2 * 2 repeated ANOVA was conducted. Gratings test results showed no effect of the groups and placebo groups compared to the experimental group showed higher concentration. After recovery of the picture memory test and a 20 minute and then repeat the measurement between the consumption of the memory group showed higher statistically. However, records for archery groups were not statistically significant effect. However, the experimental group and the placebo group recorded between the 1st and 2nd showed a statistically significant difference.

Silk fibroin is an insoluble structural protein from Bombyx mori. It can be regenerated as a fibroin solution to form a variety of biomaterials, such as gels, sponges and films, for medical applications [1]. Fibroin microsphere has been suggested as a platform for controlled drug delivery [2]. In this report, submicron scale microspheres was prepared by W/O emulsion method using sorbitan monooleate (Span80), polyoxyethylene 20 sorbitan monooleate (Tween80), decane and fibroin solution. The capsules were spherical in shape as determined by SEM with diameters in the range of 200 nm to 1200 nm, depending on the homogenization rate of W/O emulsion. Hydrophilic materials can be encapsulated by dissolving them in the fibroin solution and subsequent emulsification. Dextran and FITC-dextran were efficiently encapsulated in the fibroin microcapsule. Their core-shell structure was identified by confocal microscopy. Because of its very mild conditions of fabrication, this method is potentially advantageous for the encapsulation of heat-labile drugs such as proteins.

Alginate has been used as popular biomaterials in the forms of hydergel, microporous beads and fibers for biomedical applications. However, more versatile applications were limited by its fragility in spite of excellent biocompatability. Fiber reinforced composites have often been suggested to improve mechanical characteristics and alginate hydrogel was reported to be successfully reinforced by chitin whisker [1] and alginate fibers [2] to improve their physical properties.In this report, a concentrated calcium salt solubilizing silk fibroin was used for ionotropic curing alginate and for preparing fibroin fibers in the hydrogels. Confocal laser scanning microscope analysis indicated that the FITC stained alginate beads prepared with fibroin solution contained both short fibroin fibers and a network-like fibroin fibers of similar diameters. Methanol treatment inducing β-sheet formation increased the mechanical strength of the fibroin reinforced alginate hydrogels. A versatile method of forming fibroin-fiber networks is under development.

Silk fibroin from Bombyx mori was sulfated in an aqueous sulfuric acid as a solvent and a catalyst. Incorporation of sulfate into the fibroin molecules was verified by FT-IR and a simultaneous hydrolysis of fibroin was observed by measuring its molecular weight distribution using a gel permeation chromatography. Unlike to fibroin used as a substratum for cultivation of animal cells [1], the sulfated fibroin decreased the growth rate of 3T3 cells in DMEM/Ham' s F12(1:1) media containing 10% FBS. Heparin, a sulfated peptide, inhibited the attachment and growth of Balb/c-3T3 fibroblasts [2]. Thus, the effect of fibroin, enzymatically hydrolyzed fibroin, acid-hydrolyzed fibroin and sulfated fibroin were investigated on the attachment, metabolic activity and growth of 3T3 cells in this report. Sulfated fibroin at the concentration of 0.1 % (w/v) in the culture media reduced the specific growth rate by 20% but other fibroin peptides showed varying responses to the levels of peptides supplied. Fibroin has been used as a 3D scaffolds for cell cultivation [3]. A composite of fibroin and sulfated fibroin is under investigation as a potential extracellular matrix interacting cell adhesion and growth control.

Cellular uptake of microcapsules has been focused on the efficiency, cellular trafficking or capabilities of cargo delivery. We investigated the effect of fibroin microcapsule on morphology and cell cycle of 3T3 cells cultured in vitro using flow cytometry. Cell shrinkage and increased granularity was observed in the cells co-incubated with various concentrations of fibroin microcapsule (up to 0.33 mg/mL), while cell viability was not affected. Because granularity can be increased by aggregated cells, the morphology change in mononuclear cells was identified by flow cytometric analysis of Propidium iodide (PI) stained cells indicated that and not caused by cell aggregates. It was also shown that cells co-incubated with 0.003 and 0.033 mg/mL of microcapsules had increased G2/M phase of the cell cycle. However, the distribution of cell cycle stages was not influenced by 0.33 mg/mL of microcapsules, suggesting cell cycle freeze. Analysis of the findings shows that cellular uptake of the fibroin microcapsule reduced cell size, increased granularity, and influenced cell cycle progression.

Fibroin is a highly insoluble protein forming a fibrous protein structure by highly repeating unit of Gly-Ala-Gly-Ala-Gly-Ser giving to the crystalline nature of silk fiber [1]. It is known to aggregate and to form insoluble particles [2]. It has also been reported that insoluble aggregates are more quickly formed on oil surface [3]. Therefore, in this report, a method of forming microcapsule of various oils surrounded by fibroin coacervates was developed by using an O/W emulsion [4]. Uniform deposition of fibroin molecules on oil surface was enhanced by partial digestion using papain. The maximum yield of capsulation was obtained at pH 4 and the level of wall material fraction was 37.0% by thermogravitational analysis. A wax-based oil phase was prepared and poured into the 2% (w/v) silk fibroin yielded a creamy coacervation phase and 80% of the microcapsule was between the range of 0.5 μm and 3.8 μm.

Lactic acid bacteria (LAB) have long been used in fermentations to preserve the nutritive qualities of various foods. Recently, a number of antimicrobial metabolites, e.g. cyclic dipeptides, phenyllactic acid, proteinaceous compounds, and 3-hydroxylated fatty acids have been isolated from lactic acid bacteria.1) The antioxidative effect of lactic acid bacteria was also reported.2) This paper investigated antimicrobial activities and antioxidative effect of silk peptide transformed by lactic acid bacteria. Among the 38 lactic acid bacteria as lyophilized stock purchased, 25 of them showed good growth. I indicator strain were used for screening of antimicrobial activity and 19 strains of lactic acid bacteria showing antimicrobial activity. The antimicrobial compound from the 3 strains of lactic acid bacteria showing strong antimicrobial activity are under investigation. Free radical scavenging activities the culture supernatants were measured according to the modified method of Bilos.3) The highest radical scavenging activity was obtained in 3 lactic acid bacteria cultured under aerobic condition. Tyrosinase inhibition activites were also measured.

Silk fibroin (SF) film containing catechin was prepared and the antimicrobial activity as well as physical propertyof the film was examined. Tensile strength of the SF film decreased with increasing concentration of catechin, and water vaporpermeability of the film decreased. The film’s antimicrobial activity against Escherichia coli O157:H7 increased withincreasing catechin concentration. Sausage samples were inoculated with E. coli O157:H7 and Listeria monocytogenes, andthe sausage packaged with the SF film containing catechin had a decrease in the populations of E. coli O157:H7 and L.monocytogenes by 0.83 and 0.85 log CFU/g after 12 days of storage, respectively, compared to the control. In addition, thesausage had a better quality than the control regarding lipid oxidation. Our results indicate that sausages can be packed withthe SF film containing catechin to extend shelf life.

Controlled release technologies have been applied to intracellular delivery of DNA [1]. The DNA carriers have been engineered to release an intact DNA at the optimal time and to the targeted subcellular compartment [2]. In this report, fibroin microcapsule encapsulating DNA was prepared and investigated. The property of DNA release from the fibroin microcapsules was analyzed using shear fragmented DNAs and using different core materials. Linear DNA ranging from 3 – 10 kb was released but closed circular plasmids were not released from unmodified fibroin microspheres. Spatial distribution of the DNA in fibroin microspheres was also investigated. The release of DNA molecules, its size-dependency and cellular delivery was investigated.

Silk fibroin is an insoluble structural protein from Bombyx mori. It can be regenerated as a fibroin solution to form a variety of biomaterials, such as gels, sponges and films, for medical applications [1]. A submicron-scale fibroin microsphere with core-shell structure has been developed [2]. Cellular uptake of submicron-scale particles has been reported for polystyrene microspheres [3] and for gold nanoparticles [4]. In this report, submicron scale microspheres were prepared and its cellular uptake by 3T3 cells in vitro was investigated. Confocal laser scanning microscopy and tomographic analysis revealed that fibroinmicrospheres were successfully incorporated into the 3T3 cells in vitro and they were localized in cytoplasm. The efficiency and rates of fibroin-microspheres were investigated using fluorescence-activated cell sorting.

Silk fibroin (Bombyx mori ) is a structure protein with low solubility limiting its applications in aqueous environments. In order to improve its solubility, in this report, regenerated fibroin was sulfated using aqueous sulfuric acid. The reaction temperature and sulfuric acid concentration were varied for the investigation of optimum sulfation condition. The sulfated fibroin samples were neutralized by alkali, followed by dialysis and freeze drying. Their FT-IR spectra proved varying degree of introduction of sulfate group during the sulfation reaction, showing a strong absorption at 1145 cm-1 corresponding to the stretching vibration of S=O bond. NMR spectra indicated that sulfation occurred mainly at serine residues. This result agreed well with the previous reports1)2). The maximum yield was obtained at 70℃ and the degree of sulfation increased with sulfuric acid concentration. A sharp absorption peak at 997 cm-1 was appeared above 15% of sulfuric acid, suggesting an unidentified sulfation reaction occurs at high sulfate concentration. The ratio of sulfated groups also increased with the reaction time. The recovery yield of sulfated fibroin was around 50% in a small scale experiment in alaboratory. A successive large scale experiment produced 36.97g of sulfated fibroin with 72.5% weight yield.

Brand-new ideas with silk materials have enabled to widen applications of silkworm silk (especially, fibroin) beyond clothes to food, optical device, scaffold, gel, film, and so on. However, relatively poor biodegradation and cell adhesive ability have hindered its wide range of application for cell and tissue engineering. For this reason, many attempts to develop characteristics of fibroin as a tissue engineering material have tried. Here, we attempted to improve characteristics of silk fibroin by introduction of recombinant mussel adhesive proteins (MAPs). Mussel can strongly affix to the wet surfaces by using secretion of MAPs and maintain its adhesive force and flexibility even under harsh conditions. Previously, we successfully mass-produced recombinant MAP in bacterial expression system. We also designed and constructed RGD peptide-fused recombinant MAP as a new cell adhesive which leads better cell attachment, spreading, proliferation, and even differentiation. By co-electrospinning of MAP-RGD with silk fibroin, we fabricated nanofiber sheet as tissue engineering scaffold and investigated cell behaviors on the constructed nanofibrous scaffold. Above all, the constructed nanofibrous scaffold with silk fibroin and MAP has a lot of potentials for functionalization of biomolecules which be used for advanced bioactivity or cell and tissue engineering without complicate modification of scaffold surface.