Silk fibroin film from non-mulberry tropical tasar silkworms: A novel substrate for in vitro fibroblast culture

The silk protein fibroin, isolated from the cocoon of the domesticated mulberry silkworm, Bombyx mori, is used extensively in biomaterial design and in cell and tissue culture. We report here for the first time the potential application of fibroin obtained from the cocoon of non-mulberry tropical silkworm, Antheraea mylitta, as a substrate for in vitro cell culture. The mechanical strength of A. mylitta silk fibers indicates a stronger thread composition. The contact angle of A. mylitta fibroin films suggests that it has lower hydrophilicity and lower solubility in organic solvents compared to B. mori fibroin films. Retention of a secondary structure of fibroin in both A. mylitta and B. mori films is confirmed by Fourier transform infrared analysis. The adherence, growth and proliferation patterns of feline fibroblast cells on A. mylitta fibroin films suggest that this kind of film has a greater ability to support cell growth than B. mori fibroin films and is comparable to that of control. This study demonstrates that, as well as being non-toxic to dermal fibroblast cells, non-mulberry fibroin might be a useful alternative substrate to the more common B. mori fibroin for a variety of biomedical applications.     

Potential of biocompatible regenerated silk fibroin/sodium N-lauroyl sarcosinate hydrogels

Hydrogels are becoming widely used in biomaterial applications. The available methods for the preparation of these materials are continually growing. The gelation time (GT) of silk protein fibroin is difficult to control by physical methods. The cross-linkers used in available chemical techniques are likely to impact the biocompatibility of the resultant materials. In this paper, we demonstrate that the addition of sodium N-lauroyl sarcosinate (an amino-acid-based surfactant) accelerates the formation of hydrogels from fibroin. GT, turbidity variations, changes of viscoelasticity during the gelation process, and the mechanical properties of the products are measured. The secondary structure was probed by Fourier transform infrared spectroscopy, X-ray diffraction and the morphologies of the products were investigated by scanning electron microscopy. Transformations in the β-sheet content were monitored by the fluorescence of Thioflavine T and circular dichroism measurements. The relationship between the surface tension of sodium N-lauroyl sarcosinate and the GT was also explained. To investigate cell compatibility, fibroblast cells were seeded onto the surface of the hydrogels. The results indicate that the sodium N-lauroyl sarcosinate/fibroin GT can be controlled. This blend-hydrogel demonstrates excellent cell compatibility, good compression strength, and outstanding compression-recovery characteristics. Sodium N-lauroyl sarcosinate/silk fibroin hydrogels containing β-sheets have considerable potential as replacement materials in addressing the tissue defects involved with repair surgery.     

Silk Fibroin/Sodium Carboxymethylcellulose Blended Films for Biotechnological Applications

The potential of silk protein is increased because of its importance as natural biopolymer for biotechnological and biomedical applications. The main disadvantage of silk fibroin films is their high brittleness. Thus, we studied blends of fibroin with other polymers to improve the film properties. Considering the possible applications of films in biomedical applications, we used a natural and biodegradable polymer as the second component. This study reports the fabrication and characterization of mulberry silk protein fibroin and sodium carboxymethylcellulose (NaCMC) blended films as potential substrates for in vitro cell culture. The blended films are investigated of their chemical interactions, morphologies, thermal, mechanical properties in addition to its swelling properties and biocompatibility. The addition of NaCMC improves the elasticity of fibroin films and its thermal properties. The change of morphology, swelling behavior and increase of surface roughness of the films were also observed in the blended films. The films become insoluble on alcohol treatment and are stable for longer duration in hydrolytic medium. The blended films are cytocompatible and supported adhesion and growth of mouse fibroblast cells. The results suggest that NaCMC blended silk fibroin films are found to be potential substratum for supporting cell adhesion and proliferation.     

Evaluation of the properties of silk fibroin films from the non-mulberry silkworm Samia cynthia ricini for biomaterial design

Silk fibroin from a domesticated mulberry silkworm, Bombyx mori, is the most widely used in biomaterial design. We report for the first time the preparation of a relatively smooth (granule free) film of the nonmulberry Samia cynthia ricini fibroin for comparative evaluation of its cell-supporting properties against those of the B. mori fibroin film. The granule formation on the S. c. ricini fibroin film was successfully prevented by facilitating proper rearrangement of the protein molecules, as monitored by FT-IR, by dialysis through a stepwise decrease in the urea concentration in the dialysis media. The lower contact angle of the S. c. ricini fibroin film, compared to the B. mori fibroin film, corresponds well to its lower hydrophobic/hydrophilic amino-acid ratio and grand average of hydropathicity (GRAVY). L929 murine fibroblast cells on the granule-free S. c. ricini fibroin films exhibited greater proliferation and spreading rates than those on the B. mori fibroin films, possibly attributable to its higher content of hydrophilic and positively charged amino acids. It further suggests that fabrication, modification and/or engineering of S. c. ricini fibroin may provide a better biomaterial scaffold design than the more commonly used B. mori fibroin.     

几种丝素材料细胞毒性的实验研究

目的：研究不同交联方式的再生丝素膜(WL组，SD组，HY组)的细胞毒性及其影响细胞增殖的因素。方法：采用浸提液法，体外培养鼠胚真皮层纤维细胞，用MTT法检测细胞增殖活力和计算相对增殖率。结果：WL组，SD组再生丝素膜细胞毒性小于1级，有较强的增殖活力；HY组再生丝素细胞毒性0～2级；而用高浓度环氧交联剂交联的再生丝素膜对细胞生长有一定抑制作用；且随着环氧交联剂浓度的增加，细胞毒性增大。结论：WL组和SD组再生丝素膜无细胞毒性，HY组再生丝素膜无明显的细胞毒性，高浓度环氧剂交联的再生丝素膜对细胞有一定的毒性，有待于进一步改性。     

In Vitro and In Vivo Release of Basic Fibroblast Growth Factor Using a Silk Fibroin Scaffold as Delivery Carrier

Two different solvents were used to prepare two types of silk fibroin scaffolds via the salt-leaching technique, i.e., hexafluoroisopropanol (HFIP) and water. The in vitro release study suggests that the opposite charge between the silk fibroin and basic fibroblast growth factor (bFGF) at physiological pH rendered them to form a complex, and the difference in the solvents used to produce the silk fibroin scaffold did not affect the affinity of silk fibroin to bFGF. However, a higher degradation rate of the aqueous-derived silk fibroin scaffolds provided faster in vitro release kinetics of the bFGF, as compared to the HFIP-derived scaffolds. From the in vivo studies, the use of silk fibroin scaffolds as the carrier matrix enabled the control of the in vivo release of bFGF in a sustained fashion over two weeks, while the majority of the bFGF disappeared within one day after the injection of the bFGF in soluble form. In addition, the in vivo release of bFGF from the silk fibroin scaffolds was not affected by the mode of processing due to their similar degradation behavior in vivo.     

丝素蛋白溶液诱导成骨分化及其性能评价

BACKGROUND: Silk fibroin, as a kind of high-performance biomaterial, has been widely used to construct scaffolds in bone tissue engineering. However, whether silk fibroin itself holds osteoinductive ability has not been reported yet. OBJECTIVE: To investigate the impact of different concentrations of silk fibroin solution on the proliferation and differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. METHODS: Silk fibroin and BMSCs were respectively isolated from silkworm cocoon and rat tibia, and were identified. Then, BMSCs were cultured in different concentrations of silk fibroin solution (0.01%, 0.05% and 0.1%), and the cell proliferation and the alkaline phosphatase activity were detected at different time points. RESULTS AND CONCLUSION: FTIR spectra of the sample extracted from silkworm cocoon showed distinct absorption peaks at 1 653 (amide I), 1 530.5 (amide II) and 1 212.3 cm-1 (amide III), which could be confirmed to be silk fibroin. Thus generated BMSCs showed long fusiform or astral morphology, positive for representative markers (CD29, CD44 and CD90) relating to mesenchymal stem cells, and could differentiate into osteocytes, chondrocytes and adipocytes under specific induction conditions, which further confirmed the extracted cells were BMSCs. Compared with the control group (without silk fibroin), 0.05% silk fibroin not only significantly promoted the cell adhesion, migration and proliferation, but also enhanced the alkaline phosphatase activity (P < 0.01). With the increasing of the silk fibroin concentrations, the osteodifferentiation capacity of the BMSCs was progressively improved within the range of 0-0.05% and then declined at 0.01% of silk fibroin solutions. These results suggest that silk fibroin can promote osteogenesis, thus providing scientific evidence for developing silk fibroin-based tissue-engineered scaffolds.     

The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor

We generated germline transgenic silkworms bearing a fibroin light chain (FL) promoter-driven FL gene whose 3'-end was flanked with human basic fibroblast growth factor (bFGF) gene, FL/bFGF gene. The cocoons from transgenic worms were trypsinized to remove sericin layers, and treated with solution containing CaCl(2), ethanol, and water at a molar ratio of 1:2:8 (CaCl(2)/ethanol/water) to solubilize fibroin layers. Western blot analysis showed that the recombinant protein, r(FL/bFGF), was solubilized with CaCl(2)/ethanol/water, but not with trypsin, indicating that r(FL/bFGF) was in fibroin layers. Thus, it was concluded that the worms spun cocoons whose fibroin layers were composed of the inherent gene-derived natural fibroin (nF) and r(FL/bFGF). The mixture of nF and r(FL/bFGF) was dubbed r(FL/bFGF)nF. The solubilized r(FL/bFGF)nF was refolded using the glutathione redox system. Human umbilical vein endothelial cells (HUVECs) grew in the refolded r(FL/bFGF)nF-containing culture media, showing that bFGF in r(FL/bFGF) was biologically active. r(FL/bFGF)nF immobilized on a culture dish also supported the growth of HUVECs in bFGF-free media, suggesting the usefulness of r(FL/bFGF)nF as a new biomaterial for tissue engineering. The currently developed transgenic silkworms will be suitable for mass production of fibroins bearing a variety of biological activities.     

Preparation and characterization of silk fibroin as a biomaterial with potential for drug delivery

ABSTRACT: BACKGROUND: Degummed silk fibroin from Bombyx mori (silkworm) has potential carrier capabilities for drug delivery in humans; however, the processing methods have yet to be comparatively analyzed to determine the differential effects on the silk protein properties, including crystalline structure and activity. METHODS: In this study, we treated degummed silk with four kinds of calcium-alcohol solutions, and performed secondary structure measurements and enzyme activity test to distinguish the differences between the regenerated fibroins and degummed silk fibroin. RESULTS: Gel electrophoresis analysis revealed that Ca(NO3)2-methanol, Ca(NO3)2-ethanol, or CaCl2-methanol treatments produced more lower molecular weights of silk fibroin than CaCl2-ethanol. X-ray diffraction and Fourier-transform infrared spectroscopy showed that CaCl2-ethanol produced a crystalline structure with more silk I (alpha-form, type II beta-turn), while the other treatments produced more silk II (beta-form, anti-parallel beta-pleated sheet). Solid-State 13C cross polarization and magic angle spinning-nuclear magnetic resonance measurements suggested that regenerated fibroins from CaCl2-ethanol were nearly identical to degummed silk fibroin, while the other treatments produced fibroins with significantly different chemical shifts. Finally, enzyme activity test indicated that silk fibroins from CaCl2-ethanol had higher activity when linked to a known chemotherapeutic drug, L-asparaginase, than the fibroins from other treatments. CONCLUSIONS: Collectively, these results suggest that the CaCl2-ethanol processing method produces silk fibroin with biomaterial properties that are appropriate for drug delivery.     

Evaluation of the Properties of Silk Fibroin Films from the Non-mulberry Silkworm Samia cynthia ricini for Biomaterial Design

Silk fibroin from a domesticated mulberry silkworm, Bombyx mori, is the most widely used in biomaterial design. We report for the first time the preparation of a relatively smooth (granule free) film of the nonmulberry Samia cynthia ricini fibroin for comparative evaluation of its cell-supporting properties against those of the B. mori fibroin film. The granule formation on the S. c. ricini fibroin film was successfully prevented by facilitating proper rearrangement of the protein molecules, as monitored by FT-IR, by dialysis through a stepwise decrease in the urea concentration in the dialysis media. The lower contact angle of the S. c. ricini fibroin film, compared to the B. mori fibroin film, corresponds well to its lower hydrophobic/hydrophilic amino-acid ratio and grand average of hydropathicity (GRAVY). L929 murine fibroblast cells on the granule-free S. c. ricini fibroin films exhibited greater proliferation and spreading rates than those on the B. mori fibroin films, possibly attributable to its higher content of hydrophilic and positively charged amino acids. It further suggests that fabrication, modification and/or engineering of S. c. ricini fibroin may provide a better biomaterial scaffold design than the more commonly used B. mori fibroin.     

Engineered silk fibroin protein 3D matrices for in vitro tumor model

3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs.     

Regeneration of the femoral epicondyle on calcium-binding silk scaffolds developed using transgenic silk fibroin produced by transgenic silkworm

Genetically modified silk fibroin containing a poly-glutamic acid site, [(AGSGAG)4E8AS]4, for mineralization was produced as fibers by transgenic silkworms through systematic transformation of the silkworms. The Ca binding activity and mineralization of the transgenic silk fibroin were examined in vitro, showing that this transgenic silk fibroin had relatively high Ca binding activity compared with native silk fibroin. Porous silk scaffolds were prepared with the transgenic and native silk fibroins. Healing of femoral epicondyle defects in rabbit femurs treated with the scaffolds was examined by observing changes in images of the defects using micro-computed tomography. Earlier mineralization and bone formation were observed with scaffolds of transgenic silk fibroin compared with those of native silk fibroin. Thus, this study shows the feasibility of using genetically modified silk fibroin from transgenic silkworms as a mineralization-accelerating material for bone repair.     

Growth of human cells on a non-woven silk fibroin net: a potential for use in tissue engineering

We have examined a novel biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons for its ability to support the growth of human cells. Various human cells of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were examined for adherence and growth on the nets by confocal laser microscopy after staining of the cells with calcein-AM and by electron microscopy. All the cells readily adhered and spread over the individual fibers of the nets. Most of the cells were able to grow and survive on the nets for at least 7 weeks and growth not only covered the individual fibers of the net but generally bridged the gaps between individual fibers forming tissue-like structures. Scanning electron microscopic examination of the nets demonstrated a tight association of individual cells with the fibers and nets examined after removal of cells showed no evidence that the growth of cells in any way changed the structure of the fibers. Thus, silk fibroin nets are highly human cell-compatible and should be a useful new scaffolding biomaterial applicable for a wide range of target tissues in addition to supporting endothelial cells required for the vascularization of the newly formed tissue.     

Silk protein fibroin from Antheraea mylitta for cardiac tissue engineering

The human heart cannot regenerate after an injury. Lost cardiomyocytes are replaced by scar tissue resulting in reduced cardiac function causing high morbidity and mortality. One possible solution to this problem is cardiac tissue engineering. Here, we have investigated the suitability of non-mulberry silk protein fibroin from Indian tropical tasar Antheraea mylitta as a scaffold for engineering a cardiac patch in vitro. We have tested cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication and contractility of 3-days postnatal rat cardiomyocytes on silk fibroin. Our data demonstrate that A. mylitta silk fibroin exhibits similar properties as fibronectin, a component of the natural matrix for cardiomyocytes. Comparison to mulberry Bombyx mori silk protein fibroin shows that A. mylitta silk fibroin is superior probably due to its RGD domains. 3D scaffolds can efficiently be loaded with cardiomyocytes resulting in contractile patches. In conclusion, our findings demonstrate that A. mylitta silk fibroin 3D scaffolds are suitable for the engineering of cardiac patches.     

丝素蛋白作为组织工程材料应用于角膜的研究进展

背景：丝素蛋白纤维材料具有透明性、结构可塑性、成分单一性、力学强韧性及生物相容性等特点。 目的：综述国内外丝素蛋白应用于角膜组织工程的研究进展。 方法：由第一作者在标题和摘要中以“silk fibroin, corneal, ocular”或“丝素,角膜”为检索词,检索1980至2011年PubMed及1990至2011年CNKI数据库中关于丝素蛋白角膜的文章。 结果与结论：从天然蚕丝中提取的高分子丝素蛋白,因其良好的生物相容性、独特的力学性能、光学透明性及降解速率可控性,既可以单独应用于角膜组织结构的重建,又可与其他组织材料联合应用,成为角膜组织工程学应用的理想材料。现已证明多种角膜细胞可在丝素纤维膜上良好生长,但体外培养的细胞应用于动物模型的相关研究较少;此外丝素蛋白材料植入角膜内对其产生何种影响的研究数据较缺乏,这些均是亟待解决的问题。     

Sulfation of silk fibroin by chlorosulfonic acid and the anticoagulant activity

Silk fibroin (Bombyx mori) was sulfated using chlorosulfonic acid in pyridine. FT-IR spectra showed introduction of sulfate group by this reaction; NMR spectra indicated that sulfation occurred mainly at tyrosine and serine residues. Molecular size decreased and dispersed with sulfation. The molecular weight was estimated in around 20,000 by GPC using protein standards. Amino acid composition suggested that sulfated fibroin came from H-chain of fibroin; the crystal region of fibroin molecule remained in sulfated fibroin. The amount of sulfate groups increased with overall reaction time. The maximum amount was estimated in 1.0 mmol/g by acidimetric titration. Sulfation efficiency was calculated as 66.7%. Blood coagulation was prevented by 0.5 mg of sulfated fibroin in 1 ml of blood, while original fibroin did not show any effect. Anticoagulant activity of sulfated fibroin strongly depends on the amount of sulfate groups introduced. These results indicate that sulfate group introduction results in addition of anticoagulant function to silk fibroin. Sulfated fibroin is a new type of anticoagulant material having a protein backbone.     

丝素材料的药物吸附释放性能与调控研究

为了了解丝素材料对药物等的吸附释放性能 ,并探讨对丝素材料的吸附释放性能的调控 ,用不同离子型化合物作为药物模型 ,比较了分别用未修饰和经羧基进行酰胺化修饰后的丝素制作成的多孔丝素凝胶对不同离子型的化合物的吸附释放行为。结果表明 :经修饰后丝素蛋白质的等电点为 pH6 .0左右 ,而天然的为pH4.0左右。未修饰和经修饰的多孔丝素凝胶都随着溶液 pH的上升 ,对阳离子化合物的吸附量增加 ,释放速度减慢 ;对阴离子化合物吸附量减少 ,释放速度加快。但在相同 pH下与未修饰相比 ,经修饰的多孔丝素凝胶所吸附的阳离子化合物的释放速度加快 ,释放量也增加 ;所吸附的阴离子化合物的释放速度和释放量则明显降低。用羧基酰胺化修饰的方法 ,可在一定程度上改变丝素材料对离子型化合物的吸附释放行为     

丝素蛋白在组织工程细胞支架方面的研究进展

近年来有关丝素蛋白用做细胞培养基质材料的研究表明，丝素蛋白对多数种类细胞的体外培养表现出较高的细胞附着率和增殖率，与胶原相当，可以用作组织工程细胞支架材料，应用到组织工程皮肤、软骨、血管等诸多领域。本文就丝素蛋白用于细胞培养的研究现状及丝素蛋白在组织工程细胞支架方面的应用前景做一综述。     

人胎盘间充质干细胞促进血管生成

背景：构建组织工程化骨组织的同时,促进种子细胞与材料复合物内血液供应的重建成为研究的关键。胎盘间充质干细胞可以作为骨组织工程研究的种子细胞,研究其分化为血管内皮细胞以及促进血管生成有着重要意义。 目的：观察胎盘间充质干细胞在体外分化为血管内皮细胞以及体内促血管生成作用。 方法：分离培养人胎盘间充质干细胞,鉴定其表面抗原,经血管内皮生长因子和人碱性成纤维细胞生长因子联合体外诱导胎盘来源间充质干细胞向血管内皮细胞定向分化,诱导后细胞通过内皮细胞标志物KDR、v-WF染色鉴定。8只新西兰大白兔制成桡骨中段1.5 cm长的骨缺损模型,分别植入人胎盘间充质干细胞/丝素蛋白/羟基磷灰石和丝素蛋白/羟基磷灰石进行对照。植入后4,12周分别行大体观察、组织学观察和X射线观察,比较骨缺损修复以及血管生成情况。 结果与结论：胎盘间充质干细胞的诱导分化形态明显改变,胞体逐步回缩,立体感增强,内皮细胞标志物KDR、v-WF染色结果阳性。胎盘来源间充质干细胞与丝素蛋白/羟基磷灰石材料复合培养植入后,4周时新骨已开始形成,12周时有部分新生骨组织形成板层骨,骨小梁形成,内可见新生血管形成,而对照组支架材料降解较慢,未见新生血管。说明胎盘来源间充质干细胞体外可以分化为血管内皮细胞,与丝素蛋白/羟基磷灰石材料联合移植能够促进移植物内血管生成,较好的修复骨缺损。