再生丝素蛋白对小鼠骨髓来源树突状细胞分化、成熟的影响

体外研究再生丝素蛋白材料对小鼠骨髓来源树突状细胞分化、成熟的影响,探讨丝素蛋白的免疫原性及作为生物材料的意义。体外分离培养小鼠骨髓来源树突状细胞,接种于铺有再生丝素蛋白生物材料的细胞培养板中培养,在光学显微镜下观察细胞形态;FCM分析细胞表面相关分子的表达。同时用 MTT 法测小鼠骨髓来源树突状细胞细胞株 DC2．4在所选各种材料表面的生长增殖情况。结果表明：（1）再生柞蚕丝素蛋白和家蚕丝素蛋白均可支持小鼠骨髓来源的 DCs 生长和聚集;（2）MTT 结果表明再生柞蚕丝素蛋白与再生家蚕丝素蛋白相比可促进小鼠 BM来源 DC2．4的增殖;（3）再生柞蚕丝素蛋白和再生家蚕丝素蛋白上的小鼠 BM来源 DCs 表面低表达 CD11c、CD40、CD80、CD86、MHC -Ⅱ分子,与空白对照组 DCs 的表达谱相比无明显差别。再生柞蚕丝素蛋白与再生家蚕丝素蛋白相比,可较好地支持小鼠 BM来源 DC2．4的生长和增殖;再生丝素蛋白无刺激小鼠 BM来源 DCs 成熟的生物学效应。     

星形胶质细胞与神经元在蚕丝素纳米纤维上的联合培养

目的:研究胶质细胞结合丝素纳米纤维对神经元生长发育的作用,为神经干细胞结合丝素蛋白材料用于临床移植修复神经系统损伤提供实验依据.方法:从新生大鼠脑室下区分离细胞与取自混合培养得来的星形胶质细胞共培养在柞蚕、家蚕2种丝素蛋白溶液制成的材料上,分别选取各时间段的细胞进行β-Ⅲ-tubulin特异性免疫荧光显色,并统计不同时间段神经元在丝素材料上的复杂度.结果: 神经元与星形胶质细胞共培养在柞蚕丝素无纺布上(TSF)的复杂度明显比家蚕丝素无纺布(SF)高.结论: 柞蚕、家蚕丝素无纺布结合星形胶质细胞支持神经元的生长,具有良好的生物相容性;胶质细胞结合柞蚕丝素无纺布比家蚕丝素无纺布更适合神经元的生长发育.     

再生丝素蛋白膜作为人嗅鞘细胞移植支架的研究

探讨再生丝素蛋白膜对人嗅鞘细胞(hOECs)生物学特性的影响.人嗅鞘细胞接种在再生丝素蛋白膜上,取不同时相的细胞做下列实验.72 h固定,用免疫荧光染色技术观察人嗅鞘细胞的标志蛋白NT-3、S-100、GFAP、NGFRp75,用电子显微镜观察细胞在再生丝素蛋白膜上的生长状况;分别于48、72、96 h提取总蛋白,用免疫印迹法分析人嗅鞘细胞标志蛋白的表达情况;用MTF法分析细胞在再生丝素蛋白膜上的生长曲线.hOECs在再生丝素蛋白膜上能够很好地附着、铺展、生长,嗅鞘细胞的形态大多为梭形,嗅鞘细胞标志蛋白NT-3、NGFRp75、S-100及GFAP均呈免疫反应阳性,且免疫印迹结果与形态学结果一致;电镜结果显示,再生丝素蛋白膜的表面微纳结构适于细胞贴附、铺展;嗅鞘细胞在再生丝素蛋白膜上的生长曲线与培养板上无明显差异.再生丝素蛋白膜适合人嗅鞘细胞的贴附、增殖、生长,再生丝素蛋白膜与hOECs有较好的相容性,可以作为hOECs生长的候选组织工程支架用于修复神经损伤.     

再生丝素蛋白纳米纤维网支持和引导神经胶质细胞的生长与迁移

除了自体和异体移植外,利用生物相容性材料辅助中枢神经系统损伤后的修复成为最具开发潜力的方法之一.以来源于家蚕和柞蚕的再生丝素蛋白纳米纤维网作为星形胶质细胞的生长基质,研究星形胶质细胞在其上的粘附、生长、增殖和迁移等生命活动.结果显示星形胶质细胞在两种材料上表现出很高的相容性,星形胶质细胞在丝素蛋白纳米纤维网上具有正常的粘附、增殖和迁移等行为.更重要的是,通过实时显微摄像跟踪细胞的生长与迁移行为,发现星形胶质细胞的生长与迁移表现出很强的丝素蛋白纳米纤维依赖性,星形胶质细胞在丝素纤维上生长铺展并且沿着纤维进行迁移,纤维的走向决定着细胞的迁移轨迹.实验证明,丝素蛋白纳米纤维不仅能够支持星形胶质细胞的生长,而且对星形胶质细胞的迁移运动还有引导作用,这些特点使得再生丝素蛋白纳米纤维网成为极具开发潜力的神经组织工程替代物.     

纤维素-大豆蛋白混合膜与内皮细胞生物相容性的实验研究

目的:研究和评价纤维素-大豆蛋白混合膜与内皮细胞的生物相容性,为其作为人工生物支架材料应用于组织工程提供实验依据。方法:将内皮细胞株ECV304与不同组分、碱处理前后的纤维素-大豆蛋白混合膜共培养,分别以四氮甲唑蓝(MTT)法检测细胞活力,以扫描电镜(SEM)观察细胞形态和生长状态。结果:与阴性对照组和单纯纤维素组相比,ECV304在纤维素-大豆蛋白混合膜上生长良好,大豆蛋白加入到纤维素膜中显著提高了ECV304的细胞活力;在碱处理后的混合膜表面,细胞长出许多足突伸入膜孔隙,明显促进细胞在膜表面粘附。结论:初步表明纤维素-大豆蛋白混合膜与内皮细胞ECV304生物相容性良好,在血管组织工程领域具有应用潜能。     

医用丝素蛋白皮肤再生膜的细胞相容性评价

评价医用丝素蛋白皮肤再生膜的细胞相容性。其方法是采用细胞增殖度试验和溶血试验,对医用丝素蛋白皮肤再生膜进行细胞毒性和溶血反应的实验研究。结果表明：该再生膜无明显细胞毒性存在,溶血率为1.15%。医用丝素蛋白皮肤再生膜具有良好的细胞相容性。     

丝素蛋白与胎盘间充质干细胞生物相容性的实验研究

目的 探讨丝素蛋白（SF）材料与胎盘间充质干细胞（PMSCs）的生物相容性.方法 运用SF溶液包被的培养瓶培养PMSCs,流式细胞术分析其表型并对其定向分化潜能进行探讨;PMSCs置于SF膜材料培养后通过扫描电镜观察细胞形态变化.结果 用SF溶液包被的培养瓶培养的PMSCs,其生长特性、表面标志、多向分化潜能无明显变化;PMSCs在SF膜材料上生长良好,培养8 d时材料上细胞伸展增殖,分泌大量颗粒状、网状基质物质,材料间隙被基质填满.结论 SF材料不影响PMSCs的生长特性、表面标志和多向分化潜能,具有良好的生物相容性.     

仿生型BG-COL-HYA-PS复合支架细胞相容性

目的:研究仿生型BG-COL-PS-HYA复合支架材料与成骨细胞的相容性。方法:将小鼠胚胎成骨细胞种植于BG-COL-HYA-PS、BG-COL复合材料、58SBG支架上,用MTT法、ALP活性测定等观察细胞在材料中的生长情况。通过体外实验方法,观察其生物相容性。结果:成骨细胞在BG-COL-HYA-PS材料上能良好粘附、增殖,而在58SBG材料上粘附差、细胞逐渐死亡。MTT法结果显示:联合培养后,BG-COL-HYA-PS组的OD值为0.314±0.004,5天时达到0.621±0.002,分别为58SBG组的1.49倍和1.44倍,P<0.05。结论:仿生型BG-COL-PS-HYA复合支架具有天然骨分级结构和有良好的生物相容性,在诱导成骨细胞增殖方面性能优越,可作为骨组织工程支架材料,临床应用前景广阔。     

蚕丝丝素纤维的制备及其与骨髓间充质干细胞相容性的研究

组织工程为韧带损伤修复提供了可行途径，但韧带修复对支架材料各方面性能要求都很高。在力学性能方面，不仅要求材料有一定的强度而且需要有良好的韧性。在满足力学性能的同时，支架材料还必须兼具优良的生物相容性。蚕丝作为一种天然生物蛋白质，由于其良好的力学性能显示了在组织工程方面应用的前景。但由于丝胶存在污染问题，因此脱胶成为蚕丝在医学领域应用的首要问题。本实验首先比较了三种脱胶试剂对蚕丝力学性质的影响，选择了影响最小的碳酸钠作为脱胶试剂，进而确定了碳酸钠脱胶的最佳条件为：试剂浓度0．40%，温度90℃，时间为1h。然后在脱胶后的丝紊纤维上种植了大鼠骨髓问充质干细胞（Rat bone marrow mesenchymal stem cells，rMSCs），通过扫描电镜（SEM）、荧光显微镜检测了丝素上细胞的生长情况，结果显示蚕丝具有良好的生物相容性，细胞亲和力。为蚕丝在韧带组织工程方面的进一步应用奠定了基础。     

RGD肽对内皮细胞在聚酯材料表面粘附、增殖的影响

RGD是许多粘附蛋白结构中的高度保守序列，与细胞在生物材料表面的粘附、增殖密切相关。本研究在聚酯薄膜表面分别预衬纤维粘连蛋白和共价接枝RGD三肽，然后在不同聚酯材料上种植体外培养的人脐静脉内皮细胞，结果显示RGD可明显促进细胞在材料表面的粘附和增殖，与纤维粘连蛋白相比，RGD促进细胞粘附的作用更为明显，而在细胞增殖方面，二者的作用无显著性差异。本研究为改进生物材料的表面设计，促进心血管移植物的内皮化提供了一个切实可行的思路。     

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.     

Adhesion and function of rat liver cells adherent to silk fibroin/collagen blend films

Collagen is often used in bioartificial livers as a biomimetic coating to promote liver cell adhesion and differentiation. Animal proteins are expensive and expose the host to risks of cross-species infection due to contamination with prions. Silk fibroin (SF) is a biocompatible protein produced by Bombyx mori silk worms and possibly an alternative to collagen. We prepared SF-collagen blend films with different SF content adherent to the bottom of standard tissue culture dishes, and characterized their surface morphology by SEM, their wettability and examined them for their capacity to support rat liver cell adhesion and metabolism. Cell metabolism was characterized by estimating the rate at which cells eliminated ammonia and synthesized urea for up to 48h of culture. SF-containing films were smooth, clear and more wettable than collagen. Cells readily adhered, formed junctions and small size aggregates on all films. As many cells adhered on SF as on collagen films. Cell adhesion to high collagen content blend films could not be reliably estimated because cells dwelt in the large cavities in the film. The effect of SF on cell metabolism differed with the investigated metabolic pathway. However, cells on SF-containing films eliminated ammonia and synthesized urea at rates generally comparable to, for urea synthesis at times higher than, that of cells on collagen. These results suggest that silk fibroin is a suitable substratum for liver cell attachment and culture, and a potential alternative to collagen as a biomimetic coating.     

Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes

Membranes prepared from a protein, fibroin, isolated from domesticated silkworm (Bombyx mori) silk, support the cultivation of human limbal epithelial (HLE) cells and thus display significant potential as biomaterials for ocular surface reconstruction. We presently extend this promising avenue of research by directly comparing the attachment, morphology and phenotype of primary HLE cell cultures grown on fibroin to that observed on donor amniotic membrane (AM), the current clinical standard substrate for HLE transplantation. Fibroin membranes measuring 6.3 ± 0.5 μm (mean ± sd) in thickness and permeable to FITC dextran of a molecular weight up to 70 kDa, were used. Attachment of HLE cells to fibroin was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. Nevertheless, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (K3/K12 expression) and displayed a comparable number and distribution of ΔNp63(+) progenitor cells to that seen in cultures grown on AM. These results support the suitability of membranes constructed from Bombyx mori silk fibroin as substrata for HLE cultivation and encourage progression to studies of efficacy in preclinical models.     

Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends

Adult bone marrow derived mesenchymal stem cells are undifferentiated, multipotential cells and have the potential to differentiate into multiple lineages like bone, cartilage or fat. In this study, polyelectrolyte complex silk fibroin/chitosan blended porous scaffolds were fabricated and examined for its ability to support in vitro chondrogenesis of mesenchymal stem cells. Silk fibroin matrices provide suitable substrate for cell attachment and proliferation while chitosan are promising biomaterial for cartilage repair due to it’s structurally resemblance with glycosaminoglycans. We compared the formation of cartilaginous tissue in the silk fibroin/chitosan blended scaffolds with rat mesenchymal stem cells and cultured in vitro for 3 weeks. Additionally, pure silk fibroin scaffolds of non-mulberry silkworm, Antheraea mylitta and mulberry silkworm, Bombyx mori were also utilized for comparative studies. The constructs were analyzed for cell attachment, proliferation, differentiation, histological and immunohistochemical evaluations. Silk fibroin/chitosan blended scaffolds supported the cell attachment and proliferation as indicated by SEM observation, Confocal microscopy and metabolic activities. Alcian Blue and Safranin O histochemistry and expression of collagen II indicated the maintenance of chondrogenic phenotype in the constructs after 3 weeks of culture. Glycosaminoglycans and collagen accumulated in all the scaffolds and was highest in silk fibroin/chitosan blended scaffolds and pure silk fibroin scaffolds of A. mylitta. Chondrogenic differentiation of MSCs in the silk fibroin/chitosan and pure silk fibroin scaffolds was evident by real-time PCR analysis for cartilage-specific ECM gene markers. The results represent silk fibroin/chitosan blended 3D scaffolds as suitable scaffold for mesenchymal stem cells-based cartilage repair.     

Biodegradation behavior of silk fibroin membranes in repairing tympanic membrane perforations

Silk fibroin (SF) from silkworms has been widely studied as a biomaterial. The degradation behavior of silk biomaterials is important for medical applications, but few studies have examined long-term degradation behavior in vivo. In this study, we investigated the degradation behavior of SF membranes in vitro and in vivo. For the in vitro assay, we observed degradation of silk membranes in phosphate buffered saline, culture media, and an enzyme (proteinase K) solution. In the proteinase K solution, 80% of the silk membranes degraded within 10 days. Silk membranes exhibited no cytotoxicity toward L929 cells and rat tissues. To investigate the degradation of silk membranes in vivo, they were implanted subcutaneously in rats and harvested 19 months after surgery. Scanning electron microscopy imaging and histological analysis of silk membrane explants showed that they broke into several pieces after 16 months. Results show that silk membranes are biocompatible and display excellent long-term degradation behavior when used as biomaterials.     

Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering

The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.     

Characterization and optimization of RGD-containing silk blends to support osteoblastic differentiation

The effect of blending two silk proteins, regenerated Bombyx mori fibroin and synthetic spidroin containing RGD, on silk film material structure (beta-sheet content) and properties (solubility), as well as on biological response (osteoblast adhesion, proliferation and differentiation) was investigated. Although the elasticity and strength of silks make them attractive candidates for bone, ligament, and cartilage tissue engineering applications, silk proteins generally lack bioactive peptides for enhancing cell functions. Thus, a synthetic spider silk, spidroin, containing two RGD cell adhesive sequences (RGD-spidroin) was engineered. RGD-spidroin was blended with different ratios of fibroin and spun coat into films on glass coverslips. beta-Sheet formation, contact angle, surface topography and RGD surface presentation were characterized and correlated with cell behavior. We found that the amount of beta-sheet formation was directly related to the RGD-spidroin content of the blends after annealing, with the pure RGD-spidroin demonstrating the highest amount of beta-sheet content. The increased beta-sheet content improved film stability under culture conditions. A new visualization technique demonstrated that the RGD presentation on the film surface was affected by both the RGD-spidroin content and annealing conditions. It was determined that 10mass% RGD-spidroin was necessary to improve film stability and to achieve osteoblast attachment and differentiation.     

Electrospinning of silk fibroin nanofibers and its effect on the adhesion and spreading of normal human keratinocytes and fibroblasts in vitro

An electrospinning method was used to fabricate silk fibroin (SF) nanofiber nonwovens for cell culture of normal human keratinocytes and fibroblasts. The electrospinning of regenerated SF was performed with formic acid as a spinning solvent. For insolubilization, as-spun SF nanofiber nonwovens were chemically treated with an aqueous methanol solution of 50%. Morphology and microstructure of as-spun and chemically treated SF nanofibers were investigated by scanning electron microscopy and mercury porosimetry. As-spun SF nanofibers exhibited a circular cross-section with a smooth surface. From the image analysis, they had an average diameter of 80 nm and their diameters ranged from 30 to 120 nm. During the chemical treatment for 60 min, porosity of nonwovens composed of SF nanofibers decreased from 76.1% up to 68.1%. To assay the cytocompatibility and cell behavior onto the electrospun SF nanofibers, cell attachment and spreading of normal human keratinocytes and fibroblasts seeded on the SF nanofibers and interaction between cells and SF nanofibers were studied. Cell morphology on SF nanofibers was examined by scanning electron microscopy. Our results indicate that the SF nanofibers may be a good candidate for the biomedical applications, such as wound dressing and scaffolds for tissue engineering.     

The biocompatibility of silk fibroin films containing sulfonated silk fibroin

Sulfonation reaction may be an effective method for preparation of heparin-like materials. However, no sulfonated polymer based on protein backbone was used for improving the blood compatibility of biomaterials. In this study, the biocompatibility of new kind of composite materials films obtained by blending silk fibroin (SF) with sulfonated silk fibroin (SSF) was evaluated. The anticoagulant activity was characterized with prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT), which all increased remarkably, the clot times exceeded the measurement limit of clot detection instrument. Its platelet adhesion was also investigated as another parameter of blood compatibility. The cell compatibility of composite films was evaluated through cell morphologies on the films and cell viability by methyl thiazolyl tetrazolium (MTT) assay. Tensile strength and elongation at break of the composite films reached to 44.6 MPa and 50.3%, respectively. All these results indicated that SF/SSF composite film was a potential material for blood-contact materials and tissue engineering matrix.     

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。