丝素蛋白/聚己内酯纳米纤维支架生物相容性评价

目的:改善再生丝素蛋白的降解性及力学性能,评价丝素蛋白/聚己内酯纳米纤维支架神经生物材料的生物相容性。方法:采用静电纺丝技术制备丝素蛋白/聚己内酯纳米纤维支架。体外培养雪旺细胞并与支架及其浸提液共培养,通过荧光染色,细胞毒性试验(MTT法)检测其细胞生物相容性。将纤维支架材料在体外置于蛋白酶ⅪV溶液评价其体外降解行为;通过皮下埋植实验观察纤维材料在体内的局部组织反应。结果:丝素蛋白/聚己内酯支架材料,呈现三维网状结构。雪旺细胞具有良好的生长形态;无细胞毒性。随着丝素蛋白比例的降低,能够显著增加混合支架的降解速度。皮下移植实验未引起明显免疫排斥反应,炎症反应轻。结论:丝素蛋白/聚己内酯支架具有良好的生物相容性和生物可降解性,有望用于神经组织工程支架材料修复神经缺损。     

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。     

多肽对生物材料表面修饰的研究现状

对生物材料进行表面修饰可以提高生物材料的细胞粘附性,用多肽对生物材料进行表面修饰是一种较好的表面改性方法。本文对生物材料表面修饰用多肽的种类的选择及多肽固定的方法研究现状进行综述。     

多肽对生物材料表面修饰的研究现状

对生物材料进行表面修饰可以提高生物材料的细胞粘附性，用多肽对生物材料进行表面修饰是一种较好的表面改性方法。本文对生物材料表面修饰用多肽的种类的选择及多肽固定的方法研究现状进行综述。     

基质可湿性对人成纤维细胞吸附强度的影响

生物材料常用作生物医学装置,如血管移植物、人工皮肤、人工角膜及骨修补。重要的是这些材料与周围组织或体液的相互作用,这是研究的主要范围,另外生物材料表面的细胞粘附及分离主要依赖于材料表面的可湿性。Schanraad等人报告了疏水性表面细胞分离差,在高亲水性基质表面分离明显,内皮细胞也可吸附于可湿性聚合物表面。发现羟乙基甲基丙烯酸酯与     

丝素蛋白材料在组织工程中的新进展

背景：丝素蛋白支架已被建议运用在组织工程骨和软骨重建、肌腱重建、血管重建,神经重建以及膀胱重建等各方面。 目的：总结丝素蛋白作为支架在生物材料和组织工程领域的应用与发展。 方法：由第一作者应用计算机检索PubMed数据库及中国期刊数据库2000年1月至2011年11月有关丝素蛋白支架制备工艺,丝素蛋白支架修饰方法及丝素蛋白在组织工程中的应用等方面的文献。 结果与结论：丝素蛋白具有机械强度高、生物降解性慢、生物相容性良好、制备工艺多样等特点,支持多种细胞黏附、分化和生长,可应用于人工韧带、血管、骨、神经组织等方面。近期以丝素蛋白支架作为载体,通过多种方式添加各种生物制剂,比如各种生长因子和细胞因子,进一步扩大丝素蛋白在组织工程中的应用范围。     

结合RGD肽的聚酯材料表面粘附内皮细胞的抗剪切力研究

精氨酸-甘氨酸-天门冬氨酸(RGD)是许多粘附蛋白的高度保守氨基酸序列.生物材料表面结合RGD肽有助于内皮细胞在材料上的粘附、迁移和增殖.本研究在体外流动条件下观察结合RGD肽或纤维粘连蛋白的聚酯材料表面粘附内皮细胞的抗剪切能力,并通过观察肌动蛋白和踝蛋白的表达初步探讨影响细胞粘附稳定性的机制.结果显示材料表面结合RGD或纤维粘连蛋白可以增加细胞的粘附强度,提高抗剪切能力;而RGD和纤维粘连蛋白导致的细胞抗剪切能力增加可能与细胞内肌动蛋白和踝蛋白的表达增加有关.     

低温等离子体对丝素纤维人工血管材料蛋白吸附性能的影响

丝素纤维是一种天然蛋白质纤维,具有良好的生物相容性,在生物医用材料领域具有广阔的应用前景。该文分别采用氦气和氧气低温等离子体处理丝素纤维人工血管材料,探究不同等离子体对材料表面形貌、亲水性、力学性能及蛋白吸附性能的影响。结果表明,两种等离子体均可对丝素纤维表面产生刻蚀作用,且氧气等离子体的刻蚀作用较强。然而,氦气等离子体对改善材料表面亲水性效果较优。拉伸断裂强度结果显示,氧气等离子体对丝素纤维人工血管材料的力学性能损伤较大。蛋白吸附试验结果显示,两种等离子体均能降低血浆蛋白在材料表面的吸附,且氦气等离子体处理的效果更为显著。本研究结果表明,采用氦气等离子体处理丝素纤维人工血管材料,可能更有助于减少血细胞在材料表面的粘附,从而降低形成血栓的风险,且对材料力学性能影响不大。     

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.     

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。     

Porous Silk Fibroin Film as a Transparent Carrier for Cultivated Corneal Epithelial Sheets

Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin β1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium.     

Stem cell-based tissue engineering with silk biomaterials

Silks are naturally occurring polymers that have been used clinically as sutures for centuries. When naturally extruded from insects or worms, silk is composed of a filament core protein, termed fibroin, and a glue-like coating consisting of sericin proteins. In recent years, silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility, slow degradability and remarkable mechanical properties of the material. In addition, the ability to now control molecular structure and morphology through versatile processability and surface modification options have expanded the utility for this protein in a range of biomaterial and tissue-engineering applications. Silk fibroin in various formats (films, fibers, nets, meshes, membranes, yarns, and sponges) has been shown to support stem cell adhesion, proliferation, and differentiation in vitro and promote tissue repair in vivo. In particular, stem cell-based tissue engineering using 3D silk fibroin scaffolds has expanded the use of silk-based biomaterials as promising scaffolds for engineering a range of skeletal tissues like bone, ligament, and cartilage, as well as connective tissues like skin. To date fibroin from Bombyx mori silkworm has been the dominant source for silk-based biomaterials studied. However, silk fibroins from spiders and those formed via genetic engineering or the modification of native silk fibroin sequence chemistries are beginning to provide new options to further expand the utility of silk fibroin-based materials for medical applications.     

Silk Fibroin Processing and Thrombogenic Responses

Silkworm-derived fibroin, which constitutes the core of the silk filament, is an attractive protein–polymer for biomedical applications. Fibroin can also be processed into a variety of 2-D and 3-D formats to match morphological and structural features to specific applications. The focus of the present research was to correlate the structure of silk fibroin-derived biomaterials with plasma protein adsorption, platelet activation and inflammatory cell (THP-1 cell line) adhesion and activation. The amino-acid composition of the two types of silk studied influenced the crystallinity of the films, hydrophobicity, surface roughness and biological interactions. Protein adsorption was lower on samples with the higher crystallinity and hydrophobicity, in particular the chemotactic factors (C3a, C5a, C3b), while other proteins such as fibrinogen were comparable in terms of adsorption. As a consequence, platelets and immune cells responded differently to the various films obtained by following different processing protocols and stabilized by different methods (methanol or water vapour) in terms of their adherence, activation, and the secretion of inflammatory mediators by monocytes. The data presented here demonstrate that bioactivity can be influenced by changing the chemistry, such as the source of silk protein, or by the specific process used in the preparation of the materials used to assess biological responses.     

丝素蛋白纳米羟基磷灰石复合材料的生物安全性

BACKGROUND: Silk fibroin has excellent biocompatibility, biodegradability and unique mechanical properties. Its composite, silk fibroin/nano-hydroxyapatite, can simulate the composition and structure of nature bone tissue, contributing to remedying the insufficient mechanical properties of nano-hydroxyapatites. OBJECTIVE: To observe the biological safety of silk fibroin/nano-hydroxyapatite composites. METHODS: Silk fibroin/nano-hydroxyapetite composite biomaterial was synthesized by the coprecipitation method using silk fibroin, calcium chloride and diammonium phosphate as raw materials. According to the demands of International Standard Organization (ISO10993) and Technical Evaluation Standards of Biomedical Materials and Medical Instruments promulgated by Chinese Board of Health (GB/T 16886), experiments of cell toxicity in vitro, acute toxicity and hemolysis were investigated to evaluate the biocompatibility of silk fibroin/nano-hydroxyapetite composite. RESULTS AND CONCLUSION: L929 cells co-cultured with silk fibroin/nano-hydroxyapatite composite leaching liquor had good cell morphology, metabolism and proliferation. The leaching extract of silk fibroin/nano-hydroxyapatite composite injected into mice intraperitoneally had no significant adverse reactions. And silk fibroin/nano-hydroxyapatite composite extracts caused 2.39% blood hemolysis, less than the international standards 5%. These experimental results on cell toxicity test in vitro, acute toxicity and hemolysis met the demands of ISO10993 and GB/T, which show the biological safety of the silk fibroin/nano-hydroxyapatite composite for clinical application.     

Effect of processing on silk-based biomaterials: reproducibility and biocompatibility

Silk fibroin has been successfully used as a biomaterial for tissue regeneration. To prepare silk fibroin biomaterials for human implantation a series of processing steps are required to purify the protein. Degumming to remove inflammatory sericin is a crucial step related to biocompatibility and variability in the material. Detailed characterization of silk fibroin degumming is reported. The degumming conditions significantly affected cell viability on the silk fibroin material and the ability to form three-dimensional porous scaffolds from the silk fibroin, but did not affect macrophage activation or β-sheet content in the materials formed. Methods are also provided to determine the content of residual sericin in silk fibroin solutions and to assess changes in silk fibroin molecular weight. Amino acid composition analysis was used to detect sericin residuals in silk solutions with a detection limit between 1.0 and 10% wt/wt, while fluorescence spectroscopy was used to reproducibly distinguish between silk samples with different molecular weights. Both methods are simple and require minimal sample volume, providing useful quality control tools for silk fibroin preparation processes.     

丝蛋白应用于牙周组织工程的可行性

背景：丝蛋白是有利于表皮细胞、成纤维细胞、成骨细胞、血管内皮细胞、胶质细胞黏附和生长的一种新型生物材料。 目的：评估丝蛋白作为支架材料应用于牙周组织工程的可行性。 方法：采用组织块法培养人牙周膜细胞,将第5代细胞悬液以2×10⁷ L^-1的浓度接种到丝蛋白支架材料上复合培养,并以1%,10%,50%,100%的丝蛋白支架浸提液培养,观察人牙周膜细胞在丝蛋白上及在丝蛋白浸提液中生长状况,用MTT法测定浸提液培养人牙周膜细胞的活力。 结果与结论：扫描电镜可见人牙周膜细胞在丝蛋白支架上伸展充分,生长旺盛,不同浓度丝蛋白支架浸提液培养对人牙周膜细胞的增殖与碱性磷酸酶活性均无影响。说明丝蛋白材料具有良好的生物相容性、独特的力学性能,可作为人牙周膜细胞黏附生长的理想支架材料较好地应用于牙周组织工程中。     

编织型丝素纤维人工韧带及其体外降解性能研究

该研究制备了一种丝素纤维人工韧带,分别在PBS和蛋白酶XIV溶液中进行体外降解试验。并对其表面形态、质量损失、降解液pH及力学性能等进行了观察和测试。结果表明,PBS溶液中降解70天后,丝素纤维人工韧带表面形貌和质量损失率没有发生明显变化,力学性能基本保持不变,溶液pH稳定呈中性。而酶降解70天的丝素纤维人工韧带表面发现多根纤维断裂,质量损失率高达18.4%,丝素纤维人工韧带材料直径下降达21.8%,最大断裂强力损失了近50%。降解后溶液的pH值呈弱酸性,且溶液中存在肉眼可见的絮状物。蛋白酶XIV对天然蛋白丝素纤维人工韧带材料具有明显的降解作用。该研究可为体外评价可降解人工韧带降解速率、建立体内外降解性能之间的关系提供参考。     

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.     

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.     

静电纺胶原/丝素复合微纳米纤维的制备及细胞相容性研究

采用静电纺丝技术制备胶原/丝素复合微纳米纤维,对其理化性能进行表征并观察其细胞相容性。以六氟异丙醇(HFIP)为溶剂,将胶原和丝素以 100:0.70:30.50:50.30:70.0:100的质量比共混进行电纺。制备的五种材料经戊二醛蒸汽交联12 h。采用扫描电镜、红外光谱、X射线衍射、热重分析和拉伸力学性能测试等方法对其理化性能进行表征。材料种植成纤维细胞后,通过扫描电镜和噻唑兰(MTT)比色法观察其细胞相容性。结果显示制备的纤维平均直径在550~1 100 nm之间,随着丝素含量的增加纤维平均直径增加。交联后纤维的β化程度、结晶度和热稳定性均有一定提高,且随着丝素含量的增加提高越明显;交联后材料的力学性能优于交联前;当丝素含量为70%时,纤维膜的平均断裂强度为(8.70±1.05) MPa,高于其它配比的纤维膜。细胞在材料表面生长状态良好;丝素含量为70%组的细胞粘附和增殖高于其它组,与细胞培养板相比无显著性差异,表明其细胞相容性良好,可望成为一种新型的组织工程支架材料。