丝素蛋白的免疫学特性及细胞相容性研究进展

丝素蛋白作为一种具有良好生物相容性和低免疫原性的天然高分子材料在生物医学领域里有着广泛的应用价值。阐述了近年来对丝素蛋白在组织工程学研究中所涉及的免疫学方面及细胞相容性方面的研究概况。     

丝素蛋白生物硅化材料在骨组织工程学中的应用

制备可生物降解并具有骨再生功能的支架材料是骨组织工程当前研究的重点领域。丝素蛋白具备可用作骨组织工程支架材料的许多要素,其中通过仿生方式生物矿化制备的丝素蛋白/无机复合材料与单纯丝素相比,由于具有较好的生物相容性、生物力学性能、可生物降解性以及骨诱导和传导特性,展现出更好的应用前景。本文综述了近年丝素蛋白生物硅化制备新型骨组织工程材料的研究进展,并展望了丝素蛋白生物硅化今后的发展方向。     

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。     

胶原及丝素蛋白支架材料在脊髓组织工程中的应用

背景:以胶原及丝素蛋白材料为构架的脊髓生物支架,已被证实可以修复或部分修复受损的脊髓神经功能。目的:介绍胶原蛋白和丝素蛋白的部分特性,并对近年来其作为支架材料在脊髓组织工程中的应用及进展作一综述。方法:应用计算机检索CNKI和PubMed数据库中2003年1月至2012年10月关于胶原蛋白及丝素蛋白支架材料在脊髓损伤中应用的文章,在主题和摘要中中文以"胶原蛋白,丝素蛋白,支架材料,脊髓损伤"为检索词检索;英文以"collagen;silk fibroin;spinal cord injury"为检索词进行检索。结果与结论:胶原蛋白具有低抗原性、优良的生物相容性和生物降解性,本身及其降解产物在人体内不会引起炎症反应,但存在降解速度过快及力学性能差等缺点。丝素蛋白具有良好的生物相容性及优良的力学性能,但降解速度较慢。将胶原蛋白和丝素蛋白用静电共纺的方法制备成复合体,可以在保持良好生物相容性的基础上,提高材料的物理性能。目前,国内外已对丝素或胶原蛋白材料在神经系统修复方面的应用做了一些研究,为脊髓组织工程奠定了一些基础;又考虑到胶原和丝素材料与脊髓软组织的特性和力学性能相似,胶原/丝素蛋白复合材料有望成为脊髓组织工程理想的支架材料。     

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。     

丝素蛋白的生物相容性及临床应用

背景：皮肤损伤后的修复和重建都是棘手的问题,以皮肤细胞本身作为皮肤替代物修复缺损,以达到恢复组织器官的形态和功能,成为一种理想的途径,也是临床上难以解决的问题 目的：文章综述了丝素蛋白的生物相容性及应用的研究进展,寻找最佳人工皮肤以应用临床。 方法：应用计算机检索CNKI和PubMed数据库中1992-02/2011-03关于丝素蛋白生物相容性,在组织工程皮肤、生物材料领域应用的文章,在标题和摘要中以“丝素蛋白;生物相容性;组织工程;生物材料;应用”或“Silk fibroin;Biocompatibility;Tissue Engineering;Biological materials;Application”为检索词进行检索。选择文章内容与丝素蛋白的生物相容性及应用相关,同一领域文献则选择近期发表或发表在权威杂志文章。初检得到215篇文献,根据纳入标准选择22篇文章进行综述。 结果与结论：对丝胶蛋白特性及研究应用的了解,有利于皮肤损伤后的修复和重建,以皮肤细胞本身作为皮肤替代物修复缺损,以达到恢复组织器官的形态和功能,有良好的机械性能和理化性质及有良好的生物相容性,对表皮细胞生长具有一定的促进作用。但将其应用于临床治疗方面仍需要很长的时间,尚有一些问题需要进一步研究、解决。     

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/壳聚糖复合支架材料的细胞相容性**

背景：大量研究表明丝素蛋白、壳聚糖为天然高分子材料,具有良好的细胞生物相容性。 目的：探讨丝素蛋白/壳聚糖复合支架材料与诱导的兔骨髓间充质干细胞的生物相容性。 方法：将兔骨髓间充质干细胞分离培养、诱导后,与丝素蛋白/壳聚糖三维支架材料体外共培养,以材料的细胞毒性、细胞增殖活力、材料细胞黏附率及扫描电镜等检测评价材料的细胞相容性。 结果与结论：经诱导后的骨髓间充质干细胞在支架材料上黏附、生长良好,保持正常的分裂增殖速度;随时间的增加,细胞黏附率增加,材料组较对照组黏附率强,差异有显著性意义(P < 0.05)。扫描电镜观察发现细胞接种48 h后细胞生长良好,与支架黏附紧密,增殖分裂活跃。说明丝素蛋白/壳聚糖三维支架材料具有良好的细胞相容性。     

丝素蛋白作为药物载体材料的研究进展

近年来药物载体材料的研究已成为医学领域研究的热点。选择适宜的载体材料是获得良好给药系统的关键。丝素蛋白是一种天然蛋白质,具有良好的生物相容性和机械性能,可生物降解,无毒,易于加工制备,因而是一种优良的药物载体材料。本文基于丝素蛋白的结构和特点,综述了它作为药物载体材料的最新研究进展。     

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。     

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。     

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.     

Fibroin/collagen hybrid hydrogels with crosslinking method: preparation, properties, and cytocompatibility

Substrate stiffness is an important physical factor in the response of many cell types. Although protein-based hydrogels are widely used as cell-culture substrates because of their resemblance to the natural extracellular matrix (ECM) and complex signaling, their rigidity should be further increased to facilitate the adhesion and growth of cells. In this study, fibroin/collagen hydrogels having suitable stiffness were prepared directly by adding 1-ethyl-3-(3-dimethylaminoprophy) carbodiimide hydrochloride (EDC) in fibroin/collagen solution to induce crosslinking. The storage moduli of these crosslinked hydrogels are above 3 kPa, and even exceed 10 kPa, having stronger mechanical strength than that of previously reported protein-based hydrogels. Furthermore, the crosslinked hydrogels can maintain their configuration above 80(o)C, which proves their increased thermal stability. Although crosslinked, the hydrogels still maintain the mobility of fibroin molecules. The growth of vascular smooth muscle cells (VSMCs) in fibroin/collagen gels indicates that the crosslinking reaction has no negative influence on the biocompatibility of fibroin/collagen hydrogels. The fibroin/collagen hydrogels are more propitious to the growth of cells compared with fibroin/collagen scaffolds. Because of their inherent biocompatibility, excellent mechanical and thermal properties, and green preparation process, the fibroin/collagen hydrogels would become promising scaffolds for tissue engineering.     

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。     

Cytocompatibility and blood compatibility of multifunctional fibroin/collagen/heparin scaffolds

An applicable matrix used in tissue engineering should not only have suitable mechanical properties, porous structures and biocompatibility that facilitate the adhesion, growth and proliferation of tissue cells, but also have the ability to release bioactive factors to provide a more conducive and inductive environment for tissue growth. Because of the harsh preparation conditions and deficiency of mechanical properties, it is still difficult for fibroin and collagen matrices to possess these multifunctional properties. In this research, we successfully prepared fibroin/collagen hybrid scaffolds containing heparin that possess multifunctional properties under mild conditions. These scaffolds maintain outstanding mechanical properties and porous structures of fibroin-based scaffolds. Furthermore, the scaffolds keep the bioactivity of collagen, becoming delivering systems that release heparin slowly to make the scaffolds blood compatible. Compared with fibroin/collagen scaffolds, the scaffolds containing heparin further facilitate the growth of HepG2 cells since a more complex, dynamic environment was formed to promote the cell growth. Considering the mild aqueous preparation environment without crosslinking reaction, besides promoting the progress in blood contacting tissue engineering, our research has also opened a door to prepare various multifunctional fibroin/collagen hybrid matrices that combine the advantages of fibroin and collagen.     

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.     

Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro

Silk-based materials have been used in the field of bone or ligament tissue engineering. In order to explore the feasibility of using purified silk fibroin to construct artificial nerve grafts, it is necessary to evaluate the biocompatibility of silk fibroin material with peripheral nerve tissues and cells. We cultured rat dorsal root ganglia (DRG) on the substrate made up of silk fibroin fibers and observed the cell outgrowth from DRG during culture by using light and electron microscopy coupled with immunocytochemistry. On the other hand, we cultured Schwann cells from rat sciatic nerves in the silk fibroin extract fluid and examined the changes of Schwann cells after different times of culture. The results of light microscopy, MTT test and cell cycle analysis showed that Schwann cells cultured in the silk fibroin extract fluid showed no significant difference in their morphology, cell viability and proliferation as compared to that in plain L15 medium. Furthermore, no significant difference was found in expression of the factors secreted by Schwann cells, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and S-100, between Schwann cells cultured in the silk fibroin extraction fluid and in plain L15 medium by the aid of immunocytochemistry, RT-PCR and Western analysis. Collectively, these data indicate that silk fibroin has good biocompatibility with DRG and is also beneficial to the survival of Schwann cells without exerting any significant cytotoxic effects on their phenotype or functions, thus providing an experimental foundation for the development of silk fibroin as a candidate material for nerve tissue engineering applications.