胶原/丝素电纺纳米纤维膜体外培养肝细胞的研究

本实验采用静电纺丝技术制备胶原/丝素纳米纤维支架,对支架上培养的人肿瘤肝细胞HepG2进行形态学观察、细胞功能和代谢功能检测。胶原/丝素纳米纤维支架以六氟异丙醇(HFIP)为溶剂通过静电纺丝技术制备,5种支架材料胶原和丝素配比分别为10∶0、7∶3、5∶5、3∶7、0∶10。扫描电镜结果显示制备的纤维平均直径在550～1100 nm之间,随着丝素含量的增加纤维平均直径增加。细胞培养结果显示HepG2细胞在材料表面生长状态良好并与支架材料紧密结合。随培养时间延长,常规培养组细胞在第5 d后逐渐死亡,失去细胞功能,胶原/丝素纳米纤维支架组细胞在4～9 d内能够维持稳定状态,其尿素合成、蛋白分泌与常规培养组有明显差别,其中丝素含量为50%组的细胞状态和细胞功能高于其它组。实验表明胶原/丝素纳米纤维支架材料细胞相容性良好,较之常规培养细胞增殖效果明显,维持功能表达时间延长,有望用于改善人工肝生物反应器中的细胞活性,维持细胞功能表达。     

丝素蛋白/生物活性玻璃复合纤维膜的制备及性能表征

背景:在构建具有生物功能的引导骨再生膜时,单一材料因功能不足而无法满足临床上的需要,因此将多种材料复合已成为目前组织修复工程的一种趋势。目的:通过静电纺丝技术制备丝素蛋白/生物活性玻璃复合纤维膜,表征其理化性能和体外生物相容性。方法:将0.8 g丝素蛋白溶解于10 mL六氟异丙醇中制备静电纺丝溶液,利用静电纺丝技术制备丝素蛋白纳米纤维膜(记为SF纤维膜);将0.1,0.3,0.5,0.8 g的生物活性玻璃分别加入静电纺丝溶液中,利用静电纺丝技术制备丝素蛋白/生物活性玻璃复合纤维膜(依次记为SF/1BG、SF/3BG、SF/5BG、SF/8BG纤维膜)。表征5组纤维膜的理化性能及生物相容性。结果与结论:①扫描电镜下可见5组纤维表面光滑、连续且均匀,无串珠样结构,丝纤维之间无明显粘连,均表现出随机排布的无序多孔结构,添加生物活性玻璃后纤维膜的纤维直径减小。傅里叶红外光谱与X射线衍射检测显示,纤维膜中丝素蛋白与生物活性玻璃的化学结构稳定。SF、SF/1BG、SF/3BG、SF/5BG、SF/8BG纤维膜表面的水接触角分别为105.02°,72.58°,78.13°,79.35°,72.50°。②将骨髓间充质干细胞分别接种于5组纤维膜上,CCK-8检测显示相较于SF、SF/8BG纤维膜,SF/1BG、SF/3BG、SF/5BG纤维膜可促进骨髓间充质干细胞的增殖;活/死细胞染色显示5组纤维膜表面的细胞活力较好,其中SF/5BG纤维膜表面的细胞数量更多、分布更均匀;罗丹明-鬼笔环肽染色与扫描电镜观察显示相较于SF纤维膜,SF/5BG纤维膜更有利于骨髓间充质干细胞的黏附。将骨髓间充质干细胞分别接种于5组纤维膜上进行成骨诱导分化,SF/3BG、SF/5BG组碱性磷酸酶活性高于其他3组(P<0.05,P<0.01,P<0.001);茜素红染色显示,添加生物活性玻璃后纤维膜的钙结节形成增加,并且以SF/5BG组钙结节形成最多。③结果表明,丝素蛋白/生物活性玻璃复合纤维膜具有良好的生物安全性和生物相容性。     

胶原-壳聚糖复合材料作为组织工程支架的研究

目的 本研究考察壳聚糖对于胶原膜理化性质的影响,选择合适比例的胶原-壳聚糖复合膜作为骨髓间充质干细胞（BMSCs）载体.方法 胶原溶涨液中添加一定比例的壳聚糖,交联并冷冻干燥制备多孔组织工程支架,研究壳聚糖对胶原膜形态学、孔隙率、机械强度、降解特性等理化性质的影响,并初步探讨了胶原-壳聚糖材料作为组织工程三维支架材料与BMSCs的相容性.结果 制备的胶原-壳聚糖支架孔径分布均匀;相对于单纯胶原海绵支架,胶原-壳聚糖复合材料支架降低体外降解速度,提高支架材料的力学性能,稳定支架的结构.BMSCs种植于胶原-壳聚糖（mCol∶mCS=9∶1）支架14 d时,SEM观察到细胞通过微绒毛与支架纤维复合,细胞相容性好.结论 胶原-壳聚糖复合支架有良好的细胞相容性,作为BMSCs诱导体外支架材料具有好的研究前景.     

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/透明质酸膜与明胶海绵支架材料力学及组织相容性的比较

背景：课题组前期实验证明胶原/透明质酸膜具有良好的力学性能和组织相容性。 目的：观察复合材料胶原/透明质酸膜及明胶海绵的生物学性能。 方法：应用材料复合交联的实验方法构建胶原/透明质酸膜并测定胶原/透明质酸膜、明胶海绵支架的力学性能。将支架材料种植于兔皮下,按照2,4,6,8,12周不同时间点评价材料在体内的降解情况和组织相容性。 结果与结论：①成功制备了胶原/透明质酸膜。②胶原/透明质酸膜具有较好的韧性和抗张强度,明胶海绵的力学性能不够理想。③两种材料在体内的降解均符合生物材料的组织反应过程,胶原/透明质酸膜在体内12周可完全降解,明胶海绵约6周完全降解。④胶原/透明质酸膜与平滑肌细胞的黏附率高,细胞的增殖和代谢状况较好,而明胶海绵的细胞黏黏附和增殖率相对较低。说明胶原/透明质酸膜具有较好的生物学性能。     

Ⅱ型胶原-丝素蛋白-透明质酸复合支架的制备及力学性能检测北大核心

背景:Ⅱ型胶原、丝素蛋白和透明质酸3种天然生物可降解材料能够为细胞提供理想的微环境,已成为理想的软骨修复支架材料。目的:将Ⅱ型胶原、丝素蛋白和透明质酸3种材料制备成软骨组织工程支架,评价其理化性质及生物力学性能。方法:采用低温3D打印技术制备Ⅱ型胶原-丝素蛋白-透明质酸复合支架,检测其微观结构、孔隙率与吸水膨胀率。采用不同的应变率对该复合支架进行压缩实验,考察支架的率相关性能;在该复合支架表面施加恒定的应力水平或恒定的应变,保持3600 s,观察其蠕变应变变化及应力松弛行为。结果与结论:①Ⅱ型胶原-丝素蛋白-透明质酸复合支架呈三维多孔结构,孔径大小一致,相通性好,孔隙率为(85.1±1.6)%,吸水膨胀率为(1071.7±131.6)%;②在不同的应变率作用下,软骨支架的压缩应力-应变曲线不重合,说明软骨支架的压缩力学性能具有率相关性;随着应变率的增加,复合支架的杨氏模量增加;③当应力水平恒定时,复合支架的蠕变应变呈现先快速增加后缓慢增加的趋势,当应力水平增加时蠕变应变也增加;④当压缩应变恒定时,支架的应力随着松弛时间先快速降低后缓慢降低,随着压缩应变的增大,不同时刻的应力都增大;⑤力学性能实验表明,制备的Ⅱ型胶原-丝素蛋白-透明质酸软骨支架力学性能特征与宿主软骨组织相似,都是非线性黏弹性材料。     

纳米羟基磷灰石/胶原蛋白/丝素蛋白复合骨组织工程支架材料的生物相容性

背景:通过将两种及两种以上材料共混制备复合支架材料可以弥补各自的不足,利用各种材料的互补特性来满足组织工程对支架的要求。目的:制备纳米羟基磷灰石/胶原蛋白/丝素蛋白复合三维支架材料,并研究其细胞相容性。方法:将纳米羟基磷灰石、胶原蛋白与丝素蛋白分别按质量比为1∶1∶5、1∶2∶5、1∶3∶5的比例混合,制备纳米羟基磷灰石/胶原蛋白/丝素蛋白复合材料,测试其孔隙率、孔径大小、吸水膨胀率及压缩力学性能。将表征结果良好的质量比为1∶2∶5的纳米羟基磷灰石/胶原蛋白/丝素蛋白复合材料与MC3T3-E1细胞体外复合培养,MTT法检测复合培养2,4,6,8,12 d后的细胞活性。结果与结论:羟基磷灰石/胶原蛋白/丝素蛋白按质量1∶2∶5的比例混合更符合要求:孔径98-260μm,孔隙率为(96.72±2.78)%,吸水膨胀率为(549.37±35.29)%,生物力学试验机测定其力学性能稳定、压缩应变及弹性模量等指标适宜骨组织工程研究应用。MC3T3-E1细胞在纳米羟基磷灰石/胶原蛋白/丝素蛋白复合三维支架上生长增殖良好,表明纳米羟基磷灰石/胶原/丝素复合三维支架具有良好的细胞相容性。     

胶原/壳聚糖复合纳米纤维膜引导骨再生的细胞学研究

采用静电纺丝技术制备胶原/壳聚糖复合纳米纤维膜,研究其作为引导骨再生生物膜的细胞生物相容性及诱导成骨性。以乙酸为溶剂,聚环氧乙烯(PEO)为增塑剂,采用静电纺丝技术制备胶原纳米纤维膜及不同比例的胶原/壳聚糖复合纳米纤维膜(胶原、壳聚糖、PEO质量比5∶1∶4,5∶2∶3,5∶4∶1),电子显微镜观察4种纳米纤维膜的表面形态;将骨髓间充质干细胞种植于胶原纳米纤维膜及表面形态较好的胶原/壳聚糖纳米纤维膜上,通过MTT法、碱性磷酸酶检测、细胞内胶原检测、免疫荧光染色及茜素红染色法观察,研究其细胞生物相容性及诱导成骨性。扫描电子显微镜观察胶原纳米纤维膜及质量比为5∶1∶4的胶原/壳聚糖复合纳米纤维膜的纤维光滑,直径均一。MTT法检测显示,胶原纳米纤维膜和胶原/壳聚糖复合纳米纤维膜均可促进骨髓间充质干细胞的粘附和增殖。细胞培养14 d后,胶原/壳聚糖复合纳米纤维膜上细胞内胶原含量检测为2.02 mg/gport,高于胶原纳米纤维膜组的1.63 mg/gport胶原含量(P<0.05),且胶原/壳聚糖复合纳米纤维膜上细胞内碱性磷酸酶、骨钙素及钙化结节的形成均高于胶原纳米纤维膜组。胶原/壳聚糖复合纳米纤维膜可促进骨髓间充质干细胞的增殖和分化,有望应用于骨再生的研究。     

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。     

新型纳米支架与神经干细胞的凋亡

背景：胶原作为脊髓组织工程的良好支架有利于神经细胞及神经纤维的黏附和生长,但机械性能较差,需要在制备时提高其基本性能,还应对种子细胞的生物学行为产生积极影响。 目的：分析胶原纳米组织工程支架的性能,检测其对神经干细胞凋亡行为及相关基因表达的影响。 方法：采用电纺丝技术制备纤维定向排列及非定向排列的胶原纳米纤维膜,并对其进行表征。将新生SD大鼠脊髓源性神经干细胞分别与纤维定向排列及非定向排列的胶原纳米纤维膜共培养,并设置单独神经干细胞培养为对照,检测细胞凋亡情况及相关基因表达变化。 结果与结论：定向及非定向胶原纳米纤维膜的直径及形貌均达到纳米组织工程支架标准,溶胀系数较高,孔隙率较高,力学性能佳。与对照组比较,两胶原纳米纤维膜组神经干细胞凋亡率明显降低(P < 0.05),细胞凋亡相关基因中bcl-2基因表达量明显增加,bax及Caspase-3基因表达明显下降;两胶原纳米纤维膜组神经干细胞凋亡率差异无显著性意义。表明新型胶原纳米组织工程支架性能良好,能够抑制细胞凋亡,从基因表达水平调节神经干细胞的凋亡行为。     

载TGF-β1纳米粒的胶原/丝素蛋白复合支架的制备与表征

目的 构建一种载转化生长因子β1(TGF-β1)纳米粒的双层胶原/丝素蛋白复合支架.方法 制备载TGF-β1的壳聚糖-肝素(Ch-Hep)纳米粒,检测其形态、粒径、Zeta电位和包封率.制备不同胶原和丝素蛋白质量比(2∶8、3∶7、7∶3、8∶2、10∶0)的5种胶原/丝素蛋白复合材料,分别检测其吸水率、孔隙率、热水溶失率和生物相容性;选择其中2种综合性能良好的复合材料分别作为复合支架的疏松层和致密层,构建载TGF-β1纳米粒的双层胶原/丝素蛋白复合支架,观察其形态并进行体外释放动力学研究.结果 Ch-Hep纳米粒的平均粒径为(718.2±73.6) nm,Zeta电位为(25.5±0.8) mV,对TGF-β1的包封率为(84.82±1.57)％.随着胶原/丝素蛋白复合材料中胶原含量的增加,材料的吸水率、孔隙率逐渐增加,热水溶失率逐渐降低;5种材料对骨髓间充质干细胞(BMSCs)均有促生长和增殖的作用.综合考虑后选用质量比为3∶7和7∶3的胶原/丝素蛋白复合材料分别作为复合支架的致密层和疏松层,构建的胶原/丝素蛋白复合支架为双层结构,一侧结构致密,另一侧疏松多孔.体外释放动力学研究表明,复合支架对TGF-β1具有定向时空控制性释放作用.结论 载TGF-β1纳米粒的双层胶原/丝素蛋白复合支架对TGF-β1具有良好的时空控制释放作用,有望作为生长因子的控缓释支架材料应用于软骨组织工程.     

Genipin-crosslinked polyvinyl alcohol/silk fibroin/nano-hydroxyapatite hydrogel for fabrication of artificial cornea scaffolds—a novel approach to corneal tissue engineering

Abstract

Design of artificial corneal scaffolds substitute is crucial for replacement of impaired cornea. In this paper, porous polyvinyl alcohol/silk fibroin/nano-hydroxyapatite (PVA/SF/n-HA) composite hydrogel was prepared via the genipin (GP) cross-linking, the pore diameter of the hydrogel ranged from 8.138?nm and 90.269?nm, and the physical and physiological function of hydrogel were investigated. The resulting hydrogel exhibited favourable physical properties. With the GP content increasing, the structural regularity of PVA/SF/n-HA composite hydrogel was enhanced and the thermal stability was improved. The moisture content was slightly decreased and generally maintained at approximately 70%. The tensile strength was heightened up to 0.64?MPa, while the breaking elongation was decreased slightly. Moreover, the biofunction was investigated. The in vitro degradation test demonstrated that with the addition of GP, the stability of the composite hydrogels in protease XIV solution was promoted and the three-dimensional porosity structure of composite hydrogels was maintained as ever. And the human corneal fibroblasts (HCFs) were employed to examine the cells cytotoxicity of the PVA/SF/n-HA composite hydrogels with different GP content by CCK-8 assay. Based on confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM), HCFs had individually commendable adhesion and proliferation on PVA/n-HA/SF composite hydrogel. HCFs proliferated and grew into the pores of composite hydrogel. The results of biocompatibility experiments of composite hydrogel suggested that it was no acute toxicity, in vitro cytotoxicity was 0 or 1 grade. Overall, results from this paper, PVA/n-HA/SF composite hydrogel was a qualified medical material which conformed to the national standard, could be a promising alternative for artificial cornea scaffold material—a novel approach to corneal tissue engineering.     

Preparation and characterization of nano-hydroxyapatite/silk fibroin porous scaffolds

Novel tissue engineering scaffold materials of nano-hydroxyapatite (nHA)/silk fibroin (SF) biocomposite were prepared by freeze-drying. The needle-like nHA crystals of about 10 nm in diameter by 50-80 nm in length, which were uniformly distributed in the porous nHA/SF scaffolds, were prepared by a co-precipitation method with a size. The as-prepared nHA/SF scaffolds showed good homogeneity, interconnected pores and high porosity. XRD and FT-IR analysis suggested that the silk fibroin was in beta-sheet structure, which usually provides outstanding mechanical properties for silk materials. In this work, composite scaffolds containing as high as 70% (w/w) nHA were prepared, which had excellent compressive modulus and strength, higher than the scaffolds at low nHA content level and other porous biodegradable polymeric scaffolds often considered in bone-related tissue engineering reported previously. The cell compatibility of composite scaffolds was evaluated through cell viability by MTT assay. All these results indicated that these nHA/SF scaffold materials may be a promising biomaterial for bone tissue engineering.     

Preparation and characterization of nano-hydroxyapatite/silk fibroin porous scaffolds

Novel tissue engineering scaffold materials of nano-hydroxyapatite (nHA)/silk fibroin (SF) biocomposite were prepared by freeze-drying. The needle-like nHA crystals of about 10 nm in diameter by 50–80 nm in length, which were uniformly distributed in the porous nHA/SF scaffolds, were prepared by a co-precipitation method with a size. The as-prepared nHA/SF scaffolds showed good homogeneity, interconnected pores and high porosity. XRD and FT-IR analysis suggested that the silk fibroin was in β-sheet structure, which usually provides outstanding mechanical properties for silk materials. In this work, composite scaffolds containing as high as 70% (w/w) nHA were prepared, which had excellent compressive modulus and strength, higher than the scaffolds at low nHA content level and other porous biodegradable polymeric scaffolds often considered in bone-related tissue engineering reported previously. The cell compatibility of composite scaffolds was evaluated through cell viability by MTT assay. All these results indicated that these nHA/SF scaffold materials may be a promising biomaterial for bone tissue engineering.     

Physical-chemical and biological characterization of silk fibroin-coated porous membranes for medical applications

Membranes in artificial organs and scaffolds for tissue engineering are often coated with biomimetic molecules (e.g., collagen) to improve their biocompatibility and promote primary cell adhesion and differentiation. However, animal proteins are expensive and may be contaminated with prions. Silk fibroin (SF) made by Bombyx Mori silk worms, used as a scaffold or grafted to other polymers, reportedly promotes the adhesion and growth of many human cell types. This paper describes how commercial porous membranes were physically coated with SF, and their physical-chemical properties were characterized by SEM, AFM, tensile stress analysis and dynamic contact angle measurements. The effect of the SF coating on membrane biocompatibility and resistance to bacterial colonization is also examined. The proposed technique yields SF coats of different thickness that strengthen the membranes and make their surface remarkably more wettable. The SF coat is not cytotoxic, and promotes the adhesion and proliferation of an immortalized fibroblast cell line. Similarly to collagen, SF-coated membranes also exhibit a much better resistance to the adhesion of S. epidermidis bacteria than uncoated membranes. These preliminary results suggest that SF is a feasible alternative to collagen as a biomimetic coating for 3D scaffolds for tissue engineering or bioartificial (as well as artificial) prosthesis.     

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 bone marrow stromal cell responses on electrospun silk fibroin mats

Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700+/-50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (approximately 15 microm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.     

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。