功能化多孔聚氨酯薄膜的制备及其生物医学应用

聚氨酯(PU)因其具有良好的生物相容性、优异的力学性能、耐磨损及易加工成型等优点,广泛应用于生物医学领域,将其加以修饰改性制备多孔结构聚氨酯,有望成为组织工程支架领域的热门材料。本文中,笔者通过热致相分离法制备多孔PU薄膜,利用肝素对多孔PU薄膜进行改性;通过血液实验证明肝素改性前后多孔PU薄膜血液相容性差异;并采用血管内皮生长因子(VEGF)对薄膜进行修饰。傅里叶红外光谱、电子光谱测试和扫描电子显微镜等分析结果表明肝素改性成功。与改性前相比,修饰肝素可以赋予多孔PU薄膜优异的抗粘附特性,有效延长薄膜的体外凝血时间,降低溶血率,避免红细胞形态学异常现象和凝集现象,同时避免补体激活和血小板激活现象。因此,肝素修饰的多孔PU薄膜展示了良好的生物相容性。此外,实验结果表明:VEGF对薄膜进行修饰,可以有效提高生物活性涂层的细胞活性,促进细胞增殖。     

磷铵两性离子改性血管脱细胞支架可以有效体外抗血栓和内皮化

由于自体血管(由同一受体的血管用于血管移植材料)的有限可用性,以及非自体血管(人工制成的血管移植材料)的生长能力不足,组织工程血管越来越受到重视。文中构建了一种磷铵两性离子改性的血管脱细胞支架附以高度生物相容的骨髓源内皮祖细胞为内层的新型血管移植材料。通过一种简便的方法——共沉淀法改性血管脱细胞支架,评价其体外血小板粘附实验、溶血实验、复钙实验和细胞毒性等相关指标。磷铵两性离子改性后抗凝血活性提高,可以有效地促使类似于天然血管腔表面凹凸结构的脱细胞支架表面内皮祖细胞的附着。改性后的脱细胞支架具有与天然血管相似的力学性能,在体外可以有效地构建内皮化。研究结果为血管脱细胞支架通过改性实现体外抗血栓和内皮化方面进行了初步探索。     

许旺细胞复合改性PLA\PGA的体外生物学研究

目的：评价小鼠许旺细胞体外复合改性聚乳酸\聚羟基乙酸（PLA\PGA）的细胞活性及生物相容性。方法：转绿色荧光蛋白基 因（GFP）小鼠的许旺细胞传代培养至第2 代,然后通过MTT 检测在不同改性技术（H2O2、NaOH、NaClO4、K2CrO4及超声波）处理 的PLA\PGA 浸提液中许旺细胞的增殖情况,检测许旺细胞在PLA\PGA表面的黏附及其细胞形态。结果：于培养1 d,3 d测得在 不同改性技术处理的PLA\PGA浸提液OD值,1 天时,各浸提液组和对照组相比无显著性差异,许旺细胞的活力及增殖无影响。3 天时,经NaClO4及K2CrO4处理的PLA\PGA 与对照组相比具有统计学差异,影响许旺细胞的增殖,对许旺细胞有毒性;荧光显微 镜下观察到许旺细胞在改性PLA\PGA 表面逐渐伸展,形成伪足,最终粘附在材料表面。结论：经H2O2、NaOH 及超声波改性 PLA\PGA无细胞毒性,具有良好的生物相容性和黏附性,可以用于组织工程化神经的构筑。     

去细胞牛颈静脉构建组织工程右心带瓣管道生物相容性实验

目的:检测去细胞处理牛颈静脉的生物相容性.方法:去细胞处理牛颈静脉,与新鲜牛颈静脉分别于兔皮下包埋,术后2,4,12周取材,HE染色;进行血浆蛋白吸附和血小板粘附实验,检测血液相容性.结果:包埋早期有炎性细胞浸润,后期炎性细胞消失,以成纤维细胞为主.去细胞牛颈静脉与新鲜牛颈静脉血浆蛋白吸附和血小板粘附无显著差异.结论:去细胞牛颈静脉有良好的生物相容性.     

BaFe12O19血管内无机微粒栓塞材料的生物相容性研究

目的:本研究旨在探讨BaFe12O19微粒的生物相容性.方法:对BaFe12O19微粒进行一系列生物相容性实验,包括Ames致突变试验、细胞毒性试验、全身急性和亚急性毒性试验、溶血试验、出血和凝血时间测定、凝血试验等.结果:BaFe12O19微粒无毒性,无致突变性,对出血和凝血时间无影响,不引起溶血和凝血.结论:BaFe12O19微粒具有良好的生物相容性,有可能成为一种全新的血管内栓塞材料.     

用流式细胞术评价一种新型人工肝材料的生物相容性

目的通过用流式细胞术（FCM）观察丙稀酰胺接枝改性聚丙烯膜 （PP-g-AAm）的生物相容性,来评价FCM在检测医用生物材料的生物相容性中的作用.方法：用材料浸提液培养L929细胞24 h后用FCM检测细胞增殖周期; 用改性前、后的膜材料分别与新提取的PRP（富含血小板血浆）和PBMC（末梢血单个核细胞）孵育培养后,用FCM分别检测血小板和PBMC的激活标志物CD62P、CD63和CD69.结果：PP-g-AAm 组的PI与阴性组及空白对照组比较差异无统计学意义（P=0.063,P=0.053）,而与阳性对照组比较差异有统计学意义（P=0.002）.PP-g-AAm膜组CD62P、CD63及CD69的表达率明显少于对照组（P=0.042, P=0.004,P=0.013）.结论：PP-g-AAm无细胞毒性并具有良好的生物相容性,FCM在生物材料的生物相容性评价中有着广泛的应用价值.     

磷酸三钙涂层镁合金材料的细胞相容性研究

目的:制备磷酸三钙(β-TCP)涂层镁合金材料,评价材料表面的特性及体外的细胞生物适应性。方法 电化学法制备β-TCP涂层镁合金材料(β-TCP-Mg-AI-Zn),观测金属材料表面微观结构特性和能谱分析,小鼠颅骨源成骨细胞与材料直接接触培养,荧光染色观察材料表面细胞生长状况,检测成骨细胞增殖和碱性磷酸酶(ALP)活性。结果 β-TCP涂层Mg-AI-Zn材料表面呈多孔状,材料表面含有镁、钙和磷等元素;成骨细胞与材料直接接触培养24 h及48 h后,材料表面有大量的成骨细胞粘附、伸展、汇合;与Mg-AI-Zn材料比较,β-TCP-Mg-AI-Zn材料明显地促进细胞增殖、显著地增加成骨细胞中ALP活性 (P＜0.05)。结论 β-TCP涂层改善了Mg-AI-Zn镁合金材料表面特性及体外的细胞相容性,有望成为新一代可降解医用金属材料。     

聚己内酯静电纺丝纳米纤维支架用于小鼠诱导多能干细胞培养

干细胞联合生物支架材料体外构建功能性组织与器官,成为当前组织再生研究的重要策略,而探求具有良好生物相容性的支架材料是其关键.本研究采用扫描电镜、噻唑蓝（MTT）法、荧光显微染色等方法检测小鼠诱导多能干细胞（murine induced pluripotent stem cells, miPSCs）在聚己内酯（poly ε-caprolactone, PCL）静电纺丝纳米纤维支架上的粘附、增殖等生物学特性,探究聚己内酯纳米纤维支架与miPSCs的生物相容性. 结果显示,miPSC在PCL纳米纤维支架上具有良好粘附性并呈集落样生长,其增殖能力及干性标记物（Oct4-GFP+）的表达均不亚于标准对照组;扫描电镜显示,miPSC在PCL纳米纤维支架材料上呈现出绒毛状突起的表面结构.上述结果表明,PCL纳米纤维支架可促进miPSCs的粘附、自我增殖以及干性维持,两者具有良好的生物相容性,为下一步联合生物支架材料与干细胞构建功能性组织奠定了基础.     

AZ31B镁合金植入小鼠的生物相容性考察

目的 评价羟基磷灰石(hydroxyapatite, HAP)涂层的AZ31B镁合金（HAP/AZ31B）作为植入材料的生物安全性。方法 采用恒电压阴极电沉积法制备表面HAP涂层的HAP/AZ31B材料;判定生物材料的致突变及毒性作用;检测材料表面的腐蚀状况并进行能谱分析。结果 HAP/AZ31B材料无致突变作用,对动物组织未产生严重的炎症反应;经HAP涂层后的AZ31B材料腐蚀较慢,微核出现率为4.4‰,溶血率为0.25%。结论HAP/AZ31B表现出较好的生物安全性和相容性,有可能成为新型可降解骨支架材料。     

合浦珠母贝珍珠的生物学性能初步检测

本研究报道了合浦珠母贝珍珠的生物学性能初步检测结果。珍珠作为—种天然的生物材料。在医药、化妆品工业等领域中的应用已很广泛。本研究对合浦珠母贝珍珠进行了体外细胞毒性试验、溶血试验和经静脉全身急性毒性试验的研究。实验结果表明,合浦珠母贝珍珠具有较好的细胞相容性,其浸提液对实验动物无明显毒性。在本研究中,合浦珠母贝珍珠的浸提液对兔血红细胞的溶血指数高于对照组。     

新型锌多糖抗凝血涂层修饰聚氯乙烯导管

目的：提高体外循环聚氯乙烯导管的血液相容性。方法：采用层层自组装的方法在PVC表面形成锌离子和多糖（肝素或硫酸葡聚糖）的复合涂层来提高PVC的血液相容性。结果：傅立叶红外光谱表明锌多糖复合物成功的沉积到PVC管表面,与未修饰的PVC管相比,修饰后的PVC管具有较长的部分活化的凝血酶原时间和很少数量的血小板黏附。体系中引入硫酸葡聚糖后,表面涂层具有更好的稳定性。结论：锌多糖抗凝血涂层很好的提高了聚氯乙烯导管的血液相容性。     

Investigation of interfacial capacitance of Pt,Ti and TiN coated electrodes by electrochemical impedance spectroscopy

Electrochemical processes at the electrode-electrolyte (body fluid) interface are of ultimate importance for stimulating/sensing electrode function. A high electrode surface area is desirable for safe stimulation through double-layer charging and discharging. Pt and Pt-Ir alloys have been the most common electrode materials. The use of TiN coating as the surface layer on the electrode has found increasing interest because of its metal-like conductivity, excellent mechanical and chemical properties, and the fact that it can be deposited with a high surface area. In this work, electrochemical impedance spectroscopy (EIS), which is a sensitive and non-destructive technique and widely used for characterization of electrical properties of electrode-electrolyte interfaces, was applied to investigate pure Pt and Ti, and TiN coated electrodes exposed to a phosphate-buffered-saline (PBS) solution. Platinized Pt and Ti were also studied for comparison. The capacitance value of the electrodes in PBS was obtained through quantitative analysis of the EIS spectra. The results reveal that the capacitance of the TiN coated electrodes with a rough surface is several hundreds times higher than that of a smooth Pt surface. Platinization of Ti can also increase the capacitance to the same extent as platina. EIS has been shown to be a powerful technique for characterization of stimulating/sensing electrodes.     

区域选择性改性的纤维素和壳聚糖衍生物及其血液相容性

通过对纤维素和壳聚糖的区域选择性改性,将内皮细胞表面硫酸乙酰肝素（ES—HS）分子结构中对其血液相容性有重要影响的官能团引入纤维素和壳聚糖的分子结构中,并将其通过离子键固定在部分阳离子化的纤维素膜上,以期模拟ES—HS的血液相容性。血小板吸附结果表明,6位改性的纤维素衍生物的吸附程度较高。在五种壳聚糖衍生物中,2位的NS03／NAc为6／4的衍生物表现出最低的血小板吸附。当保持壳聚糖2位的NS03／NAc值不变时,对6位进行完全磺酸酯化,也可有效减少血小板的吸附。     

肺癌患者外周血血小板在肺癌血行转移中的作用研究

目的：观察肺癌患者外周血血小板对肺癌分期和血行转移的影响。方法：选择在我院呼吸科初诊的原发性肺癌患168例,分析其外周血小板计数与肺癌病理类型和分期的关系,并在模拟流体状态下,体外研究活化血小板对培养的肺癌细胞和内皮细胞相互作用的影响。结果：肺腺癌中外周血血小板计数增高现象最为明显,占37.09%（23/62）（P〈0.05）,鳞癌占22.64%,小细胞癌占22.70%,其他占14.20%。其中有远处血行转移者血小板增多（20.24%）较无明显转移者（7.14%）相差显著（P〈0.01）。同时体外研究显示流体状态对肺癌细胞粘附存在影响,而活化血小板增强了肺癌细胞与内皮细胞的相互作用。结论：血小板活化与肺癌尤其是肺腺癌的血行转移密切相关;活化血小板增强了肺癌细胞与内皮细胞的相互作用是血小板促进肺癌血行转移的重要机制之一。     

Tailoring of the titanium surface by immobilization of heparin/fibronectin complexes for improving blood compatibility and endothelialization: an in vitro study

To improve the blood compatibility and endothelialization simultaneously and to ensure the long-term effectiveness of the cardiovascular implants, we developed a surface modification method, enabling the coimmobilization of biomolecules to metal surfaces. In the present study, a heparin and fibronectin mixture (Hep/Fn) covalently immobilized on a titanium (Ti) substrate for biocompatibility was investigated. Different systems [N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide, electrostatic] were used for the formation of Hep/Fn layers. Atomic force microscopy (AFM) showed that the roughness of the silanized Ti surface decreased after the immobilization of Hep/Fn. Fourier transform infrared spectroscopy (FTIR), Toluidine Blue O (TBO) test, and immunochemistry assay showed that Hep/Fn mixture was successfully immobilized on Ti surface. Blood compatibility tests (hemolysis rate, APTT, platelet adhesion, fibrinogen conformational change) showed that the coimmobilized films of Hep/Fn mixture reduced blood hemolysis rate, prolonged blood coagulation time, reduced platelets activation and aggregation, and induced less fibrinogen conformational change compared with a bare Ti surface. Endothelial cell (EC) seeding showed more EC with better morphology on pH 4 samples than on pH 7 and EDC/NHS samples, which showed rounded and aggregated cells. Systematic evaluation showed that the pH 4 samples also had much better blood compatibility. All results suggest that the coimmobilized films of Hep/Fn can confer excellent antithrombotic properties and with good endothelialization. We envisage that this method will provide a potential and effective solution for the surface modification of cardiovascular implant materials.     

Proteomic profile of the saliva and plasma protein layer adsorbed on Ti–Zr alloy: the effect of sandblasted and acid-etched surface treatment

Abstract

Titanium–zirconium (Ti–Zr) alloy has been widely used as a biomaterial for implant devices, and it is commonly treated by sandblasting followed by acid etching (SLA) to improve biological responses. Although protein adsorption is the first biological response, the effect of this SLA treatment on the proteomic profile of proteins adsorbed from saliva and blood plasma has not been tested. In this study, the proteomic profile was evaluated by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Streptococcus sanguinis was used to test whether the protein layer affects bacterial adhesion. SLA treatment affected the proteomic profile, showing exclusive proteins adsorbed from saliva (14) and plasma (3). However, both groups exhibited close patterns of intensity for common proteins, molecular functions and biological processes mediated by proteins. Interestingly, Ti–Zr_SLA showed higher bacterial adhesion (～1.9 fold over) for the surface coated with plasma proteins. Therefore, SLA treatment of Ti–Zr alloy changed the proteomic profile, which may affect bacterial adhesion.     

Ti2448合金种植体表面不同纳米管径生物活性膜对成骨细胞早期黏附的影响

观察Ti2448合金表面不同纳米管径生物活性膜对成骨细胞早期黏附的影响,筛选出能够抵抗细菌在种植体表面黏附和定植的最适管径,为研制开发具有抗菌性能的种植体提供实验基础。采用阳极氧化法,设定不同氧化电压,在Ti2448合金表面生成具有不同纳米管径的生物活性膜。通过MTT实验检测不同管径纳米管表面在不同时间点黏附的MG-63细胞数量差异;通过免疫荧光法观察接种24h后不同管径纳米管表面黏附细胞形态学差异。结果显示,细胞接种3h和24h后,30nm组的OD值显著高于对照组及其他实验组(P0.05);随着纳米管径的增加,细胞骨架蛋白的铺展范围逐渐减小,细胞由多角形渐变为梭形、椭圆形、圆形。研究结果表明,30nm管径生物活性膜更有利于成骨细胞的早期黏附,可能更能抵抗细菌在钛种植体上的黏附与定植。     

Enhanced endothelial cell density on NiTi surfaces with sub-micron to nanometer roughness

The shape memory effect and superelastic properties of NiTi (or Nitinol, a nickel-titanium alloy) have already attracted much attention for various biomedical applications (such as vascular stents, orthodontic wires, orthopedic implants, etc). However, for vascular stents, conventional approaches have required coating NiTi with anti-thrombogenic or antiinflammatory drug-eluting polymers which as of late have proven problematic for healing atherosclerotic blood vessels. Instead of focusing on the use of drug-eluting anti-thrombogenic or anti-inflammatory proteins, this study focused on promoting the formation of a natural antithrombogenic and anti-inflammatory surface on metallic stents: the endothelium. In this study, we synthesized various NiTi substrates with different micron to nanometer surface roughness by using dissimilar dimensions of constituent NiTi powder. Endothelial cell adhesion on these compacts was compared with conventional commercially pure (cp) titanium (Ti) samples. The results after 5 hrs showed that endothelial cells adhered much better on fine grain (< 60 microm) compared with coarse grain NiTi compacts (< 100 microm). Coarse grain NiTi compacts and conventional Ti promoted similar levels of endothelial cell adhesion. In addition, cells proliferated more after 5 days on NiTi with greater sub-micron and nanoscale surface roughness compared with coarse grain NiTi. In this manner, this study emphasized the positive pole that NiTi with sub-micron to nanometer surface features can play in promoting a natural anti-thrombogenic and anti-inflammatory surface (the endothelium) on a vascular stent and, thus, suggests that more studies should be conducted on NiTi with sub-micron to nanometer surface features.     

石墨化炭/炭复合材料＋羟基磷灰石涂层（C/C＋HA）复合骨植入材料研究

目的：通过建立兔股骨缺损的动物实验模型,对采用等温化学气相沉积法和等离子喷涂技术所制备的石墨化炭/炭复合材料＋羟基磷灰石涂层（C/C＋HA）复合骨植入材料进行骨植入实验的的生物相容性进行评价,探索该复合材料作为植入机体骨组织的可行性依据。方法：采用骨科钻在实验动物股骨髁上钻孔的方法建立骨缺损的动物实验模型,将待研究比较的实验材料分别植入实验动物的股骨髁内,持续观察8周,在术后第2、4、8周时应用X线照片、组织学染色和扫描电镜技术,分别观察所研究材料在机体内对骨缺损愈合及其对机体的影响,进行组间比较和相关性分析。结果：石墨化炭/炭复合材料＋羟基磷灰石涂层（C/C＋HA）复合骨植入材料的骨植入实验生物相容性良好,材料与骨组织结合牢固,界面中成骨细胞生长明显,且炭颗粒脱落现象少,未见炎症细胞浸润。植入动物体内的材料在植入期未引起机体局部的炎症浸润反应且表面脱落的碳颗粒在机体组织中也未引起局部严重的炎症反应。在实验动物植入材料后的连续8周观察期中,组织学观察显示：表面涂有HA的炭/炭复合材料对骨组织形态改建上表现良好,其与骨组织接界处所形成的纤维结缔组织膜层厚度明显比未涂HA的材料要小,与骨组织结合更为紧密和牢固;碳颗粒出现脱落游离的现象明显减少。结论：在炭/炭复合材料表面涂以HA生物涂层对骨的形态改建和促进骨小梁生长等方面具有良好的作用,是一种具有发展潜力的骨修复材料。     

The Effect of Heparin-VEGF Multilayer on the Biocompatibility of Decellularized Aortic Valve with Platelet and Endothelial Progenitor Cells

The application of polyelectrolyte multilayer films is a new, versatile approach to surface modification of decellularized tissue, which has the potential to greatly enhance the functionality of engineered tissue constructs derived from decellularized organs. In the present study, we test the hypothesis that Heparin- vascular endothelial growth factor (VEGF) multilayer film can not only act as an antithrombotic coating reagent, but also induce proliferation of endothelial progenitor cells (EPCs) on the decellularized aortic heart valve. SEM demonstrated the adhesion and geometric deformation of platelets. The quantitative assay of platelet activation was determined by measuring the production of soluble P-selectin. Binding and subsequent release of heparin and VEGF from valve leaflets were assessed qualitatively by laser confocal scanning microscopy and quantitatively by ELISA methods. Human blood derived EPCs were cultured and the adhesion and growth of EPCs on the surface modified valvular scaffolds were assessed. The results showed that Heparin-VEGF multilayer film improved decellularized valve haemocompatibility with respect to a substantial reduction of platelet adhesion. Release of VEGF from the decellularized heart valve leaflets at physiological conditions was sustained over 5 days. In vitro biological tests demonstrated that EPCs achieved better adhesion, proliferation and migration on the coatings with Heparin-VEGF multilayer film. Combined, these results indicate that Heparin-VEGF multilayer film could be used to cover the decellularized porcine aortic valve to decrease platelet adhesion while exhibiting excellent EPCs biocompatibility.