钛合金表面纳米结构对钙磷矿化物体外沉积及成骨细胞矿化功能的影响

背景：通过改变金属材料置入假体表面物理性质,可以增强细胞的成骨性能,表面纳米化处理是当前研究的一个重要方向。目的：通过测定纳米钛合金和未处理微米钛合金表面钙磷沉积量及成骨细胞在两种材料表面的钙化功能,评估材料表面纳米结构对钛合金生物相容性的影响。方法：采用表面机械研磨处理(SMAT)制备了表面纳米结构的Ti6Al4V钛合金,将钛合金表面的微米级晶粒转变为纳米尺寸的晶粒。实验分为微米合金和纳米合金组,将试样浸入模拟体液中浸泡21 d。分离新生乳鼠颅盖骨成骨细胞进行培养,将其分别接种到纳米表面和微米表面钛合金上进行共培养14 d。观察两组合金表面粗糙度、润湿角、材料表面钙磷沉积量、成骨细胞钙结节形成的情况。结果与结论：①基于SMAT技术的表面纳米钛合金表面得到粗糙表面,其组成的晶粒则为纳米量级,表面粗糙度(Ra)由 132.5 nm增加到4019.3 nm;接触角由57.26°减小到22.4°;表面能由39.4 mJ/m2增加到67.3 mJ/m2。②在无成骨细胞的模拟体液中沉积7,14,21 d之后,纳米Ti6Al4V合金表面的钙磷元素沉积量显著增加。③纳米钛合金表面形成的钙结节面积显著大于微米钛合金表面,培养14 d后前者约为后者的3倍。结果提示,表面纳米钛合金能明显促进成骨细胞的矿化,并增加表面钙磷的沉积,具有较好的生物相容性。关键词：纳米结构;Ti6Al4V;成骨细胞;矿化;钙磷沉积;纳米生物材料doi:10.3969/j.issn.1673-8225.2010.12.016     

生物素化聚乙二醇/聚乳酸纳米粒子的体外主动靶向行为

背景：目前研究的大部分高分子药物载体没有靶向性,在应用上有局限性,只有几个国外课题组报道生物素化聚乙二醇/聚乳酸(Biotin-PEO-PLA)纳米粒子的体外靶向行为,国内没有这方面的研究报道。 目的：分析Biotin-PEO-PLA纳米粒子作为靶向药物载体的可行性。 方法：透析法制备包埋紫杉醇的Biotin-PEO-PLA纳米粒子并表征;通过高效液相色谱研究包埋紫杉醇的Biotin-PEO-PLA纳米粒子的体外释放行为;利用细胞毒性法比较研究生物素-亲和素三步法实施的包埋紫杉醇的Biotin-PEO-PLA纳米粒子对OVCAR-3(表面表达CA-125抗体)和SKOV-3(表面不表达CA-125抗体)细胞的体外靶向行为。 结果与结论：包埋在Biotin-PEO-PLA纳米粒子中的紫杉醇的释放呈现初期的快速释放以及随后的缓慢释放。利用三步法处理的OVCAR-3细胞存活率明显低于SKOV-3细胞,表明通过Biotin-PEO-PLA/avidin/biotinylated MAB X306与OVCAR-3细胞表面CA-125抗原的特异性相互作用,包埋紫杉醇的Biotin-PEO-PLA纳米粒子被更为有效地传递进了OVCAR-3细胞。     

二元镁合金在细胞培养基中的腐蚀特性

背景：镁和镁合金将可能成为新型骨科内植物材料,系统的研究镁合金相关金属离子对骨组织中的关键性细胞成人骨髓间充质干细胞（hBMSCs）增殖的影响对于新型镁合金的开发十分关键。 目的：从生物学角度探索理想的镁合金元素构成 方法：制备纯镁和8种二元镁合金（Mg-Al、Mg-Ca、Mg-Mn、Mg-Sn、Mg-Si、Mg-Y、Mg-Zn、Mg-Zr）样品;采用α-MEM细胞培养基（含10%FBS）模拟体内环境下制备标准浸提液,并检测pH值;电感耦合等离子体原子发射光谱仪(ICP-AES)检测浸提液中镁和添加元素含量;分离、鉴定、培养健康成人骨髓间充质干细胞;alamarBlue法分别检测培养达１、３、５、７天时100%、50%、25%浓度的纯镁和８种镁合金浸提液对hBMSCs增殖的影响,判断８种二元镁合金的生物相容性。 结果与结论：Mg-Ca、Mg-Y合金耐腐蚀性较差浸提液中Mg2 浓度分别达到408 ?37.9 mg/L 和351 ?15.3 mg/L,pH值分别为8.87?.19和8.84?.15;其次是Mg-Zr合金;其它二元合金耐腐蚀性与纯镁相当。纯镁和8种二元镁合金100%含量的浸提液均对hBMSCs的增殖具有显著抑制作用,当Mg2 浓度低于110mg／L,pH值7.35-7.65时纯镁和8种二元镁合金对hBMSCs的增殖无显著影响。     

316L不锈钢和NiTi合金血管支架的血液相容性*★

学术背景：临床广泛使用的316L不锈钢、NiTi合金血管支架，有较好的耐腐蚀性和血液相容性。但随着人们对这两种材料血液相容性的深入研究，也发现了它们在血液中有离子渗出、表面血栓等问题，如何提高316L、NiTi合金血管支架的血液相容性已成为人们研究的重要课题。 目的：介绍316L不锈钢、NiTi合金血管支架血液相容性研究现状，探讨金属支架表面生物化处理的方向。 检索策略：应用计算机检索PubMed数据库、Science Direct数据库和中文科技期刊CNKI数据库1996-01/2007-09的相关文章，检索词为“NiTi合金血管支架、316L血管支架、表面改性、血液相容性”，限定文章语言种类为中文和英文。共检索到论文1 759篇，其中英文909篇，中文850篇。纳入标准:①与NiTi合金、316L不锈钢有关的表面改性、血液相容性研究。②材料表面因素与蛋白吸附、血液相容性关系。剔除内容重复性的研究，并以近3年文献为主，最终确定了33篇。 文献评价：在确定的文献中，关于316Ｌ、NiTi合金血管支架应用中存在血栓问题的文献6篇，腐蚀问题的文献6篇，影响材料抗凝血性表面因素的文献8篇，提高316Ｌ、NiTi合金支架血液相容性途径的文献13篇。 资料综合：现用的血管支架材料316L、NiTi合金在血液中仍有离子析出、血栓产生。通过降低材料表面的粗糙度，改变生物材料表面化学成分和化学组成，使材料表面呈负电性，降低材料表面的张力与表面能几方面因素提高血管支架材料316L、NiTi合金的血液相容性。 结论：在没有开发出新的血管支架材料前，对现有支架材料316L、NiTi合金表面进行生物化改性处理是提高支架血液相容性的一条途径。     

In vitro behavior of granule cells from Staggerer and Weaver mutants of mice.

The survival, in monolayer culture, of cerebellar granule cells from the cerebellar mutants Staggerer and Weaver was studied to examine whether the observed in vivo granule cell degeneration is intrinsic or environmentally induced. Granule cells in vitro can be identified by means of a combination of criteria including their size, shape, nuclear morphology, relative proportion of the total cell population, and failure to take up gamma-aminobutyric acid (GABA) in the presence of several other cell types which do. Cultures from both Staggerer and Weaver cerebella had surviving granule cells for at least 3 weeks in vitro. Therefore, since the mice used as the source of the cells were 7 days of age, the degeneration observed in vivo cannot be a case of irreversibly programmed cell death determined before postnatal day 7. While the behavior of Weaver granule cells is essentially the same as littermate controls, cells from Staggerer cerebella both clump less and survive considerably longer than those from wild-type. The role of intrinsic granule cell differences vs. primary changes in some other cell type is discussed.     

In vitro analyses of diamond-like carbon coated stents. Reduction of metal ion release, platelet activation, and thrombogenicity

Coronary artery stents can induce platelet activation by shear forces, contact to the biomaterial, and release of metal ions. This activation is one important trigger for thrombosis. Coating of stents is a possible approach to prevent this side effect. The purpose of this study was to evaluate in vitro the biocompatibility of stents coated with diamond-like carbon (DLC). Semiquantitative energy-dispersive X-ray microanalyses showed a complete coverage of the DLC stents. Flow cytometric analyses revealed a significantly higher increase of mean channel fluorescence intensity for the activation-dependent antigens CD62p and CD63 in non-coated compared to DLC-coated stents (p<0.05). Atomic adsorption spectrophotometry analyses revealed a significant release of nickel and chromium metal ions by non-coated stents over a storage period of 96 hours in human plasma (p<0.05). In contrast, only minimal concentrations of released ions could be detected in the case of DLC-coated stents. Similar observations were made with inductively coupled plasma mass spectrometry analyses. Here, high concentrations of molybdenum and manganese ions were released from non-coated stents (p<0.05), while release of these ions from DLC-coated stents was virtually undetectable (p=0.1 for molybdenum and p=0.4 for manganese). Coating of intracoronary stents with diamond-like carbon significantly improves biocompatibility. This biocompatible coating may therefore contribute to a reduction in thrombogenicity.     

In vitro electrical activity in the suprachiasmatic nucleus following splitting and masking of wheel-running behavior

Djungarian hamsters (Phodopus sungorus sungorus) were chronically exposed to constant light (60 lux) in order to generate a split rhythm wheel-running behavior. The animals were killed and coronal hypothalamic slices prepared for extracellular recording from the left and right suprachiasmatic nuclei (SCN). Nine hamsters which exhibited a split in overt behavior also had bimodal peaks (280 cells) of SCN firing frequency (5.4 ± 1.1 and 5.8 ± 0.7 Hz); these peaks were about 180° antiphase. Two troughs in firing frequency were also apparent (1.8 ± 0.4 and 2.4 ± 0.5 Hz) and coincided with the projected time of wheel-running activity. Differences in circadian pattern of electrical activity between the right and left SCN were not observed. When wheel-running activity in 5 hamsters was suppressed with high intensity (500 lux) constant light, the SCN firing profile (154 cells) failed to exhibit a daily rhythm. Firing frequency was consistently high (5.8 ± 1.4 Hz) throughout the 24-h period. These results indicate that in vitro electrical activity of the SCN is related to overt behavior in Djungarian hamsters, in that high electrical activity occurs when locomotor activity is minimal.     

In vitro analysis of the origin,migratory behavior,and maturation of cortical pyramidal cells

In cerebral cortex of rat and monkey, the neuropeptide somatostatin (SOM) marks a population of nonpyramidal cells (McDonald et al. [1982] J. Neurocytol. 11:809-824; Hendry et al. [1984] J. Neurosci. 4:2497:2517; Laemle and Feldman [1985] J. Comp. Neurol. 233:452-462; Meineke and Peters [1986] J. Neurocytol. 15:121-136; DeLima and Morrison [1989] J. Comp. Neurol. 283:212-227) that represent a distinct type of gamma-aminobutyric acid (GABA) -ergic neuron (Gonchar and Burkhalter [1997] Cereb. Cortex 7:347-358; Kawaguchi and Kubota [1997] Cereb. Cortex 7:476-486) whose synaptic connections are incompletely understood. The organization of inhibitory inputs to the axon initial segment are of particular interest because of their role in the suppression of action potentials (Miles et al. [1996] Neuron 16:815:823). Synapses on axon initial segments are morphologically heterogeneous (Peters and Harriman [1990] J. Neurocytol. 19:154-174), and some terminals lack parvalbumin (PV) and contain calbindin (Del Rio and DeFelipe [1997] J. Comp. Neurol. 342:389-408), that is also expressed by many SOM-immunoreactive neurons (Kubota et al. [1994] Brain Res. 649:159-173; Gonchar and Burkhalter [1997] Cereb. Cortex 7:347-358). We studied the innervation of pyramidal neurons by SOM neurons in rat and monkey visual cortex and examined putative contacts by confocal microscopy and determined synaptic connections in the electron microscope. Through the confocal microscope, SOM-positive boutons were observed to form close appositions with somata, dendrites, and spines of intracortically projecting pyramidal neurons of rat area 17 and pyramidal cells in monkey striate cortex. In addition, in rat and monkey, SOM boutons were found to be associated with axon initial segments of pyramidal neurons. SOM axon terminals that were apposed to axon initial segments of pyramidal neurons lacked PV, which was shown previously to label axo-axonic terminals provided by chandelier cells (DeFelipe et al. [1989] Proc. Natl. Acad. Sci. USA 86:2093-2097; Gonchar and Burkhalter [1999a] J. Comp. Neurol. 406:346:360). Electron microscopic examination directly demonstrated that SOM axon terminals form symmetric synapses with the initial segments of pyramidal cells in supragranular layers of rat and monkey primary visual cortex. These SOM synapses differed ultrastructurally from the more numerous unlabeled symmetric synapses found on initial segments. Postembedding immunostaining revealed that all SOM axon terminals contained GABA. Unlike PV-expressing chandelier cell axons that innervate exclusively initial segments of pyramidal cell axons, SOM-immunoreactive neurons innervate somata, dendrites, spines, and initial segments, that are just one of their targets. Thus, SOM neurons may influence synaptic excitation of pyramidal neurons at the level of synaptic inputs to dendrites as well as at the initiation site of action potential output.     

抑肽酶/氨甲环酸改良可注射性纤维蛋白凝胶的体外实验

背景：可注射性纤维蛋白凝胶为软骨缺损组织工程完全再生修复的临床应用带来了新的方向，其降解速度的调控是其中的关键问题之一。 目的：在可注射性纤维蛋白凝胶中引入不同浓度的抑肽酶和氨甲环酸，观察其对纤维蛋白凝胶支架降解速率的影响。 设计、时间及地点：体外细胞学实验，于2008-02/08在广东省构建与检测实验室进行。 材料：以纤维蛋白原、凝血酶及氯化钙制备纤维蛋白凝胶。 方法：取3周龄新西兰幼兔的关节软骨制取软骨细胞，体外单层培养、扩增后植于标准纤维蛋白凝胶支架和改良纤维蛋白凝胶支架（加入抑肽酶7 500，12 500，17 500 MIU/L及氨甲环酸15，20，25 g/L复合液）上，进行体外培养、扩增6周。 主要观察指标：支架降解情况。 结果：支架细胞复合物体外培养3周时，标准组已完全崩解，改良各组体积尚不到原来1/2。培养6周时仍能保持其一定的外形，具有一定的厚度及弹性。不同浓度的抑肽酶和氨甲环酸，在体外环境下均显著地减缓了纤维蛋白胶的降解速度，低于1 2500 MIU/L的抑肽酶和20 g/L氨甲环酸对软骨细胞的繁殖、表型的维持、基质的分泌无明显不良影响，而高于此浓度的抑肽酶和氨甲环酸的加入明显抑制了细胞的增殖、表型的维持和基质的分泌。 结论：通过调节纤维蛋白凝胶中抑肽酶和氨甲环酸的含量，可实现对纤维蛋白凝胶降解速度的调控。     

口腔修复用钛锆铌锡合金机械性能及体内外的生物毒性***☆

背景：在口腔修复领域广泛应用的纯钛和Ti-6Al-4V合金存在强度低,耐磨性较差,加工性能不理想,易出现卡环折断现象等缺点,并且含有人体有害的Al及V元素。因此,研制机械性能优良,生物学性能良好的口腔修复用钛合金,对于克服纯钛及钛合金修复体的缺陷,扩大口腔钛合金修复体临床应用范围,提高齿科修复体的质量及制作水平具有重要意义。 目的：评价口腔修复用钛锆铌锡(Ti-12.5Zr-3Nb-2.5Sn)合金机械性能及体内外生物毒性。 设计、时间及地点：对比观察实验,于2008-04/2009-02在天津市材料复合与功能化重点实验室、天津医科大学附属口腔医院口腔生物材料实验室及天津市现代医药开发研究所完成。 材料：纯度为99.9%的钛板,纯度为99.9%的锆板,纯度为99.9%的铌棒和纯度为99.9%的颗粒状锡由宝鸡市胜超有色金属材料有限公司提供。 方法：制备Ti-Zr-Nb-Sn合金,测试合金的机械性能和显微硬度,观察拉伸试样的金相组织、物相结构及断口形貌。参照 ISO7406 技术报告中的相关标准,对Ti-Zr-Nb-Sn合金进行细胞毒性实验和急性全身毒性试验。 主要观察指标：机械性能,合金金相结构,端口形貌,细胞相对增殖率,急性毒性反应。 结果：Ti-12.5Zr-3Nb-2.5Sn抗拉强度为(652.0±34.5) MPa,屈服强度为(590.0±29.8) MPa,延伸率为(28.3±2.4)%,弹性模量为(93.8±7.9) GPa,维氏硬度为(315.8±13.0) HV,XRD分析显示合金显微组织主要为α相,金相组织表现为细小的针状α相结构,合金断口呈韧窝状形貌,体现出良好的韧性断裂特征。细胞毒性试验及急性全身毒性试验结果表明本研究制备的钛锆铌锡合金细胞相对增殖度为124%,分级为0级,无细胞毒性;未见任何急性毒性反应。 结论：Ti-12.5Zr-3Nb-2.5Sn合金具有良好的机械性能和生物安全性,可满足口腔修复材料的临床应用要求。 关键词：钛锆铌锡合金;机械性能;生物安全性