金属学报, 2023, 59(4): 577-584 DOI: 10.11900/0412.1961.2022.00553

研究论文

CuNi-Ti合金抗支架内再狭窄与耐蚀性能的影响

许林杰1,2, 刘徽1, 任玲1, 杨柯,1

1中国科学院金属研究所 沈阳 110016

2中国科学技术大学 材料科学与工程学院 沈阳 110016

Effect of Cu on In-Stent Restenosis and Corrosion Resistance of Ni-Ti Alloy

XU Linjie1,2, LIU Hui1, REN Ling1, YANG Ke,1

1Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016, China

2School of Materials Science and Engineering, University of Science and Technology of China, Shenyang 110016, China

通讯作者: 杨柯,kyang@imr.ac.cn,主要从事新型医用金属材料研发及应用研究、先进钢铁材料研究以及储氢合金研究

责任编辑: 毕淑娟

收稿日期: 2022-10-31   修回日期: 2022-12-07  

基金资助: 国家重点研发计划项目(2022YFC2406003)
国家自然科学基金项目(81873918)
国家自然科学基金项目(82272099)
辽宁省自然科学基金计划项目(2021020399-JH2/103)
辽宁省自然科学基金计划项目(2022-YGJC-34)

Corresponding authors: YANG Ke, professor, Tel:(024)23971628, E-mail:kyang@imr.ac.cn

Received: 2022-10-31   Revised: 2022-12-07  

Fund supported: National Key Research and Development Program of China(2022YFC2406003)
National Natural Science Foundation of China(81873918)
National Natural Science Foundation of China(82272099)
Natural Science Foundation of Liaoning Province(2021020399-JH2/103)
Natural Science Foundation of Liaoning Province(2022-YGJC-34)

作者简介 About authors

许林杰,男,1999年生,硕士生

摘要

基于合金化策略,通过向医用Ni-Ti合金中添加适量Cu制备生物功能化Ni-Ti-Cu合金,并利用OM、SEM、XRD、表面自由能测试、电化学实验以及体外细胞实验等方法,探究Ni-Ti-Cu合金抑制支架内再狭窄作用以及耐蚀性能。结果表明,与Ni-Ti合金相比,Ni-Ti-Cu合金由等轴奥氏体组织向细板条马氏体组织转变,其表面自由能显著降低,在模拟人体血液中的耐蚀性能提高。此外,Ni-Ti-Cu合金浸提液还可显著促进人脐静脉内皮细胞的增殖、迁移及成血管能力。与Ni-Ti合金相比,Ni-Ti-Cu合金可降低血液凝固速率,表现出更优异的抗凝血性能,具有抑制支架内再狭窄发生的应用潜力。

关键词: Ni-Ti合金; Cu; 支架内再狭窄; 耐蚀性能

Abstract

Ni-Ti alloys are used widely as a self-expanding vascular stent material because of their unique shape memory effect and superelasticity. However, after implantation, there is a risk of in-stent restenosis (ISR) because of insufficient endothelialization and coagulation problems. As a biological functional metal element, the proper addition of Cu endows vascular stent materials, such as stainless steel and cobalt-based alloys, with significant endothelialization promotion and anticoagulant effect, which can effectively inhibit the occurrence of ISR. Based on the alloying strategy, a biofunctional Ni-Ti-Cu alloy was prepared by adding the proper amount of Cu into medical Ni-Ti alloys. The inhibition effect of ISR and corrosion resistance of the Ni-Ti-Cu alloy were studied via OM, SEM, XRD, surface free energy test, electrochemical test, and in vitro cell experiment. Results showed that compared with the Ni-Ti alloy, the Ni-Ti-Cu alloy promoted the transformation of an equiaxed austenite grain structure to fine lath martensite, reduced the surface free energy, and improved corrosion resistance in simulated human blood. In addition, the extract of the Ni-Ti-Cu alloy could promote the proliferation, migration, and tube formation of human umbilical vein endothelial cells. Furthermore, compared with the Ni-Ti alloy, the Ni-Ti-Cu alloy decreased the blood coagulation rate, presenting better anticoagulation ability, which has an application potential for inhibiting the occurrence of ISR.

Keywords: Ni-Ti alloy; Cu; in-stent restenosis; corrosion resistance

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本文引用格式

许林杰, 刘徽, 任玲, 杨柯. CuNi-Ti合金抗支架内再狭窄与耐蚀性能的影响[J]. 金属学报, 2023, 59(4): 577-584 DOI:10.11900/0412.1961.2022.00553

XU Linjie, LIU Hui, REN Ling, YANG Ke. Effect of Cu on In-Stent Restenosis and Corrosion Resistance of Ni-Ti Alloy[J]. Acta Metallurgica Sinica, 2023, 59(4): 577-584 DOI:10.11900/0412.1961.2022.00553

Ni-Ti合金以其独特的形状记忆效应[1]和超弹性[2]广泛用作自膨式介入支架材料,如血管支架[3]、尿路支架[4]、肠道支架[5]、下肢动脉支架[6]等。但是,当支架植入人体内血管后,仍有15%~20%的患者会发生不同程度的支架内再狭窄问题[7],这可能会直接导致支架植入手术的失败。此外,在生理环境中,由于体液腐蚀导致的Ni-Ti合金支架中的Ni2+释放,还可能会造成一定的毒性反应[8]。研究表明,支架内再狭窄可通过支架表面的促内皮化[9]和抗凝血[10,11]处理来解决,Ni2+潜在的毒性危害也可通过进一步提高合金的耐蚀性能得到改善[8]

有研究表明,采用等离子沉积[12]、水热处理[13]等表面改性方法在Ni-Ti合金表面构建功能性涂层,可以促进支架表面的内皮化,以抑制发生支架内再狭窄,同时还能够提高Ni-Ti合金的耐腐蚀性能,减少Ni2+的释放。例如,Yang等[12]采用等离子沉积技术在Ni-Ti合金表面沉积TiN涂层,改善了内皮细胞功能,对Ni2+释放有一定阻止作用。Zhao等[13]通过水热处理法在Ni-Ti合金表面构建TiO2与Ti4Ni2O组成的纳米纺锤体,增强了内皮细胞的迁移能力,在促进内皮细胞功能表达的同时,减少了Ni2+的释放。然而,这些涂层往往存在与合金基体材料的结合力差,因而在支架使用过程中易发生剥落等问题[14,15]。因此,发展自身具有抑制支架内再狭窄功能并进一步提高耐蚀性能的新型Ni-Ti合金具有重要的临床应用价值。

Cu作为一种金属中常见的合金元素,同时还具有促血管内皮化、抗凝血、促成骨等多重生物功能。大量研究已经证实,包括316L-Cu不锈钢[16]、L605-Cu钴基合金[17]在内的含Cu金属材料均具有优异的促内皮化与抗凝血性能,并在动物体内表现出显著的抑制支架内再狭窄功能。因此,在现有Ni-Ti合金中适量添加Cu,有望赋予材料自身促内皮化与抗凝血功能。此外,已有研究[18~23]表明,Ni-Ti-Cu合金不仅具有良好的超弹性,而且还能保持良好的细胞相容性与抗菌性能[24],这使Ni-Ti-Cu合金具有临床应用的巨大潜力。

然而,对于Cu元素的加入是否有利于提高Ni-Ti合金的耐蚀性能,目前还存在争议[24~30]。例如,Li等[24]的研究结果是Ni-Ti-Cu合金的耐蚀性能优于Ni-Ti合金;而Cheng等[26]的结果则是Cu的加入降低了Ni-Ti合金的耐蚀性能,但Cu会提高Ni-Ti合金的再钝化能力。基于上述分析,将Cu加入到Ni-Ti合金中有望发展出具有抑制支架内再狭窄功能且耐蚀性能较高的新型血管支架材料。

本工作基于合金化策略,在医用Ni-Ti合金成分的基础上制备了Ni-Ti-Cu合金,并以商用Ni-Ti合金作为对照组,采用电化学实验分析添加Cu对Ni-Ti合金在模拟人体血液中腐蚀行为的影响。通过细胞增殖实验、划痕实验、Transwell实验及小管生成实验,系统研究了Ni-Ti-Cu合金对人脐静脉内皮细胞(HUVECs)分化的促进作用。此外,还采用动态凝血实验,进一步评估了Ni-Ti-Cu合金的抗凝血性能。旨在全面展示Ni-Ti-Cu合金的抑制支架内再狭窄功能及耐蚀性能,为新型含铜Ni-Ti合金血管支架材料的开发提供科学依据。

1 实验方法

1.1 材料制备

采用真空感应炉分别制备Ni-Ti和Ni-Ti-Cu合金,合金的化学成分分别为:50.48Ni-49.52Ti和42.79Ni-50.92Ti-6.29Cu (原子分数,%)。合金在真空熔炼炉中反复熔炼6次而成,以保证合金成分的均匀性。熔炼后的合金锭在1000℃均匀化退火12 h,并在850℃进行热锻,再冷拔为直径7 mm的圆棒,最后切割成1 mm厚的圆片。所有样品在实验前均用SiC砂纸逐级打磨至2000#,用去离子水冲洗后,再用酒精清洗,干燥后得到实验用样品。

1.2 显微组织与相组成表征

通过SSX-550扫描电子显微镜(SEM)观察Ni-Ti与Ni-Ti-Cu合金的微观形貌,工作电压为20 kV。采用D/max 2500 X射线衍射仪(XRD)分析Ni-Ti与Ni-Ti-Cu合金显微组织中的相组成,辐射源为Cu靶Kα 射线,工作电压为40 kV,电流为40 mA,扫描速率为10°/min。用Jade 6.0软件对XRD数据进行相组成分析。

1.3 表面自由能测试

采用JC-2000C型静滴接触角测量仪对样品的表面自由能进行测试。利用微量进样器将去离子水、甘油(C3H8O3)、溴代萘(C10H7Br)缓缓滴于样品表面,当液滴与样品表面接触后,立即采集图像并计算样品的接触角(θ),基于OWRK/Fowkes方法计算样品的表面自由能。表面自由能按照Young-Dupre方程进行计算[31]

γlv1+cosθ=2(γsvpγlvp)1/2+2(γsvdγlvd)1/2

表面自由能由极性分量与色散分量组成:

γsv=γsvp+γsvd

式中,γsvγlv分别为固-气界面与液-气界面的表面自由能,γsvpγsvdγlvpγlvd分别为γsvγlv的极性分量与色散分量。

1.4 电化学实验

电化学实验在Reference 600TM上完成,电解质溶液采用模拟人体血液[32]。首先进行开路电位(OCP)测试,之后进行电化学阻抗谱(EIS)测试,从105 Hz下降到10-2 Hz。EIS测试结束后,以0.5 mV/s的扫描速率进行动电位测量,范围为-0.5~2.0 V。使用Gamry Echem Analyst软件分析测试结果,以获得腐蚀电位(Ecorr)、腐蚀电流密度(icorr)和点蚀电位(Epit)。利用ZsimDemo软件对EIS进行电路拟合,以获得电路图中元器件参数。所有测试至少重复3次,以确保结果的可重复性。

1.5 体外生物学实验

1.5.1 细胞培养

使用HUVECs作为模型细胞,在含抗生素(100 U/mL青霉素、100 μg/mL链霉素)和10%胎牛血清的RPMI.1640培养基中于37℃、5%CO2 (体积分数)饱和湿度条件下进行培养。当细胞生长至趋近融合时,使用胰蛋白酶消化,收集细胞并调整细胞浓度至2 × 104 cell/mL,于无菌条件下在样品表面接种细胞进行培养。

1.5.2 浸提液制备

样品经121℃高温高压蒸汽灭菌30 min后,于无菌条件下制备浸提液。以1.25 cm2/mL的浸提比例将样品浸泡于RPMI.1640培养基中,在37℃浸泡72 h后,取出准备好的浸提液即刻进行实验。

1.5.3 细胞增殖实验

将Ni-Ti合金与Ni-Ti-Cu合金样品分别置于48孔板中,设置4个平行样。每孔加入1 × 104个细胞,在37℃、5%CO2条件下分别培养1和3 d。培养结束后,弃去培养板内培养基,加入300 μL含10% CCK-8试剂的培养基,再在37℃、5%CO2条件下培养2 h。取出样品,在Multidkan GO酶标仪上测量450 nm波长处各孔的吸光度(optical density,OD)。

1.5.4 划痕实验

在孔板背面绘制平行线,保证每孔有3条直线穿过。每孔加入5 × 104个细胞,在37℃、5%CO2条件下培养至铺满孔底90%面积。用200 μL枪头垂直于上述平行线轻轻绘制垂线,随后经磷酸盐缓冲溶液(PBS)缓慢冲洗后,加入1 mL无血清的浸提液。在加入浸提液0和24 h后,对同一位置进行拍摄。使用Photoshop软件计算前后2个时间点照片中的划痕区域面积,计算相对迁移面积比率。

1.5.5 Transwell实验

利用Transwell实验可以研究不同材料浸提液对内皮细胞个体迁移能力的影响。实验前,内皮细胞于无血清培养液中饥饿培养24 h。向Transwell上室每孔加入2 × 104个细胞,下室加入600 μL含20%血清的样品浸提液,于37℃、5%CO2下培养24 h。培养结束后,取出Transwell小室,弃去孔中的培养液,用棉签轻轻擦掉上层迁移的细胞,经PBS清洗2次后,在4%多聚甲醛固定30 min。将小室适当风干后,使用0.1%结晶紫染色20 min,经PBS清洗3次后,在FV10-ASW激光共聚焦荧光显微镜下随机挑选5个视野进行拍照并记数。

1.5.6 小管生成实验

将Matrigel基质胶置于4℃溶解,向96孔板中每孔加入50 μL基质胶,于37℃细胞培养箱中孵育30 min进行固化。将内皮细胞消化后,每孔加入100 μL浓度为5 × 105 cell/mL的细胞悬液,并加入100 μL含10%血清的浸提液,于37℃、5%CO2下培养3 h生成小管。在显微镜下随机选取5个视野进行观察拍摄。使用Wimasis Wim Tube软件定量分析小管长度和节点数。

1.5.7 动态凝血实验

采用动态凝血实验评价样品的抗凝血性能。将100 μL抗凝人血滴加到实验样品表面,加入10 μL浓度为0.2 mol/L的CaCl2溶液,启动凝血。分别计时10、30、50、70、90 min后,用2 mL去离子水缓慢流注于样品表面,并轻轻混匀。随后,吸取100 μL的流注液于96孔板中,使用酶标仪在545 nm波长处测量流注液的OD,并绘制动态凝血时间曲线,分析样品表面的血液凝固情况。

1.6 统计学分析

实验数据采用平均值和标准差来表示,每种材料测试3个以上的样品。采用SPSS 13.0软件和GraphPad Prism软件对数据进行统计学分析以得到方差pp < 0.05时表示数据具有显著性差异,p < 0.01时表明数据差异性极显著。

2 实验结果与讨论

Ni-Ti和Ni-Ti-Cu合金的XRD谱如图1a所示。可以看出,Cu的加入使Ni-Ti合金中的B2奥氏体相与Ni4Ti3相转变为B19'单斜马氏体相、B19正交马氏体相以及CuNi2Ti相。表明添加Cu提高了Ni-Ti合金的马氏体相变起始温度(Ms),这与Ni-Ti合金中的Ni被加入的Cu所取代有关[24]。Ni-Ti与Ni-Ti-Cu合金的SEM像如图1bc所示。由图可见,Ni-Ti合金为等轴的奥氏体晶粒组织(图1b),Ni-Ti-Cu合金的晶粒内部分布着细板条状的马氏体(图1c),可见Cu的加入直接导致Ni-Ti-Cu合金的显微组织发生了改变。

图1

图1   Ni-Ti和Ni-Ti-Cu合金的XRD谱与SEM像

Fig.1   XRD spectra (a) and SEM images of Ni-Ti (b) and Ni-Ti-Cu (c) alloys


植入材料的表面润湿性及其他表面特征(如表面形貌、表面电荷、表面官能团等),对材料与人体组织之间界面上发生的一系列生物学反应具有重要影响,包括蛋白质吸附、与软组织的相互作用以及细菌生物膜的形成[33]。因此,测试了Ni-Ti和Ni-Ti-Cu合金与不同液体的接触角,并基于OWRK/Fowkes方法计算出表面自由能,结果如表1所示。可见,Ni-Ti和Ni-Ti-Cu合金与3种液体的接触角均小于90°,都是亲水性表面。Ni-Ti-Cu合金具有更低的极性力分量与色散力分量,因此具有更低的总表面自由能。有研究[34]表明,具有超亲水表面与超疏水表面的材料都具有良好的血液相容性。Ni-Ti与Ni-Ti-Cu合金均为一般的亲水性表面,因此应该具有相近的血液相容性,但具有较低表面自由能的材料更易于获得优异的抗凝血性能[35]

表1   不同样品与去离子水、甘油和溴代萘的接触角及表面自由能测量结果

Table 1  Measurement results of contact angle and surface free energy of different samples with deionized water, glycerin, and bromonaphthalene

Alloyθ1 / (°)θ2 / (°)θ3 / (°)γsvp / (mJ·m-2)γsvd / (mJ·m-2)
Ni-Ti53.44 ± 1.9455.53 ± 3.2718.59 ± 0.3711.94 ± 1.3139.31 ± 0.83
Ni-Ti-Cu58.36 ± 3.6065.73 ± 3.5819.12 ± 0.888.99 ± 1.1237.56 ± 1.08

Note:θ1─contact angle of deionized water, θ2─contact angle of glycerol, θ3─contact angle of bromonaphthalene, γsvp─polarity component of surface free energy, γsvd─dispersion component of surface free energy

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通过电化学实验探究Ni-Ti-Cu合金在模拟人体血液中的耐蚀性能。图2a中的OCP测试结果表明,Ni-Ti-Cu合金较Ni-Ti合金具有更高的开路电位(Eocp),表明Ni-Ti-Cu合金在热力学上发生腐蚀的倾向较低。图2b的动电位极化曲线结果表明,Ni-Ti与Ni-Ti-Cu合金均发生了点蚀,且均表现出活化-钝化-过钝化的特征。EcorricorrEpit的测试结果如表2所示。可见,Ni-Ti-Cu合金的EcorrEpit均高于Ni-Ti合金,icorr低于Ni-Ti合金,表明在动力学上,Ni-Ti-Cu合金耐局部腐蚀与均匀腐蚀的能力均优于Ni-Ti合金,这可能是由于Ni-Ti-Cu合金表面上形成了更为致密的钝化膜[36]。随后,对2种合金进行EIS测试,结果如图2cd所示。对EIS结果进行拟合,拟合电路图和参数示于图2c插图和表2中,拟合电路图中的RsQc1Rc1分别表示溶液电阻、常相位角元件和钝化膜电阻。结果表明,Ni-Ti与Ni-Ti-Cu合金的Nyquist图(图2c)在整个测试频率范围内(105~10-2 Hz)均表现为单一的容抗弧,表明2者均为具有单个时间常数的典型电容行为。这可以通过Bode图(图2d)验证,其中2种合金的相位角在宽频率范围内(102~10-1 Hz)均接近90°。此外,Ni-Ti-Cu合金的阻抗弧半径大于Ni-Ti合金,2种合金的RsQc1相近,但Ni-Ti-Cu合金的Rc1较Ni-Ti合金高出1个数量级,表明Ni-Ti-Cu合金的钝化膜具有更加优异的保护能力。以上电化学实验结果表明,Ni-Ti-Cu合金在模拟人体血液中的耐蚀性能要优于Ni-Ti合金。由此可见,Cu的添加提高了Ni-Ti-Cu合金的耐蚀能力。

图2

图2   Ni-Ti和Ni-Ti-Cu合金的电化学测试结果

Fig.2   Electrochemical test results of Ni-Ti and Ni-Ti-Cu alloys

(a) open circuit potential curves (Eocp—open circuit potential)

(b) potentiodynamic polarization curves (i—current density, E—potential)

(c) Nyquist diagram and fitting circuit diagram (Inset shows the equivalent circuit diagram. Z'—real impedance, Z''—imaginary impedance, R.E.—reference electrode, W.E.—working electrode, Rs—solution resistance, Rc1—passive film resistance, Qc1—constant-phase element)

(d) Bode diagram (|Z|—impedance modulus)


表2   从动电位极化曲线与EIS图中获得的电化学参数

Table 2  Electrochemical parameters obtained from polarization curves of driven potential and EIS diagram

AlloyEcorr / mVicorr / (nA·cm-2)Epit / mVRs / (Ω·cm2)Rc1 / (104 Ω·cm2)Qc1 / (μΩ-1·s n ·cm-2)
Ni-Ti-399 ± 6299.7 ± 13.4828 ± 3013.73 ± 1.646.41 ± 1.2052.3 ± 1.5
Ni-Ti-Cu-283 ± 5259.5 ± 16.3862 ± 2511.79 ± 1.2267.32 ± 3.4251.5 ± 1.2

Note:Ecorr—corrosion potential, icorr—corrosion current density, Epit—pitting potential, n—dispersion index

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Bricen˜o等[37]的研究表明,Ni-Ti合金中的马氏体相占比越多,奥氏体相占比越少,其耐蚀性能越好,因而会释放更少的Ni2+。本工作的实验结果也证实,Cu的添加会使Ni-Ti-Cu合金的Ms点升高,这会导致合金中的奥氏体相更易于转变为马氏体相。XRD谱分析结果也表明,Cu的添加促进Ni-Ti合金由奥氏体相转变为马氏体相,并且Ni-Ti-Cu合金的耐蚀能力较Ni-Ti合金也得到了提高。此外,Marattukalam等[38]的研究表明,具有低的表面自由能的Ni-Ti-Cu合金,其腐蚀电位会向更高的方向移动,腐蚀电流密度也会降低,因而低表面自由能为Ni-Ti-Cu合金提供了更好的腐蚀保护。本工作也证实,相比于Ni-Ti合金,Ni-Ti-Cu合金具有更低的表面自由能,因而其耐蚀能力同样得到了提高。

此外,还研究了Ni-Ti-Cu合金对HUVECs的作用,从而评价其是否具有促血管内皮化功能。利用细胞增殖实验评估了Ni-Ti合金中添加Cu对HUVECs增殖的影响。实验结果表明,随着培养时间的延长,Ni-Ti合金的OD由1 d的1.40增加至3 d的2.27,相对增长率62.14%。Ni-Ti-Cu合金的OD由1 d的1.45增加至3 d的2.53,相对增长率74.48%,所有样品表面上的细胞数目均出现增长,Ni-Ti-Cu合金样品表面的增长更为明显。由此可见,与Ni-Ti合金相比,Ni-Ti-Cu合金促进内皮细胞增殖的作用更强,并且无细胞毒性。

随后,本工作进一步研究Ni-Ti-Cu合金浸提液对内皮细胞迁移能力的影响。通过划痕实验评价不同样品浸提液对单层细胞长满后的横向迁移的影响,即对细胞群体运动的影响。采用Transwell实验分析不同样品浸提液对单个细胞迁移能力的影响。图3a1a2b1b2给出了划痕实验与Transwell实验的结果。如图3a1a2所示,与不同样品浸提液培养后,内皮细胞均迁移至划痕位置之间的空白区域,见图中黑色线所示。内皮细胞的相对迁移面积比率统计结果表明,Ni-Ti合金的相对迁移面积比率为25.83%,Ni-Ti-Cu合金的相对面积迁移比率为34.51%,Ni-Ti-Cu合金浸提液促进了内皮细胞的横向迁移。图3b1b2的结果显示,Ni-Ti合金浸提液向下室迁移的内皮细胞数目约为35,而Ni-Ti-Cu合金则约为69。因此与Ni-Ti合金相比,内皮细胞与Ni-Ti-Cu合金浸提液共培养后,向Transwell下室迁移的细胞数目明显增多。划痕实验与Transwell实验结果均表明,Cu在Ni-Ti合金中的添加显著增大了内皮细胞的迁移能力。

图3

图3   Ni-Ti和Ni-Ti-Cu合金的体外细胞实验结果

Fig.3   In vitro cell test results of Ni-Ti (a1-c1) and Ni-Ti-Cu (a2-c2) alloys

(a1, a2) scratch (b1, b2) Transwell (c1, c2) tube formation


血管支架植入人体后,血管的快速修复能够减少新生内膜的生成和血小板吸附,进而会降低血栓及支架内再狭窄发生率。因此本工作还研究了Cu的添加对Ni-Ti合金的血管生成能力的影响。由于内皮细胞在基质胶中可以形成类似管腔的结构,因此采用小管生成实验,研究不同材料浸提液对内皮细胞成血管能力的影响,实验结果如图3c1c2所示。可见,与Ni-Ti合金相比,Ni-Ti-Cu合金浸提液能促进内皮细胞的小管形成。统计结果显示,与Ni-Ti和Ni-Ti-Cu合金浸提液共培养后的内皮细胞形成的毛细血管状结构的节点数分别为134和156,血管长度分别为22.74和25.79 mm。与Ni-Ti-Cu合金浸提液共培养后的内皮细胞形成的毛细血管状结构的节点数及血管长度均明显增多,表明Ni-Ti-Cu合金能够加速内皮细胞形成网络结构。

动态凝血实验可以用来评估材料的抗凝血能力。图4为Ni-Ti和Ni-Ti-Cu合金的动态凝血实验结果。可见在相同的凝血时间内,Ni-Ti-Cu合金稀释液的OD更高,说明血液中的血红蛋白更多,即材料表面上凝固的血液更少。进一步对OD与时间进行线性拟合,结果表明,Ni-Ti合金的凝血时间为61.78 min,而Ni-Ti-Cu合金的凝血时间则延长至79.06 min,相比Ni-Ti合金增加近30%,说明Ni-Ti-Cu合金能够显著降低血液凝固速率,具有更优的抗凝血能力。

图4

图4   Ni-Ti和Ni-Ti-Cu合金的动态凝血实验结果

Fig.4   Results of dynamic coagulation experiment of Ni-Ti and Ni-Ti-Cu alloys (y—fitted value of absorbance of residual free hemoglobin in streamer solution measured at 545 nm wavelength, x—dynamic coagulation time, R2—fitting correlation coefficient)


以上实验结果表明,Ni-Ti-Cu合金较Ni-Ti合金具有更为优异的促进血管内皮化功能,这与Cu的添加密切相关。已有研究[39,40]表明,Cu可以提高心肌血管中的内皮生长因子(VEGF)水平,因而会促进内皮细胞的增殖与迁移。本工作研究结果证实,在Ni-Ti合金中添加适量Cu可促进HUVECs的增殖和迁移。动态凝血实验结果表明,Ni-Ti-Cu合金的抗凝血能力也优于Ni-Ti合金。这是因为表面自由能较低的表面对血液中某些成分的吸附能力减弱,因此会表现出更优的抗凝血性能[41]。表面能测试结果证明,Cu的添加使Ni-Ti合金的表面自由能下降,由此可见,Ni-Ti-Cu合金具有更为优异的抗凝血作用,因而更有利于降低血栓的发生率。

3 结论

(1) 与Ni-Ti合金相比,Ni-Ti-Cu合金的Ms点提高,使其显微组织由等轴奥氏体晶粒组织向细板条马氏体组织转变。此外,由于添加Cu,Ni-Ti-Cu合金的表面自由能下降。

(2) 在显微组织变化和较低的表面自由能的共同作用下,与Ni-Ti合金相比,Ni-Ti-Cu合金的耐局部腐蚀与均匀腐蚀能力均得到提高。

(3) Ni-Ti-Cu合金具有更优的促进人脐静脉内皮细胞增殖、迁移和成血管的能力,因而促内皮化作用更强。与Ni-Ti合金相比,Ni-Ti-Cu合金显著降低血液凝固速率,具有更优的抗凝血能力。

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[J]. Int. J. Electrochem. Sci., 2013, 8: 10320

[本文引用: 1]

Zhang X Y, Zhao Y H, Gao W W, et al.

Study of TiCuN/ZrN multilayer coatings with adjustable combination properties deposited on TiCu alloy

[J]. Vacuum, 2022, 202: 111202

DOI      URL     [本文引用: 1]

Toker S M, Canadinc D, Maier H J, et al.

Evaluation of passive oxide layer formation-biocompatibility relationship in NiTi shape memory alloys: Geometry and body location dependency

[J]. Mater. Sci. Eng., 2014, C36: 118

[本文引用: 1]

Zhao J.

Study on biological functions of Cu-bearing stainless steel for urethral system

[D]. Shenyang: Institute of Metal Research, Chinese Academy of Sciences, 2017

[本文引用: 1]

赵 静.

含铜不锈钢在泌尿系统中的生物医学功能研究

[D]. 沈阳: 中国科学院金属研究所, 2017

[本文引用: 1]

Yang H J, Yang K, Zhang B C.

Study of in vitro anticoagulant property of the La added medical 316L stainless steel

[J]. Acta Metall. Sin., 2006, 42: 959

[本文引用: 1]

The in vitro anticoagulant property of a medical grade 316L stainless steel with 0.05% La addition was systematically studied. The results showed that, compared with the conventionally used medical 316L stainless steel, the La added 316L steel possesses less platelets adhesion and less activation of the platelets, and their kinetic clotting time and the plasma recalcification time become longer, which reveals that the activation extent of the latter on intrinsic coagulation factors is smaller than the former and the anticoagulant property of the latter is better. Through measurement of the contact angles of the steels, and calculations of the surface tension of the steels and the interfacial tension between the steels and the blood, the anticoagulant mechanism of the steels was analyzed from the viewpoint of surface energy.

杨化娟, 杨 柯, 张炳春.

含La医用316L不锈钢的体外抗凝血性能研究

[J]. 金属学报, 2006, 42: 959

[本文引用: 1]

系统研究了在医用316L不锈钢中加入0.05%La后的体外抗凝血性能. 结果表明, 与传统医用316L不锈钢相比, 含La医用316L不锈钢较少地粘附和激活血小板, 且动态凝血时间及血浆复钙时间均延长, 表明其对内源性凝血因子的激活程度下降, 抗凝血性能提高. 通过测量材料的接触角, 计算了表面张力及材料与血液之间的界面张力, 从表面能的角度分析了材料的抗凝血机理.

Yu X X, Hong Z, Jiang H W, et al.

Surface wettability of water and blood on diversified nanocone‐shaped ZnO films modified with n‐dodecyl mercaptan

[J]. Surf. Interface Anal., 2022, 54: 1211

DOI      URL     [本文引用: 1]

Qiu P, Gao P P, Wang S Y, et al.

Study on corrosion behavior of the selective laser melted NiTi alloy with superior tensile property and shape memory effect

[J]. Corros. Sci., 2020, 175: 108891

DOI      URL     [本文引用: 1]

Briceño J, Romeu A, Espinar E, et al.

Influence of the microstructure on electrochemical corrosion and nickel release in NiTi orthodontic archwires

[J]. Mater. Sci. Eng., 2013, C33: 4989

[本文引用: 1]

Marattukalam J J, Singh A K, Datta S, et al.

Microstructure and corrosion behavior of laser processed NiTi alloy

[J]. Mater. Sci. Eng., 2015, C57: 309

[本文引用: 1]

Liu H, Zhang X Y, Jin S J, et al.

Effect of copper-doped titanium nitride coating on angiogenesis

[J]. Mater. Lett., 2020, 269: 127634

DOI      URL     [本文引用: 1]

Jin S J, Qi X, Zhang B, et al.

Evaluation of promoting effect of a novel Cu-bearing metal stent on endothelialization process from in vitro and in vivo studies

[J]. Sci. Rep., 2017, 7: 17394

DOI      PMID      [本文引用: 1]

Drug eluting stents (DES) have been extensively applied nowadays and reduce the incidence of instent restenosis (ISR) greatly as compared with bare metal stents (BMS). However, the development of DES is hindered by the risk of late stent thrombosis (LST) due to delayed re-endothelialization, while endothelialization is an important process related to ISR and LST after implantation. 316L is a traditional stent material without bioactivity and have a high risk of ISR. Cu is recognized for angiogenesis stimulation in these years. Hence a copper bearing 316L stainless steel (316L-Cu) was prepared and evaluated about its effect on endothelialization in this paper. Compared with traditional 316L, it was proved that 316L-Cu increased the proliferation of co-cultured human umbilical vein endothelial cells (HUVECs) at first day. Moreover, HUVECs stretched better on the surface of 316L-Cu. It also improved the expression of angiogenesis related genes and tube formation ability in vitro. 316L-CuBMS, DES and 316L-BMS were implanted in swine to evaluate the re-endothelialization ability in vivo. And 316L-Cu-BMS showed the best effect on endothelialization with good biosafety. Consequently, 316L-Cu is a kind of promising BMS material for coronary field.

Hong Z, Yu X X, Jiang H W, et al.

Influence of surface morphology and surface free energy on the anticoagulant properties of nanocone‐shaped ZnO films

[J]. J. Appl. Polym. Sci., 2022, 139: 52005

DOI      URL     [本文引用: 1]

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