CN112126926B - 钛表面修饰纳米结构同步加载生物活性锌离子的制备方法 - Google Patents

钛表面修饰纳米结构同步加载生物活性锌离子的制备方法 Download PDF

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CN112126926B
CN112126926B CN202010825715.6A CN202010825715A CN112126926B CN 112126926 B CN112126926 B CN 112126926B CN 202010825715 A CN202010825715 A CN 202010825715A CN 112126926 B CN112126926 B CN 112126926B
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邱憬
朱文卿
汤泽华
张文思
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Affiliated Stomatological Hospital of Nanjing Medical University
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Abstract

钛表面修饰纳米结构同步加载生物活性锌离子的制备方法，步骤A）将纯钛打磨抛光；步骤B）将打磨抛光后的纯钛加入氢氧化钠溶液中，60‑80℃水浴条件下碱化反应10‑20min后再清洗；步骤C）将碱化后的钛片置于醋酸锌、磷酸氢二铵混合溶液中，醋酸锌与磷酸氢二铵溶液的体积比为1:10‑10:1，70‑90℃水热溶液反应0.5至2小时，清洗后烘干，得到含锌纳米钛材料。本发明材料表面可增强钛与骨的相容和结合能力，有利于钛表面成骨细胞的增殖和分化，以提高钛的骨整合能力。

Description

钛表面修饰纳米结构同步加载生物活性锌离子的制备方法
技术领域
本发明属于生物医用材料领域。具体涉及一种钛表面修饰纳米结构同步加载生物活性锌离子的制备方法。
背景技术
钛及其合金因其优异的力学性能、良好的生物相容性、稳定性和良好的骨整合潜能，被广泛应用于骨科及口腔科医疗领域，如骨固定装置和种植体。人们已经对钛表面修饰进行了大量的尝试，以期诱导骨的快速生长。碱热处理是最常用的处理之一，可形成改性微米或纳米钛表面，促进钛表面成骨细胞碱性磷酸酶和胶原合成，诱导骨形成。
锌是一种能促进细胞增殖、分化的重要微量元素，对成骨细胞分化和骨组织发育有直接影响，并作为一种信号分子影响细胞内的信号通路。[Masayoshi Yamaguchi,etal.Role of nutritional zinc in the prevention of osteoporosis.[J].Mol CellBiochem (2010) 338:241–254]有学者开发了一种含锌离子的碱热处理方法来制备含锌改性钛植入体表面。从锌修饰的钛表面释放的锌离子可以刺激骨髓间充质细胞的增殖、成骨细胞标记基因表达和钙沉积，对成骨细胞分化和骨形成有显著影响，且锌改性钛表面的剪切强度比对照组大5倍。文献[Kazuyuki Yusa, D.D.S., PhD., etal.Eluted zinc ionsstimulate osteoblast differentiation and mineralization in human dental pulpstem cells for bone tissue engineering.[J].Archives of Oral Biology 71 (2016)162–169]但其工艺较复杂，处理时间长，且表面微结构直径较粗。
现有技术在形成纳米级微结构和沉积锌离子的方法较复杂，需经过酸蚀、碱化先形成纳米结构，再通过含锌盐溶液水浴加载锌离子，处理时间长，制备表面粒度较粗。
发明内容
解决的技术问题：本发明针对上述现有技术的不足，提供一种钛表面修饰纳米结构同步加载生物活性锌离子的制备方法，无需先制备纳米结构表面再加载锌离子。本发明制备的钛样品具有纳米结构以及锌元素修饰，无需氢氟酸酸蚀，仅通过两步水热法同步完成锌离子沉积和纳米结构修饰，工艺简单，易于加工，制备成本低廉。
技术方案：钛表面修饰纳米结构同步加载生物活性锌离子的制备方法，包括如下步骤：步骤A）将纯钛打磨抛光；步骤B）将打磨抛光后的纯钛加入氢氧化钠溶液中，60-80℃水浴条件下碱化反应10-20min后再清洗；步骤C）将碱化后的钛片置于醋酸锌、磷酸氢二铵混合溶液中，醋酸锌与磷酸氢二铵溶液的体积比为1:10-10:1，70-90℃水热溶液反应0.5至2小时，清洗后烘干，得到含锌纳米钛材料。
优选的，步骤A）中纯钛打磨抛光依次使用标号为 600 # ，800 # ，1200 # ，1500# 的砂纸。
优选的，步骤B）中氢氧化钠溶液的浓度为10 wt.%。
优选的，步骤B）中所述的纯钛在70℃水浴温度下进行氢氧化钠水热反应，反应时间为15min。
优选的，步骤C）中醋酸锌/磷酸氢二铵混合液按体积比为2.15 wt.%醋酸锌:1.30wt.%磷酸氢二铵＝3:2。
优选的，步骤C）中所述的醋酸锌、磷酸氢二铵混合液反应溶液是在80℃水浴温度下进行，反应时间为0.5-2h。
有益效果：本发明工艺简单，无需传统酸蚀步骤即可同步形成钛表面纳米结构并加载生物活性锌离子，成本低廉，钛表面兼具了纳米结构以及锌元素修饰。三种经不同混合液反应时间形成的含锌纳米钛表面均可增强钛与骨的相容和结合能力，有利于钛表面成骨细胞的增殖和分化，以提高钛的骨整合能力。其中，15min处理组表面结构粒度更致密，亲水性、蛋白吸附率更佳，其表面成骨细胞增殖率和ALP活性更高，且处理时间最短。
附图说明
图1为本发明实施例1所制得的样本扫描电镜图(a：50000倍；b：150000倍)；
图2为本发明实施例2所制得的样本扫描电镜图(a：50000倍；b：150000倍)；
图3为本发明实施例3所制得的样本扫描电镜图(a：50000倍；b：150000倍)；
图4为本发明实施例1、2、3所测得的XPS图谱；
图5为本发明实施例1、2、3为实验组，纯钛为对照组，所测得的4组钛片的水接触角及表面能；
图6为本发明实施例1、2、3为实验组，纯钛为对照组，每组加入α-MEM于37℃孵育24小时，所测得的蛋白吸附率；
图7为以本发明实施例1、2、3为实验组，纯钛为对照组，将MC3T3-E1成骨细胞系接种于其表面培养1、3、6天后，所测得细胞增殖CCK-8值柱状图，*表示存在显著性差异；
图8为以本发明实施例1、2、3为实验组，纯钛为对照组，将MC3T3-E1成骨细胞系接种于其表面培养7天后，所测得的碱性磷酸酶(ALP)定量图；
图9为以本发明实施例1、2、3为实验组，纯钛为对照组，将MC3T3-E1成骨细胞系接种于其表面培养7天后，所测得的Runx2、Osterix得的蛋白表达水平。
具体实施方式
下面的实施例可使本专业技术人员更全面地理解本发明，但不以任何方式限制本发明。
实施例1
一种钛表面修饰纳米结构同步加载生物活性锌离子的制备方法，包括以下步骤：
步骤A）纯钛预处理：使用标号依次为600 # 、800 # 、1200 # 、1500 # 的砂纸由粗到细将纯钛试样逐级打磨抛光。然后用双蒸水、无水75%乙醇、双蒸水超声清洗各5min，干燥待用；
步骤B）将打磨抛光后的纯钛在70℃水浴下进行氢氧化钠溶液碱化反应15min，双蒸水、75%乙醇、双蒸水各超声清洗5min；
步骤C）将步骤B）碱化处理过的纯钛试样在醋酸锌、磷酸氢二铵混合液中80℃水热溶液反应30min，混合液配方为2.15 wt.%醋酸锌:1.30 wt.%磷酸氢二铵按体积比＝3:2。双蒸水、75%乙醇、双蒸水各超声清洗5min，80℃烘干备用。
实施例2
一种钛表面修饰纳米结构同步加载生物活性锌离子的制备方法，包括以下步骤：
步骤A）纯钛预处理：使用标号依次为600 # 、800 # 、1200 # 、1500 # 的砂纸由粗到细将纯钛试样逐级打磨抛光。然后用双蒸水、无水75%乙醇、双蒸水超声清洗各5min，干燥待用；
步骤B）将打磨抛光后的纯钛在70℃水浴下进行氢氧化钠溶液碱化反应15min，双蒸水、75%乙醇、双蒸水各超声清洗5min；
步骤C）将步骤B）碱化处理过的纯钛试样在醋酸锌、磷酸氢二铵混合液中80℃水热溶液反应1h，混合液配方为2.15 wt.%醋酸锌:1.30 wt.%磷酸氢二铵按体积比＝3:2。双蒸水、75%乙醇、双蒸水各超声清洗5min，80℃烘干备用。
实施例3
一种钛表面修饰纳米结构同步加载生物活性锌离子的制备方法，包括以下步骤：
步骤A）纯钛预处理：使用标号依次为600 # 、800 # 、1200 # 、1500 # 的砂纸由粗到细将纯钛试样逐级打磨抛光。然后用双蒸水、无水75%乙醇、双蒸水超声清洗各5min，干燥待用；
步骤B）将打磨抛光后的纯钛在70℃水浴下进行氢氧化钠溶液碱化反应15min，双蒸水、75%乙醇、双蒸水各超声清洗5min；
步骤C）将步骤B）碱化处理过的纯钛试样在醋酸锌、磷酸氢二铵混合液中80℃水热溶液反应2h，混合液配方为2.15 wt.%醋酸锌:1.30 wt.%磷酸氢二铵按体积比＝3:2。双蒸水、75%乙醇、双蒸水各超声清洗5min，80℃烘干备用。
用扫描电子显微镜观察实施例1、2、3，制得扫描电镜图片如图1、2、3(a：50000倍；b：150000倍)。结果表明实施例1、2、3均具有纳米结构，其表面微观结构呈纳米棒状，其中实例1纳米结构更致密。
用X射线能谱仪测定实施例1、2、3所含主要元素，制得XPS图谱如图4。结果表明实施例1、2、3均含有锌元素，且实施例1、2、3相比对照组C元素含量减少、Ti元素和O元素含量增加。
实施例1、2、3为实验组，纯钛为对照组，检测4组钛片的水接触角及表面能，图5(a：水接触角；b：表面能)结果显示实施例1、2、3的水接触角小于对照组，表面能大于对照组，表明实施例1、2、3较对照组具有更好的表面亲水性，且实例1亲水性最佳。
实施例1、2、3为实验组，纯钛为对照组，每组加入α-MEM于37℃孵育24小时，使培养基中的血清蛋白吸附于钛片上，采用BCA试剂盒检测蛋白吸附率，图6结果表明实施例1、2、3表面较对照组具有更好的蛋白吸附性，且实例1蛋白吸附性最佳。
实施例1、2、3为实验组，纯钛为对照组，将MC3T3-E1成骨细胞系在其表面培养1、3、6天后，采用CCK－8试剂盒检测细胞增殖。图7结果表明，实施例1、2、3的表面较对照组具有更好的促进细胞增殖的作用，其中实例1最佳。
实施例1、2、3为实验组，纯钛为对照组，将MC3T3-E1成骨细胞系在其表面培养7天后裂解细胞，采用碱性磷酸酶(ALP)试剂盒检测成骨细胞ALP活性。图8结果表明，实施例1、3的表面较对照组具有更好的促进细胞成骨、分化的作用。
实施例1、2、3为实验组，纯钛为对照组，将MC3T3-E1成骨细胞系接种于其表面培养7天后，使用RIPA 裂解液于裂解细胞，提取细胞总蛋白，通过蛋白印迹法（Western Blot）检测细胞中 Runx2、Osterix的蛋白表达水平，以GAPDH为内参。图9结果表明实施例1、2、3的表面较对照组表面均能明显提高细胞成骨分化相关蛋白表达量。

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1.钛表面修饰纳米结构同步加载生物活性锌离子的制备方法，其特征在于，包括如下步骤：步骤A）纯钛预处理：使用标号依次为600 # 、800 # 、1200 # 、1500 # 的砂纸由粗到细将纯钛试样逐级打磨抛光；然后用双蒸水、无水75%乙醇、双蒸水超声清洗各5min，干燥待用；步骤B）将打磨抛光后的纯钛在70℃水浴下进行氢氧化钠溶液碱化反应15min，双蒸水、75%乙醇、双蒸水各超声清洗5min；步骤C）将步骤B）碱化处理过的纯钛试样在醋酸锌、磷酸氢二铵混合液中80℃水热溶液反应30min，混合液配方为2.15 wt.%醋酸锌:1.30 wt.%磷酸氢二铵按体积比＝3:2；双蒸水、75%乙醇、双蒸水各超声清洗5min，80℃烘干备用。
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