CN115522207A - 一种在喷砂酸蚀钛表面构建pll/cpp-acp自组装多层膜的制备方法 - Google Patents
一种在喷砂酸蚀钛表面构建pll/cpp-acp自组装多层膜的制备方法 Download PDFInfo
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Abstract
本发明公开了一种在喷砂酸蚀钛表面构建PLL/CPP‑ACP自组装多层膜的制备方法，属于材料领域。该方法是将纯钛打磨抛光，喷砂，超声清洗并干燥；干燥后的纯钛采用氢氟酸/硝酸混合液常温酸蚀，之后超声清洗干燥，干燥后再经浓盐酸/浓硫酸混合酸75～85℃水浴温度下酸蚀，超声清洗并干燥，使钛表面形成微米结构，得到酸蚀钛材料；将酸蚀钛材料加入氢氧化钠溶液中进行碱化，碱化后的钛片置于PLL溶液中，之后将PLL预处理后的钛片交替、反复浸入CPP‑ACP溶液和PLL溶液中，即可得到目标产品。本发明工艺简单，仅氢氧化钠碱热处理及自组装技术即可形成钛表面纳米结构并加载生物活性钙离子，所需设备简单，成本低廉，钛表面兼具纳米结构以及钙元素修饰。
Description
技术领域
本发明属于生物材料领域。具体涉及一种在喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法。
背景技术
钛及钛合金材料因具有良好的耐腐蚀性、生物相容性和机械性能，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。然而钛材料具有生物惰性，植入骨组织时缺乏显著的生物活性，因此快速完成骨结合仍然是一个巨大的挑战。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。层层自组装技术是制备厚度可控、分子结构可控的生物活性多层膜的最有效、最有前途的方法之一，具有巨大的商业化潜力[F.X.Xiao,M.Pagliaro,Y.J.Xu,B.Liu,Layer-by-layer assemblyof versatile nanoarchitectures with diverse dimensionality:a new perspectivefor rational construction of multilayer assemblies,Chemical Society Reviews45(11)(2016)3088-3121]。
CPP含有的-ser(P)-ser(P)-ser(P)-glu-glu-序列，与二价金属离子具有高亲和力，因此可以稳定ACP形成CPP-ACP复合物，为稳定的钙磷再矿化***。CPP-ACP作为钙、磷的储存库，可以显著提高对这两种离子的生物利用率，并使溶液中维持较高浓度的钙离子、磷酸根离子或离子对[N.L.Huq,K.J.Cross,E.C.Reynolds,Molecular modelling of themultiphosphorylated casein phosphopeptide alphaS1-casein(59-79)based on NMRconstraints,The Journal of dairy research 71(1)(2004)28-32.]。钙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，并且时骨钙素和胶原蛋白表达等多种细胞活动的有效调节因子[E.Gabusi,C.Manferdini,F.Grassi,A.Piacentini,L.Cattini,G.Filardo,E.Lambertini,R.Piva,N.Zini,A.Facchini,G.Lisignoli,Extracellular calcium chronically induced human osteoblastseffects:Specific modulation of osteocalcin and collagen type XV,Journal ofcellular physiology 227(8)(2012)3151-3161].
现有技术在形成微米、纳米级微结构和沉积钙离子的方法较复杂，需要的设备较为复杂，技术敏感性高，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，提供一种在喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，无需复杂的设备，技术敏感性低。本发明制备的钛样品具有纳米结构以及钙元素修饰，工艺简单，易于加工，制备成本低廉。
技术方案：
一种在喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，该方法包括如下步骤：
步骤A)：将纯钛打磨抛光，喷砂，超声清洗并干燥；
步骤B)：将步骤A)干燥后的纯钛采用氢氟酸/硝酸混合液常温酸蚀5～15min，之后超声清洗干燥，干燥后再经浓盐酸/浓硫酸混合酸75～85℃水浴温度下酸蚀25～35min，超声清洗并干燥，使钛表面形成微米结构，得到酸蚀钛材料；
步骤C)：将酸蚀钛材料加入氢氧化钠溶液中，60-80℃水浴条件下碱化反应10-14h后再清洗；
步骤D)：将步骤C)碱化后的钛片置于PLL(poly(L-lysine)，多聚左旋赖氨酸)溶液中，4-6℃孵育10-12h，得到PLL预处理后的钛片；
步骤E)：将PLL预处理后的钛片交替、反复浸入CPP-ACP(caseinphosphopeptides-amorphous calcium phosphate，酪蛋白磷酸肽-无定形磷酸钙)和PLL溶液中，循环10次，每次浸泡10-20min，浸泡后荡洗，室温干燥，得到含钙纳米钛材料。
本发明技术方案中：步骤A)中纯钛试样抛光使用标号依次为600#，800#，1200#，1500#的砂纸由粗砂到细砂将纯钛逐级打磨抛光。
本发明技术方案中：步骤A)中纯钛试样喷砂使用80目Al2O3砂，压力0.4MPa，距离1.5cm，喷砂15s。
本发明技术方案中：步骤B)中氢氟酸/硝酸混合液中水：氢氟酸溶液：硝酸溶液的体积比为1000：1～3：3～5；氢氟酸溶液的体积浓度为0.22％，硝酸溶液的体积浓度为0.57％。
本发明技术方案中：步骤B)中浓盐酸/浓硫酸混合酸中水：浓盐酸溶液：浓硫酸溶液体积比为4～7：0.5～1.5：0.5～1.5，盐酸溶液的体积浓度为37％，硫酸溶液的体积浓度为98％。
本发明技术方案中：步骤C)中氢氧化钠溶液的浓度为1～3mol/L。
本发明技术方案中：步骤D)和步骤E)中所述的PLL多聚左旋赖氨酸的浓度为2.5mg/mL，步骤E)中CPP-ACP溶液的质量浓度为0.5-3.0％。
有益效果：本发明工艺简单，仅氢氧化钠碱热处理及自组装技术即可形成钛表面纳米结构并加载生物活性钙离子，所需设备简单，成本低廉，钛表面兼具纳米结构以及钙元素修饰。PLL/CPP-ACP钛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。其中，当CPP-ACP的浓度为3.0％时，钛表面结构更致密，亲水性更佳，其表面成骨细胞增殖和黏附性更强，更有利于成骨细胞成骨相关蛋白的表达。
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(40000倍)；
图2为本发明实施例2所制得的样本扫描电镜图(40000倍)；
图3为本发明实施例1、2所测得的XPS图谱；
图4为以本发明实施例1、2为实验组，喷砂酸蚀(SLA)钛表面为对照组，所测得的3组钛片的水接触角；
图5为以本发明实施例1、2为实验组，喷砂酸蚀(SLA)钛表面为对照组，将MC3T3-E1成骨细胞系接种于其表面培养8h后，所拍摄的细胞黏附图像；
图6为以本发明实施例1、2为实验组，喷砂酸蚀(SLA)钛表面为对照组，将MC3T3-E1成骨细胞系接种于其表面培养1、3、5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7为以本发明实施例1、2为实验组，喷砂酸蚀(SLA)钛表面为对照组，将MC3T3-E1成骨细胞系接种于其表面培养7天后，所测得的Runx2、OPN、OCN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
一种在喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，包括以下步骤：
步骤A)将纯钛打磨抛光(纯钛试样抛光使用标号依次为600#，800#，1200#，1500#的砂纸由粗砂到细砂将纯钛逐级打磨抛光)，喷砂(纯钛试样喷砂使用80目Al2O3砂，压力0.4MPa，距离1.5cm，喷砂15s)，超声清洗并干燥待用；
步骤B)将步骤A)处理的纯钛材料用氢氟酸/硝酸混合液(氢氟酸/硝酸混合液中双蒸水：氢氟酸溶液：硝酸溶液的体积比为1000：2：4；氢氟酸溶液的体积浓度为0.22％，硝酸溶液的体积浓度为0.57％)常温酸蚀10min，双蒸水超声清洗干燥，再经浓盐酸/浓硫酸混合酸液(浓盐酸/浓硫酸混合酸中双蒸水：盐酸溶液：硫酸溶液体积比为6：1：1，盐酸溶液的体积浓度为37％，硫酸溶液的体积浓度为98％)80℃水浴温度下酸蚀30min，双蒸水超声清洗并干燥；
步骤C)将步骤B)喷砂酸蚀处理的钛材料加入氢氧化钠溶液(氢氧化钠溶液的浓度为2mol/L)中，70℃水浴条件下碱化反应12h后再清洗；
步骤D)将步骤C)碱化后的钛片置于PLL(poly(L-lysine)，多聚左旋赖氨酸，浓度为2.5mg/mL)溶液中，5℃孵育11h。
步骤E)将步骤D)PLL处理过的钛材料交替浸入质量浓度为0.5％的CPP-ACP和PLL(浓度为2.5mg/mL)溶液，循环次数为10次，每次浸泡15min，浸泡后双蒸水荡洗2min，室温干燥备用。
实施例2
一种在喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，包括以下步骤：
步骤A)将纯钛打磨抛光(纯钛试样抛光使用标号依次为600#，800#，1200#，1500#的砂纸由粗砂到细砂将纯钛逐级打磨抛光)，喷砂(喷砂使用80目Al2O3砂，压力0.4MPa，距离1.5cm，喷砂15s)，超声清洗并干燥待用；
步骤B)将步骤A)处理的纯钛材料用氢氟酸/硝酸混合液(氢氟酸/硝酸混合液中双蒸水：氢氟酸溶液：硝酸溶液的体积比为1000：2：4；氢氟酸溶液的体积浓度为0.22％，硝酸溶液的体积浓度为0.57％)常温酸蚀10min，双蒸水超声清洗干燥，再经浓盐酸/浓硫酸混合酸液(浓盐酸/浓硫酸混合酸中双蒸水：盐酸溶液：硫酸溶液体积比为6：1：1，盐酸溶液的体积浓度为37％，硫酸溶液的体积浓度为98％)80℃水浴温度下酸蚀30min，双蒸水超声清洗并干燥；
步骤C)将步骤B)喷砂酸蚀处理的钛材料加入氢氧化钠溶液氢氧化钠溶液的浓度为2mol/L)中，70℃水浴条件下碱化反应12h后再清洗；
步骤D)将步骤C)碱化后的钛片置于PLL(poly(L-lysine)，多聚左旋赖氨酸，浓度为2.5mg/mL)溶液中，5℃孵育11h。
步骤E)将步骤D)PLL处理过的钛材料交替浸入质量浓度为3.0％的CPP-ACP和PLL浓度为2.5mg/mL)溶液，循环次数为10次，每次浸泡15min，浸泡后双蒸水荡洗2min，室温干燥备用。
用扫描电子显微镜观察实施例1、2制得扫描电镜图片如图1、2(40000倍)。结果表明实施例1、2均具有纳米结构，其表面微观结构呈纳米丛状，其中实例2纳米结构更致密，并且有一层薄膜覆盖于纳米结构表面。
实施例1、2为实验组，喷砂酸蚀(SLA)钛表面为对照组，用X射线能谱仪测定三组所含主要元素，制得XPS图谱如图3。结果表明实施例1、2均含有钙元素，其中实施例2的含量更高。实施例1、2相比对照组Ti元素含量减少、P元素和C元素含量增加。
实施例1、2为实验组，喷砂酸蚀(SLA)钛表面为对照组，检测3组钛片的水接触角及表面能，图4结果显示实施例1、2的水接触角小于对照组，表明实施例1、2较对照组具有更好的表面亲水性，且实例2亲水性最佳。
实施例1、2为实验组，喷砂酸蚀(SLA)钛表面为对照组，将MC3T3-E1成骨细胞系在其表面培养8h后，激光共聚焦显微镜观察显示(图5)，对照组黏附细胞较少，形态较为缩窄，实施例1、2组黏附细胞数量最多，铺展充分，细胞伪足多而长。结果表明实施例1、2组具有促进MC3T3-E1细胞黏附的作用，且实例2效果更佳。
实施例1、2为实验组，喷砂酸蚀(SLA)钛表面为对照组，将MC3T3-E1成骨细胞系在其表面培养1、3、5天后，采用CCK－8试剂盒检测细胞增殖。图6结果表明，实施例2的表面较对照组具有更好的促进细胞增殖的作用，实施例1的效果不明显。
实施例1、2为实验组，喷砂酸蚀(SLA)钛表面为对照组，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、OPN、OCN的蛋白表达水平，以GAPDH为内参。图7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，且实例2效果更明显。
Claims (7)
1.一种在喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，其特征在于包括如下步骤：
步骤A)：将纯钛打磨抛光，喷砂，超声清洗并干燥；
步骤B)：将步骤A)干燥后的纯钛采用氢氟酸/硝酸混合液常温酸蚀5～15min，之后超声清洗干燥，干燥后再经浓盐酸/浓硫酸混合酸75～85℃水浴温度下酸蚀25～35min，超声清洗并干燥，使钛表面形成微米结构，得到酸蚀钛材料；
步骤C)：将酸蚀钛材料加入氢氧化钠溶液中，60-80℃水浴条件下碱化反应10-14h后再清洗；
步骤D)：将步骤C)碱化后的钛片置于PLL溶液中，4-6℃孵育10-12h，得到PLL预处理后的钛片；
步骤E)：将PLL预处理后的钛片交替、反复浸入CPP-ACP溶液和PLL溶液中，循环10次，每次浸泡10-20min，浸泡后荡洗，室温干燥，得到目标产品。
2.根据权利要求1所述在喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，其特征在于：步骤A)中纯钛试样抛光使用标号依次为600#，800#，1200#，1500#的砂纸由粗砂到细砂将纯钛逐级打磨抛光。
3.根据权利要求1所述在喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，其特征在于：步骤A)中纯钛试样喷砂使用80目Al2O3砂，压力0.4MPa，距离1.5cm，喷砂15s。
4.根据权利要求1所述喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，其特征在于：步骤B)中氢氟酸/硝酸混合液中水：氢氟酸溶液：硝酸溶液的体积比为1000：1～3：3～5；氢氟酸溶液的体积浓度为0.22％，硝酸溶液的体积浓度为0.57％。
5.根据权利要求1所述喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，其特征在于：步骤B)中浓盐酸/浓硫酸混合酸中水：浓盐酸溶液：浓硫酸溶液体积比为4～7：0.5～1.5：0.5～1.5，盐酸溶液的体积浓度为37％，硫酸溶液的体积浓度为98％。
6.根据权利要求1所述喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，其特征在于步骤C)中氢氧化钠溶液的浓度为1～3mol/L。
7.根据权利要求1所述喷砂酸蚀钛表面构建PLL/CPP-ACP自组装多层膜的制备方法，其特征在于步骤D)和步骤E)中所述的PLL溶液的浓度为2.5mg/mL，步骤E)中CPP-ACP溶液的质量浓度为0.5-3.0％。
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