CN111632192A - 兼具抗菌、促成骨分化及矿化的载碘钛合金植入物及其制备方法 - Google Patents
兼具抗菌、促成骨分化及矿化的载碘钛合金植入物及其制备方法 Download PDFInfo
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- CN111632192A CN111632192A CN202010565315.6A CN202010565315A CN111632192A CN 111632192 A CN111632192 A CN 111632192A CN 202010565315 A CN202010565315 A CN 202010565315A CN 111632192 A CN111632192 A CN 111632192A
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- titanium alloy
- iodine
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- alloy implant
- antibacterial
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Abstract
本发明提供了一种兼具抗菌、促成骨分化及矿化的载碘钛合金植入物及其制备方法，本发明通过共价接枝及氧化还原反应的方法将碘涂层固定到钛合金上，制得的碘涂层钛片表现出抗菌性能，同时能够促进骨髓间充质干细胞成骨分化，并在模拟体液中促进钙磷矿化层生成；本发明制备工艺简单易行，具有高效、低成本、重复性佳的优势，并可有效改善目前钛合金植入物应用于骨科、口腔颌面外科以及神经外科临床的不足，降低其植入体内后的术后早期感染的发生率及因过度的炎症反应引发植入物松动等相关并发症。
Description
技术领域
本发明涉及骨科、口腔颌面外科、神经外科植入物技术领域，具体地说，涉及一种兼具抗菌、促成骨分化及矿化的载碘钛合金植入物及其制备方法。
背景技术
钛合金因其优良的生物相容性及其接近骨弹性模量的生物力学特性，使其作为骨修复植入物的代表在临床上广泛应用。然而优秀的骨修复植入物不应仅着眼于骨修复重建，更应该立足于预防复杂的手术并发症以达到最佳的手术效果。骨感染作为最严重的骨科手术并发症之一，局部乃至全身广泛的感染炎症反应及继发的骨缺损及骨坏死极大的延缓了患者的术后康复，造成了大量的医疗经济负担。因此，研发兼具抗菌、促成骨分化、矿化的钛合金植入物应用于骨修复手术迫在眉睫。
钛合金抗菌生物涂层技术因其制备方法多样，选取材料广泛，缓释抗菌，生物相容性可调等特点受到了广泛关注。具有抗菌性能的金属如银、锌、铜；抗菌有机物如季铵盐类以及抗菌肽等均成为钛合金抗菌涂层的研究重点。然而上述抗菌物质因缺乏大量临床实验数据及临床应用经验使其作为植入物涂层的安全性及组织相容性受到质疑，限制了其钛合金涂层产品的临床转化。
聚维酮碘是一种具有抗菌活性的含碘络合物，对细菌、芽孢、病毒及真菌均具有强效杀灭活性。其抗菌谱广、安全性高、刺激性小等优点使其成为外科手术消毒清创的首选，具有悠久的临床实践历史。因此，将聚维酮碘作为抗菌涂层材料生产制造钛合金骨科植入物具有极大的转化前景。
既往日本科学家以聚维酮碘为电解液，通过电镀工艺在钛合金表面涂覆碘涂层并以此方法制备外固定骨钉、脊柱钉棒系统等骨科植入物。相关临床实验显示载碘涂层的钛合金植入物具有较好的抗菌效果及成骨效果。这无疑为载碘涂层钛合金的进一步研究奠定了基础。然而因电镀工艺设备成本高昂、电解液制备条件严格、电镀条件影响因素繁多使其在经济发展各异的地区扩大生产应用受到了一定的制约。
因此，本发明受贻贝粘附蛋白启发，将制备条件简易的聚多巴胺作为表面改性物质引入钛合金涂层制备。聚多巴胺在钛合金表面粘附形成稳定涂层后与聚维酮碘水溶液或碘化钾溶液中的聚维酮碘相结合，制得表面碘密度可调的植入物，并表征其抗菌、促成骨分化及矿化的骨修复生物学特性。
发明内容
本发明的目的是针对钛合金植入物应用于骨科、口腔颌面外科以及神经外科临床的不足，提供一种兼具抗菌、促成骨分化及矿化的载碘钛合金植入物及其制备方法。
本发明的技术方案如下：
一种兼具抗菌、促成骨分化及矿化的载碘钛合金植入物，该植入物以钛合金为基体，钛合金基体表面引入有聚多巴胺涂层，聚多巴胺涂层上共价结合有碘元素；所述碘元素的形式包括：聚维酮碘、碘离子、碘酸根离子、碘离子与碘酸根离子氧化还原反应形成的碘单质；
所述载碘钛合金植入物的制备方法为：
(a)将钛合金基体浸入碱溶液中，60-80℃下反应10-18h，之后取出洗涤，干燥，备用；
所述碱溶液由碱性物质溶于水中得到，浓度为5-10M，所述碱性物质为氢氧化钠或氢氧化钾；
(b)将经过步骤(a)处理的钛合金基体浸入多巴胺Tris-HCl溶液中，pH＝8-9，25-37℃下反应6-12h，之后取出洗涤，干燥，得到涂覆有聚多巴胺的钛合金植入物；
所述多巴胺Tris-HCl溶液中多巴胺的浓度为1-5mg/mL；
(c)将步骤(b)所得涂覆有聚多巴胺的钛合金植入物浸入聚维酮碘的碘化钾溶液中，25-37℃下反应6-12h，之后取出洗涤，干燥，备用；
所述聚维酮碘的碘化钾溶液由聚维酮碘溶于0.1-0.2M碘化钾水溶液中得到，其中聚维酮碘的质量分数为3-5％；
(d)将步骤(c)准备好的钛合金植入物浸入碘酸钾水溶液中，pH＝3-5，25-37℃下反应3-12h，之后洗涤，干燥，得到所述载碘钛合金植入物；
所述碘酸钾水溶液的浓度为0.1-0.2M。
本发明载碘钛合金植入物可用于制备骨科、口腔颌面外科、神经外科植入物，具体例如：钛合金外固定架骨钉、骨折接骨板、髓内钉、脊柱内固定器、人工关节假体、肿瘤型假体、个性化增材制造(3D打印)钛合金系列关节假体，包含金属垫块、加强臼杯；口腔颌面外科的钛合金种植体、种植钉及口腔接骨板；神经外科的颅骨修补钛合金网、颅骨接骨板等钛合金植入物。
本发明载碘钛合金植入物具有抗感染和促进成骨分化及骨矿化的特点，所述抗感染主要针对骨科感染常见菌，包括革兰氏阳性菌和革兰氏阴性菌，其中革兰氏阳性菌包括金黄色葡萄球菌、耐药金黄色葡萄球菌、表皮葡萄球菌及耐药表皮葡萄球菌等，革兰氏阴性菌包括大肠杆菌、铜绿假单胞菌等。
本发明的优点在于：
1、本发明使用碘涂层对钛合金植入物进行修饰，得到的载碘钛合金植入物不仅具有抗感染的功能，还具有促进骨髓间充质干细胞成骨分化，并在模拟体液中促进钙磷矿化层生成的功能。本发明可有效改善目前钛合金植入物应用于骨科、口腔颌面外科以及神经外科临床的不足，降低其植入体内后的术后早期感染的发生率及引发植入物松动等相关并发症。
2、本发明是利用聚多巴胺经典的吸附作用将碘与钛合金共价连接，同时利用碘酸盐的强氧化性进一步增加钛合金植入物载碘量，工艺简单，反应条件适宜，具有高效、低成本、重复性佳的优势，获得的载碘钛合金植入物在抗菌、促成骨分化及矿化的效果十分显著。
附图说明
图1：实施例1中载碘钛片Ti-I表面电镜表征及元素EDS分析结果。
图2：实施例1中载碘钛片Ti-I浸泡于Tris-HCl缓冲液中碘离子的释放曲线。
图3：实施例1中空白钛片Ti与载碘钛片Ti-I表面培养金黄色葡萄球菌(ATCC25923)后的洗脱培养的抗菌效果。
图4：实施例1中空白钛片Ti与载碘钛片Ti-I表面接种人源骨髓间充质干细胞1天后的活死染色，绿色表示存活细胞，红色表示死亡细胞。
图5：实施例1中载碘钛片浸泡于SBF模拟体液中14天后的矿化电镜表征及元素EDS分析。
图6：实施例1中空白钛片Ti与载碘钛片Ti-I表面接种人源骨髓间充质干细胞3天后成骨相关基因Runx2及Sp7的表达情况，载碘钛片Ti-I组成骨基因高表达，*P＜0.05。
具体实施方式
下面结合附图通过具体实施例对本发明作进一步的说明，但本发明的保护范围并不仅限于此。
实施例1载碘钛合金植入物的制备(一)
1、钛片表面修饰聚多巴胺(Ti-PDA)
首先将钛合金Ti6AL4V(浙江科惠医疗公司)浸没于5M的NaOH溶液中，持续磁力搅拌状态下80℃下反应18h。取出钛片用去离子水超声清洗3次，每次5min，通风橱干燥。随后将钛片浸入到2mg/ml的多巴胺Tris-HCl(pH＝8.5)溶液中，室温下反应12h，去离子水超声清洗3次，通风橱干燥。
2、钛片表面共价键载碘(Ti-I)
用0.1M的碘化钾溶液为溶剂，配制3％的聚维酮碘/碘化钾(PVP-I2/KI)溶液，室温下持续磁力搅拌状态30min。随后将Ti-PDA浸没于PVP-I2/KI，室温下反应12h。去离子水超声清洗3次，通风橱干燥。随后将钛片浸入0.2M碘酸钾(KIO3)溶液中(pH＝5)，室温下反应10h后终止反应，去离子水超声清洗3次，通风橱干燥后即得所述的载碘钛合金植入物。
3、载碘钛合金植入物的理化性质表征
图1将Ti-I进行电子显微镜观察，可见碘均匀接枝于钛片表面，质量百分比达1.58％，原子百分比达0.35％。图2将Ti-I钛片浸没于pH＝7.4的Tris-HCl缓冲液中，碘可稳定持续释放14天，且最高累积碘浓度超过1.2ppm。
4、载碘钛合金植入物的抗菌实验
本研究应用金黄色葡萄球菌(ATCC 25923)作为实验细菌，但不仅限于金黄色葡萄球菌，其他革兰氏阳性菌及阴性菌均适用于本研究。
选取普通钛片(Ti)和载碘钛片(Ti-I)，直径10mm，高度2mm。将两组钛片环氧乙烷低温消毒后置于24孔板中，每组设3个复孔。将金黄色葡萄球菌(ATCC 25923)菌液(106CFU/ml)按每孔500μl分别接种于上述各组钛片上37℃下静态培养24h。无菌PBS漂洗3次以去除未粘附的细菌。应用超声震荡技术洗脱钛片表面黏附的细菌，然后行琼脂糖凝胶培养皿涂板，培养箱内37℃下培养24h后菌落计数拍照。
图3为两组钛片表面活菌涂板后的定性结果。研究可得Ti-I相较于Ti能够明显抑制金黄色葡萄球菌的生长，表现出良好的抗菌效果。
5、载碘钛合金植入物的生物相容性实验
本研究应用大鼠骨髓间充质干细胞(rBMSCs)作为实验细胞，但不仅限于rBMSCs，其他种属的各类细胞均适用于本研究。
选取普通钛片(Ti)和载碘钛片(Ti-I)，直径10mm，高度2mm。将两组钛片环氧乙烷低温消毒后置于24孔板中，每组设3个复孔。将rBMSCs细胞悬液(105/ml)按每孔500μl分别接种于上述各组钛片上，培养箱内37℃下培养24h后无菌PBS漂洗3次。随后用细胞活死试剂盒(南京凯基)染色，应用激光共聚焦显微镜(CLSM，德国)进行观察。其中钙黄绿素使活细胞发出绿色荧光，碘化丙啶使死细胞发出红色荧光。
图4为两组钛片表面细胞培养后的活死染色荧光照片。研究可得Ti-I相较于Ti均可保持rBMSCs的活性，体现出良好的生物相容性。
6、载碘钛合金植入物的自矿化性能
将Ti-I钛片浸没于pH＝7.4的SBF缓冲液中，37℃静置14天后，去离子水超声清洗3次，通风橱干燥。随后应用电子显微镜及能谱检测行表面结构及元素分布分析。
图5为Ti-I在SBF中浸泡14天后的表面结构，可见类羟基磷灰石的结晶形成。能谱分析显示Ti-I表面呈现出钙、磷元素沉积，其中钙元素质量百分比为1.92％，磷元素百分比为1.55％。从而得知，Ti-I具有良好的自矿化性能。
7、载碘钛合金植入物的成骨性能
选取普通钛片(Ti)和载碘钛片(Ti-I)，直径10mm，高度2mm。将两组钛片环氧乙烷低温消毒后置于24孔板中，每组设3个复孔。将rBMSCs细胞悬液(105/ml)按每孔500μl分别接种于上述各组钛片上，培养箱内37℃下培养3天后无菌PBS漂洗3次。利用Trizol法提取细胞RNA，qPCR检测成骨相关基因Runx2及Sp7的表达情况。
图6为两组钛片表面细胞培养后的成骨相关基因Runx2及Sp7表达情况。Ti-I相较于Ti能够显著提升Runx2和Sp7表达，表现出良好的成骨性能。
实施例2本发明的载碘钛合金植入物的制备(二)
1、钛片表面修饰聚多巴胺(Ti-PDA)
首先将钛合金Ti6AL4V(浙江科惠医疗公司)浸没于5M的NaOH溶液中，持续磁力搅拌状态下80℃下反应10h。取出钛片用去离子水超声清洗3次，每次10min，通风橱干燥。随后将钛片浸入到2mg/ml的多巴胺Tris-HCl(pH＝9)溶液中，室温下反应10h，去离子水超声清洗3次，通风橱干燥。
2、钛片表面共价键载碘(Ti-I)
用0.1M的碘化钾溶液为溶剂，配制5％的聚维酮碘/碘化钾(PVP-I2/KI)溶液，室温下持续磁力搅拌状态1h。随后将Ti-PDA浸没于PVP-I2/KI，室温下反应10h。去离子水超声清洗3次，通风橱干燥。随后将钛片浸入0.1M碘酸钾(KIO3)溶液中，用醋酸将pH调至3，室温下反应12h后终止反应，去离子水超声清洗3次，通风橱干燥后即得所述的载碘钛合金植入物。
对比例
目前已有报道的载碘钛合金多采用以碘溶液作为电解液阳极氧化的方法进行。该研究以H2SO4溶液作为电解液首先在钛片表面制备阳极氧化膜；再以PVP-I2溶液为电解液，电压70-150V，形成含碘阳极氧化膜，蒸馏水冲洗，压缩空气吹干，灭菌备用。
然而电镀工艺设备成本高昂、电镀条件影响因素繁多限制了其进一步的发展。本发明利用仿贻贝类聚多巴胺的粘附性能在温和实验条件下于钛合金表面稳定的负载聚维酮碘涂层，并利用碘化钾、碘酸钾的氧化还原反应梯度调控钛合金表面碘含量，可针对不同毒力的细菌制备梯度碘负载量的钛合金植入物。不仅方便中小企业的大规模生产，同时使开展临床转化研究的医疗科研人员有能力制备合格产品，有利于医疗器械临床转化的实验验证，搭建由实验室向临床导向的桥梁。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (6)
1.一种兼具抗菌、促成骨分化及矿化的载碘钛合金植入物，其特征在于，该植入物以钛合金为基体，钛合金基体表面引入有聚多巴胺涂层，聚多巴胺涂层上共价结合有碘元素；所述碘元素的形式为：聚维酮碘、碘离子、碘酸根离子、碘离子与碘酸根离子氧化还原反应形成的碘单质；
所述载碘钛合金植入物的制备方法为：
(a)将钛合金基体浸入碱溶液中，60-80℃下反应10-18h，之后取出洗涤，干燥，备用；
(b)将经过步骤(a)处理的钛合金基体浸入多巴胺Tris-HCl溶液中，pH＝8-9，25-37℃下反应6-12h，之后取出洗涤，干燥，得到涂覆有聚多巴胺的钛合金植入物；
(c)将步骤(b)所得涂覆有聚多巴胺的钛合金植入物浸入聚维酮碘的碘化钾溶液中，25-37℃下反应6-12h，之后取出洗涤，干燥，备用；
(d)将步骤(c)准备好的钛合金植入物浸入碘酸钾水溶液中，pH＝3-5，25-37℃下反应3-12h，之后洗涤，干燥，得到所述载碘钛合金植入物。
2.如权利要求1所述兼具抗菌、促成骨分化及矿化的载碘钛合金植入物，其特征在于，制备方法步骤(a)中，所述碱溶液由碱性物质溶于水中得到，浓度为5-10M，所述碱性物质为氢氧化钠或氢氧化钾。
3.如权利要求1所述兼具抗菌、促成骨分化及矿化的载碘钛合金植入物，其特征在于，制备方法步骤(b)中，所述多巴胺Tris-HCl溶液中多巴胺的浓度为1-5mg/mL。
4.如权利要求1所述兼具抗菌、促成骨分化及矿化的载碘钛合金植入物，其特征在于，制备方法步骤(c)中，所述聚维酮碘的碘化钾溶液由聚维酮碘溶于0.1-0.2M碘化钾水溶液中得到，其中聚维酮碘的质量分数为3-5％。
5.如权利要求1所述兼具抗菌、促成骨分化及矿化的载碘钛合金植入物，其特征在于，制备方法步骤(d)中，所述碘酸钾水溶液的浓度为0.1-0.2M。
6.如权利要求1所述兼具抗菌、促成骨分化及矿化的载碘钛合金植入物作为骨科、口腔颌面外科、神经外科植入物的应用。
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| CN113769162A (zh) * | 2021-09-14 | 2021-12-10 | 武汉大学 | 一种用于治疗感染性骨缺损的负载聚乙烯吡咯烷酮碘的仿生矿化微球的制备方法 |
| CN114344562A (zh) * | 2022-01-13 | 2022-04-15 | 吉林大学第一医院 | 一种仿骨小梁结构钛合金支架的生物活性改善方法和应用 |
| CN115814168A (zh) * | 2022-12-13 | 2023-03-21 | 广东省人民医院 | 一种以聚多巴胺涂层为载体的医用钛合金器件及其制备方法 |
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| CN113769162A (zh) * | 2021-09-14 | 2021-12-10 | 武汉大学 | 一种用于治疗感染性骨缺损的负载聚乙烯吡咯烷酮碘的仿生矿化微球的制备方法 |
| CN113769162B (zh) * | 2021-09-14 | 2022-09-13 | 武汉大学 | 一种用于治疗感染性骨缺损的负载聚乙烯吡咯烷酮碘的仿生矿化微球的制备方法 |
| CN114344562A (zh) * | 2022-01-13 | 2022-04-15 | 吉林大学第一医院 | 一种仿骨小梁结构钛合金支架的生物活性改善方法和应用 |
| CN115814168A (zh) * | 2022-12-13 | 2023-03-21 | 广东省人民医院 | 一种以聚多巴胺涂层为载体的医用钛合金器件及其制备方法 |
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