CN110791798B - 一种在仿生多孔钛合金表面构建壳聚糖-多聚胆碱磷酸/明胶涂层的方法 - Google Patents
一种在仿生多孔钛合金表面构建壳聚糖-多聚胆碱磷酸/明胶涂层的方法 Download PDFInfo
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Abstract
一种在仿生多孔钛合金表面构建壳聚糖‑多聚胆碱磷酸/明胶涂层的方法,首先制备胆碱磷酸单体,并对壳聚糖进行多聚胆碱磷酸改性,然后制备电泳溶液,将电泳溶液置于电泳容器在进行电泳沉积,获得带壳聚糖‑多聚胆碱磷酸/明胶涂层的多孔钛合金。本发明制备的带壳聚糖‑多聚胆碱磷酸/明胶涂层的多孔钛合金兼具抑制非特异性蛋白吸附和促进细胞粘附的功能,所获得多聚胆碱磷酸(PMCP),较之单层CP,该PMCP的亲水性、细胞粘附性及抗非特异性蛋白吸附性进一步增强。
Description
技术领域
本发明属于医疗器械领域,具体涉及一种在仿生多孔钛合金表面构建壳聚糖-多聚胆碱磷酸/明胶涂层的方法。
背景技术
大段骨缺损的修复一直是创伤骨科、骨肿瘤领域的一项难题。近年来,随着3D打印快速发展,采取新兴的电子束熔融、选区激光融化快速成型技术可有效制备具有复杂外部构型和内部微孔隙结构的多孔钛合金,大幅简化了先前多孔钛合金的制备工艺、降低了成本,为解决大段骨缺损修复的难题提供新的方法和参考。
但是,钛合金为生物惰性金属,缺乏生物活性,大段多孔钛合金植入机体后,其内部孔隙常存在骨长入不佳的问题。多孔钛合金内部孔隙骨组织长入主要由周边孔隙爬行替代而来,而此爬行替代过程通常比较缓慢,在此过程中,部分内部孔隙由蛋白、纤维组织填充,且该过程领先于骨组织的爬行替代,由此阻碍了骨组织的进一步长入,引起内部孔隙骨长入不佳和最终的内植物松动。因此,在多孔钛合金与骨组织进行有效整合之前,抑制非特异性蛋白的吸附可有效抑制蛋白、纤维组织的填充,为多孔材料孔隙内部骨长入提供条件。
针对以上问题,学者们所尝试的普遍做法是在材料表面复合聚乙二醇或者两性离子群以抑制非特异性蛋白吸附。上述做法的缺点在于其抑制非特异性蛋白吸附的同时,抑制了蛋白吸附介导的细胞粘附,而细胞粘附在多孔材料孔隙内部骨长入的过程中至关重要。
近几年,文献报道了一种两性离子胆碱磷酸(Choline Phosphate,CP),这种新型的两性离子CP可与细胞膜表面的磷酸胆碱(Phosphate Choline,PC)形成两组独特的CP-PC配对,对细胞膜有着强烈的粘附效应。同时,CP为两性离子,该两性离子聚合物可形成静电介导的水合作用,在聚合物链表面形成紧密的疏水层可抵抗非特异性蛋白的吸附。
将多孔钛合金表面进行明胶改性后可有效复合生物大分子物质,可达到增强其生物活性或载药性能。传统的钛合金表面明胶涂层改性方法为:将多孔钛合金浸入NaOH溶液中水化,清洗后浸入5%明胶溶液负压处理得到改性后的多孔钛合金。该方法的不足在于钛合金与明胶的结合为简单的物理接触结合,很难产生牢靠的结合力,其载药及缓释性能较差。
近年来,文献报道了一种使用电泳沉积法(Electrophoretic Deposition,EPD)在钛合金表面构建壳聚糖(Chitosan,CS)/明胶(Gelatin,G)载药系统的方法,取得了良好的效果。壳聚糖由于含有(1.4)-2-氨基-2-脱氧-β-D-葡聚糖分子结构,带阳性电荷,可以通过EPD技术复合于钛合金表面。通过该方法构建的载药涂层厚度均匀、可调整,且结合力牢靠,且对于复杂的多孔材料孔隙结构内部,该方法仍可获得理想效果。
CS含有大量的氨基,采取原子转移自由基聚合(Atom Transfer RadicalPolymerization,ATRP)技术易与单体MCP结合,获得CS-PMCP。PMCP分子是水溶性分子,若将其作为单一材料用于修复骨缺损时,其降解速度将远大于骨的新生速度,细胞粘附作用短暂,因此,有必要构建PMCP缓释载药系统,在支架材料植入体内后,PMCP获得平稳、缓慢释放的效果。将PMCP复合至多孔钛合金表面有望解决多孔钛合金内部孔隙骨长入不佳的难题。但是,如何将极具细胞粘附效应的PMCP分子复合至多孔钛合金表面是该设想的瓶颈问题。
发明内容
本发明的目的在于克服上述现有技术的缺点,提供了一种在仿生多孔钛合金表面构建壳聚糖-多聚胆碱磷酸/明胶涂层的方法。
为达到上述目的,本发明采用的技术方案是:
1)制备胆碱磷酸单体,并对壳聚糖进行多聚胆碱磷酸改性
在-20℃取5mL溶剂四氢呋喃向其中加入8mmol缚酸剂三乙胺、8mmol甲醇和8mmol的2-氯-2-氧-1,3,2-二氧磷杂环戊烷,而后用减压抽滤的获得中间产物2-甲氧基-2-氧-1,3,2-二氧磷杂环戊烷,取8mmol中间产物2-甲氧基-2-氧-1,3,2-二氧磷杂环戊烷向其中加入10mL甲基丙烯酸二甲氨基乙酯和10mL乙腈对2-甲氧基-2-氧-1,3,2-二氧磷杂环戊烷进行开环反应,反应结束后在以四氢呋喃溶液为沉淀剂沉淀得单体胆碱磷酸;
按壳聚糖:2-溴异丁酰溴为1:2的摩尔比混合反应得壳聚糖-Br,再按单体胆碱磷酸:壳聚糖-Br:CuBr2:N,N,N’,N”,N”-五甲基二乙烯三胺为50:1:2:2的摩尔比,即以壳聚糖-Br作为大分子引发剂与单体胆碱磷酸通过原子转移自由基聚合得含壳聚糖-多聚胆碱磷酸复合物;
2)壳聚糖-多聚胆碱磷酸和明胶电泳沉积复合至多孔钛合金
2-1)电泳溶液的制备
取1.2mg含壳聚糖-多聚胆碱磷酸复合物溶于150mL摩尔浓度为0.04mol/L的HCl溶液中,再向其中加入含壳聚糖-多聚胆碱磷酸复合物质量30~70%的A型明胶在60℃磁力搅拌均匀后得混合溶液,NaOH溶液室温下调整混合溶液的pH值为4.0得到电泳溶液;
2-2)电泳容器及电极的设计制备
将圆片状多孔钛合金放置于电泳容器的平台上作为阴极,与之平行的铂金电极作为阳极,二者距离为50mm;
或在圆柱形电泳容器中央设置为阴极,多孔钛合金支撑棒竖直放置在阴极之上,容器外壁设计为铂金阳极;
将电泳溶液置于电泳容器在进行电泳沉积,获得改性的多孔钛合金。
所述壳聚糖的脱乙酰度大于95%。
所述步骤2-1)NaOH溶液的浓度为0.1mol/L。
所述多孔钛合金采取电子束熔融技术或选区激光熔化快速成型技术制备的多孔钛合金。
所述多孔钛合金置于硝酸、氢氟酸和水的摩尔比为1∶4∶5的酸液中侵蚀1~2秒去除多孔钛合金孔隙内填充的粉末,减薄小梁厚度,增加小梁表面粗糙度,然后在65℃浸泡至摩尔比为1∶1的盐酸与硫酸的混合溶液中30分钟,后依次用丙酮、乙醇及蒸馏水超声清洗后用蒸馏水清洗干净。
所述的电泳沉积加载电压为3.5V,时间为3分钟,沉积完毕后,断开电源,取出多孔钛合金材料,蒸馏水冲洗,37℃隔水式恒温培养箱保存过夜后等离子消毒。
本发明制备的带壳聚糖-多聚胆碱磷酸/明胶涂层的多孔钛合金兼具抑制非特异性蛋白吸附和促进细胞粘附的功能,所获得多聚胆碱磷酸(PMCP),较之单层CP,该PMCP的亲水性、细胞粘附性及抗非特异性蛋白吸附性进一步增强。
具体实施方式
本发明的制备方法如下:
1)制备胆碱磷酸单体,并对壳聚糖进行多聚胆碱磷酸改性
在-20℃取5mL溶剂四氢呋喃向其中加入8mmol缚酸剂三乙胺、8mmol甲醇和8mmol的2-氯-2-氧-1,3,2-二氧磷杂环戊烷,而后用减压抽滤的获得中间产物2-甲氧基-2-氧-1,3,2-二氧磷杂环戊烷,取8mmol中间产物2-甲氧基-2-氧-1,3,2-二氧磷杂环戊烷向其中加入10mL甲基丙烯酸二甲氨基乙酯和10mL乙腈对2-甲氧基-2-氧-1,3,2-二氧磷杂环戊烷进行开环反应,反应结束后在以四氢呋喃溶液为沉淀剂沉淀得单体胆碱磷酸;
按脱乙酰度大于95%的壳聚糖:2-溴异丁酰溴为1:2的摩尔比混合反应得壳聚糖-Br,再按单体胆碱磷酸:壳聚糖-Br:CuBr2:N,N,N’,N”,N”-五甲基二乙烯三胺为50:1:2:2的摩尔比,即以壳聚糖-Br作为大分子引发剂与单体胆碱磷酸通过原子转移自由基聚合得含壳聚糖-多聚胆碱磷酸复合物;
2)壳聚糖-多聚胆碱磷酸和明胶电泳沉积复合至多孔钛合金
2-1)电泳溶液的制备
取1.2mg含壳聚糖-多聚胆碱磷酸复合物溶于150mL摩尔浓度为0.04mol/L的HCl溶液中,再向其中加入含壳聚糖-多聚胆碱磷酸复合物质量30~70%的A型明胶在60℃磁力搅拌均匀后得混合溶液,用0.1mol/L的NaOH溶液室温下调整混合溶液的pH值为4.0得到电泳溶液;
2-2)电泳容器及电极的设计制备
将圆片状多孔钛合金放置于电泳容器的平台上作为阴极,与之平行的铂金电极作为阳极,二者距离为50mm;
或棒体状多孔钛合金主要用于体内实验,对于此类体积较大材料,采取上述平行电极可能存在生物活性物质沉积不均匀等弊端。因此,可设计制备圆柱形沉积容器,在圆柱形电泳容器中央设置为阴极,多孔钛合金支撑棒竖直放置在阴极之上,容器外壁设计为铂金阳极;
将电泳溶液置于电泳容器加载电压为3.5V,时间为3分钟,沉积完毕后,断开电源,取出多孔钛合金材料,蒸馏水冲洗,37℃隔水式恒温培养箱保存过夜后等离子消毒获得改性的多孔钛合金。
所述多孔钛合金采取电子束熔融技术或选区激光熔化快速成型技术制备的多孔钛合金。
所述多孔钛合金置于硝酸、氢氟酸和水的摩尔比为1∶4∶5的酸液中侵蚀1~2秒去除多孔钛合金孔隙内填充的粉末,减薄小梁厚度,增加小梁表面粗糙度,然后在65℃浸泡至摩尔比为1∶1的盐酸与硫酸的混合溶液中30分钟,后依次用丙酮、乙醇及蒸馏水超声清洗后用蒸馏水清洗干净。
Claims (6)
1.一种在仿生多孔钛合金表面构建壳聚糖-多聚胆碱磷酸/明胶涂层的方法,其特征在于:
1)制备胆碱磷酸单体,并对壳聚糖进行多聚胆碱磷酸改性
在-20℃取5mL溶剂四氢呋喃向其中加入8mmol缚酸剂三乙胺、8mmol甲醇和8mmol的2-氯-2-氧-1,3,2-二氧磷杂环戊烷,而后用减压抽滤的获得中间产物2-甲氧基-2-氧-1,3,2-二氧磷杂环戊烷,取8mmol中间产物2-甲氧基-2-氧-1,3,2-二氧磷杂环戊烷向其中加入10mL甲基丙烯酸二甲氨基乙酯和10mL乙腈对2-甲氧基-2-氧-1,3,2-二氧磷杂环戊烷进行开环反应,反应结束后在以四氢呋喃溶液为沉淀剂沉淀得单体胆碱磷酸;
按壳聚糖:2-溴异丁酰溴为1:2的摩尔比混合反应得壳聚糖-Br,再按单体胆碱磷酸:壳聚糖-Br:CuBr2:N,N,N’,N”,N”-五甲基二乙烯三胺为50:1:2:2的摩尔比,即以壳聚糖-Br作为大分子引发剂与单体胆碱磷酸通过原子转移自由基聚合得含壳聚糖-多聚胆碱磷酸复合物;
2)壳聚糖-多聚胆碱磷酸和明胶电泳沉积复合至多孔钛合金
2-1)电泳溶液的制备
取1.2mg含壳聚糖-多聚胆碱磷酸复合物溶于150mL摩尔浓度为0.04mol/L的HCl溶液中,再向其中加入含壳聚糖-多聚胆碱磷酸复合物质量30~70%的A型明胶在60℃磁力搅拌均匀后得混合溶液,NaOH溶液室温下调整混合溶液的pH值为4.0得到电泳溶液;
2-2)电泳容器及电极的设计制备
将圆片状多孔钛合金放置于电泳容器的平台上作为阴极,与之平行的铂金电极作为阳极,二者距离为50mm;
或在圆柱形电泳容器中央设置为阴极,多孔钛合金支撑棒竖直放置在阴极之上,容器外壁设计为铂金阳极;
将电泳溶液置于电泳容器在进行电泳沉积,获得带壳聚糖-多聚胆碱磷酸/明胶涂层的多孔钛合金。
2.根据权利要求1所述的在仿生多孔钛合金表面构建壳聚糖-多聚胆碱磷酸/明胶涂层的方法,其特征在于:所述壳聚糖的脱乙酰度大于95%。
3.根据权利要求1所述的在仿生多孔钛合金表面构建壳聚糖-多聚胆碱磷酸/明胶涂层的方法,其特征在于:所述步骤2-1)NaOH溶液的浓度为0.1mol/L。
4.根据权利要求1所述的在仿生多孔钛合金表面构建壳聚糖-多聚胆碱磷酸/明胶涂层的方法,其特征在于:所述多孔钛合金采取电子束熔融技术或选区激光熔化快速成型技术制备的多孔钛合金。
5.根据权利要求1所述的在仿生多孔钛合金表面构建壳聚糖-多聚胆碱磷酸/明胶涂层的方法,其特征在于:所述多孔钛合金置于硝酸、氢氟酸和水的摩尔比为1:4:5的酸液中侵蚀1~2秒去除多孔钛合金孔隙内填充的粉末,然后在65℃浸泡至摩尔比为1:1的盐酸与硫酸的混合溶液中30分钟,后依次用丙酮、乙醇及蒸馏水超声清洗后用蒸馏水清洗干净。
6.根据权利要求1所述的在仿生多孔钛合金表面构建壳聚糖-多聚胆碱磷酸/明胶涂层的方法,其特征在于:所述的电泳沉积加载电压为3.5V,时间为3分钟,沉积完毕后,断开电源,取出多孔钛合金材料,蒸馏水冲洗,37℃隔水式恒温培养箱保存过夜后等离子消毒。
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