CN113262331B - 一种促内皮化的密网支架及其制备方法 - Google Patents
一种促内皮化的密网支架及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种促内皮化的密网支架及其制备方法,支架包括密网支架基底以及键接于密网支架基底表面的有机小分子涂层,有机小分子涂层的厚度为5~1000nm。在制备该支架时,先对密网支架基底进行清洗,然后采用处理液对密网支架基底进行处理,在其表面形成一层二氧化钛基层,再用紫外线对密网支架基底进行照射,最后将经过紫外照射的密网支架基底浸没于有机小分子酸溶液中,反映一定时间后即得。本发明针对密网支架的促内皮要求构建了二氧化钛‑有机小分子涂层,该涂层能使密网支架能够快速内皮化,进而减少血栓的形成,抑制长期炎症的发生且防止再狭窄甚至闭塞。
Description
技术领域
本发明属于医疗器械植入物技术领域,具体涉及一种促内皮化的密网支架及其制备方法。
背景技术
由于动脉病变或损伤导致的动脉壁扩张或膨胀形成的动脉瘤是一种常见的血管疾病,动脉瘤的致死致残率极高,严重威胁到患者的生命健康安全。目前针对动脉瘤的治疗方法主要由外科夹闭手术、弹簧圈栓塞术和支架植入三种。外科手术存在创伤大且易发生并发症。弹簧圈栓塞术的适用范围窄,对大动脉瘤,假性动脉瘤等的治疗效果差、风险高。利用密网支架或是覆膜支架的植入引导血流重建是相对较新的方法。通过血管介入手术将密网支架放置到血管瘤处引导血流重建,降低血流对动脉瘤的冲刷和剪切作用,能大大降低动脉瘤破裂的风险。且在密网支架的阻隔作用下动脉瘤内的血供变差,动脉瘤会逐渐萎缩直至一个安全的水平。密网支架相对于外科夹闭手术和弹簧圈栓塞治疗具有更广的适用范围、更高的安全性等。
然而,密网支架也存在缺点和不足。如在体内的长期存在容易造成内皮化延缓和并发症的产生,导致再狭窄甚至闭塞;支架与血流的大面积接触容易吸附更多的蛋白和血小板等,容易导致血小板的粘附和激活,同时也不利于内皮细胞的爬移,容易造成再狭窄。目前针对这些问题的研究主要是通过设计不同结构的密网支架(如分段式,分段可降解式等)来改善促进内皮细胞的粘附和抗凝;此外,大量患者需要体外服用抗凝药物进行治疗。但是这些方法都存在一定的风险。因此,设计快速内皮化和抗凝的密网支架迫在眉睫。
发明内容
针对上述现有技术,本发明提供一种促内皮化的密网支架及其制备方法,以解决现有密网支架容易造成动脉再狭窄的问题。
为了达到上述目的,本发明所采用的技术方案是:提供一种促内皮化的密网支架及其制备方法,本发明中的密网支架包括密网支架基底以及键接于密网支架基底表面并具有促内皮化功能的有机小分子酸,有机小分子酸在密网支架基底表面形成厚度为5~1000nm的涂层。
在上述技术方案的基础上,本发明还可以做如下改进。
进一步,密网支架基底为镍钛合金密网支架。
进一步,有机小分子酸为阿魏酸、苹果酸、抗坏血酸或氯原酸。
本发明中的促内皮化的密网支架经过以下步骤制得:
S1:对密网支架基底进行清洗并干燥;
S2:将经过S1处理后的密网支架基底浸没于温度为45~55℃的处理液中,并用氢氟酸将处理液的pH调节至3.5~4.0,浸泡0.5~6h后取出密网支架基底;所述处理液由氟钛酸铵溶液和硼酸溶液混合而成,其中氟钛酸铵的浓度为0.05~0.2M,硼酸的浓度为0.2~0.5M;
S3:将经过S2处理后的密网支架基底置于紫外下照射4~8h;
S4:将经过紫外照射后的密网支架基底浸没于浓度为0.5~2mg/mL有机小分子酸溶液中,于37~45℃下反应0.5~2h,取出,清洗并干燥,即得。
本发明的机理是:通过化学转化法对镍钛合金密网支架进行处理,在支架表面得到一层二氧化钛基层,再通过紫外照射增加其表面活性羟基,以利于后续阿魏酸等有机小分子的组装和沉积。基于自组装和共价接枝的原理,阿魏酸等有机小分子和二氧化钛表面的活性羟基进行共价接枝,形成具有纳米尺度的有机小分子功能涂层。
促内皮化的密网支架的制备方法在上述技术方案的基础上还可以做如下进一步的改进。
进一步,将密网支架基底置于去离子水中,超声清洗三次,每次5min;然后置于无水乙醇中,超声清洗三次,每次5min;再置于丙酮中,超声清洗三次,每次5min;最后真空干燥箱干燥。
进一步,处理液的温度为50℃,pH为3.88,密网支架基底在处理液中的浸泡时间为3h。
进一步,处理液中氟钛酸铵的浓度为0.1M,硼酸的浓度为0.3M。
进一步,S3中紫外照射时间为6h。
进一步,有机小分子酸为阿魏酸、苹果酸、抗坏血酸或氯原酸。
进一步,S4中反应温度为40℃,反应时间为1h。
本发明的有益效果是:
1.本发明构筑了纳米尺度的改性功能层,包含应用广泛的二氧化钛,天然有机小分子,并基于自组装和共价接枝的原理,将两种组分结合在一起,形成了纳米尺度涂层;并且,区别于传统载药涂层体系,本发明中通过共价接枝固定在表面的有机小分子涂层更加稳定牢固,可以实现表面长效功能化,并不涉及药物释放动力学,因此更加简单有效。
2.本发明中采用阿魏酸、苹果酸、抗坏血酸或氯原酸等有机小分子酸在密网支架基底表面形成涂层,不仅能够促进内皮细胞的粘附和迁移,有利于快速内皮化,而且小分子涂层相较于高聚物涂层分子量更小,不会降解产生有害的低聚物等,再者,小分子形成的涂层更薄,也更容易对厚度进行控制。
附图说明
图1为镍钛合金密网支架基底键接阿魏酸涂层前后的扫描电镜形貌图;
图2为镍钛合金密网支架基底键接阿魏酸涂层后的红外光谱图;
图3为镍钛金属片及镍钛合金密网支架基底键接阿魏酸涂层前后亲水性演示结果;
图4为镍钛金属片及镍钛合金密网支架基底键接阿魏酸涂层前后内皮细胞粘附荧光染色图;
图5为镍钛金属片及镍钛合金密网支架基底键接阿魏酸涂层前后内皮细胞活性检测图;
图6为镍钛合金密网支架基底键接阿魏酸涂层前后内皮细胞迁移结果。
具体实施方式
下面结合实施例对本发明的具体实施方式做详细的说明。
实施例1
一种促内皮化的密网支架,包括镍钛合金密网支架基底以及键接于镍钛合金密网支架基底表面的阿魏酸涂层,阿魏酸涂层的厚度为400nm左右。
本实施例中的促内皮化的密网支架经过以下步骤制得:
S1:将密网支架基底置于去离子水中,超声清洗三次,每次5min;然后置于无水乙醇中,超声清洗三次,每次5min;再置于丙酮中,超声清洗三次,每次5min;最后真空干燥箱干燥;
S2:将经过S1处理后的镍钛合金密网支架基底浸没于温度为50℃的处理液中,并用氢氟酸将处理液的pH调节至3.88,浸泡3h后取出密网支架基底;处理液由氟钛酸铵溶液和硼酸溶液混合而成,其中氟钛酸铵的浓度为0.1M,硼酸的浓度为0.3M;
S3:将经过S2处理后的密网支架基底置于紫外下照射6h;
S4:将经过紫外照射后的密网支架基底浸没于浓度为1mg/mL阿魏酸溶液中,于40℃下反应1h,取出,清洗并干燥,即得。
实施例2
一种促内皮化的密网支架,包括镍钛合金密网支架基底以及键接于镍钛合金密网支架基底表面的苹果酸涂层,苹果酸涂层的厚度为100nm左右。
本实施例中的促内皮化的密网支架经过以下步骤制得:
S1:将密网支架基底置于去离子水中,超声清洗三次,每次5min;然后置于无水乙醇中,超声清洗三次,每次5min;再置于丙酮中,超声清洗三次,每次5min;最后真空干燥箱干燥;
S2:将经过S1处理后的镍钛合金密网支架基底浸没于温度为45℃的处理液中,并用氢氟酸将处理液的pH调节至3.5,浸泡1h后取出密网支架基底;处理液由氟钛酸铵溶液和硼酸溶液混合而成,其中氟钛酸铵的浓度为0.05M,硼酸的浓度为0.5M;
S3:将经过S2处理后的密网支架基底置于紫外下照射4h;
S4:将经过紫外照射后的密网支架基底浸没于浓度为2mg/mL苹果酸溶液中,于37℃下反应0.5h,取出,清洗并干燥,即得。
实施例3
一种促内皮化的密网支架,包括镍钛合金密网支架基底以及键接于镍钛合金密网支架基底表面的抗坏血酸涂层,抗坏血酸涂层的厚度为800nm左右。
本实施例中的促内皮化的密网支架经过以下步骤制得:
S1:将密网支架基底置于去离子水中,超声清洗三次,每次5min;然后置于无水乙醇中,超声清洗三次,每次5min;再置于丙酮中,超声清洗三次,每次5min;最后真空干燥箱干燥;
S2:将经过S1处理后的镍钛合金密网支架基底浸没于温度为55℃的处理液中,并用氢氟酸将处理液的pH调节至4.0,浸泡6h后取出密网支架基底;处理液由氟钛酸铵溶液和硼酸溶液混合而成,其中氟钛酸铵的浓度为0.2M,硼酸的浓度为0.2M;
S3:将经过S2处理后的密网支架基底置于紫外下照射5h;
S4:将经过紫外照射后的密网支架基底浸没于浓度为0.5mg/mL抗坏血酸溶液中,于45℃下反应2h,取出,清洗并干燥,即得。
结果分析
以实施例1制得的促内皮化的密网支架为例,对采用本发明方案制得的密网支架的性能进行详细说明。
采用扫描电镜对实施例1制得的促内皮化的密网支架的微观结构进行观察,结果如图1所示。从扫描结果可以看出,未改性的镍钛密网支架表明光滑平整,有些许细小的划痕,改性之后的支架表面呈致密分布的纳米颗粒状,涂层整体覆盖完整,说明了涂层成功构建在了镍钛密网支架的表面。
采用FTIR对实施例1制得的促内皮化的密网支架的化学组分进行分析,结果如图2所示。从FTIR谱图的结果可知,改性后的支架表面出现了Ti-O键的特征峰,说明了二氧化钛基底层成功的构建在了密网支架表面,出现的C=O、C-O、C-H、C=C以及-OH特征峰充分表明了阿魏酸小分子成功的接枝到了二氧化钛的表面。
通过UP水滴对实施例1制得的促内皮化的密网支架的表面亲疏水性进行演示,结果如图3所示。通过照片可知,改性之前的镍钛金属片以及镍钛合金密网支架都是疏水的,而改性之后的样品表面的水滴铺展更开,充分说明阿魏酸小分子涂层提高了支架的亲水性。
另外,考察实施例1制得的促内皮化的密网支架的体外内皮细胞粘附和活性情况。
1.采用HUVECs细胞进行促内皮能力的验证,内皮细胞使用添加10%FBS的F-12DMEM(Hyclone)培养基培养。细胞培养至单层铺满约80%后,进行细胞接种;
2.细胞接种步骤为:
(1)实验前准备:实验所需耗材进行高温灭菌处理;
(2)灭菌处理的实验器材(玻璃吸管、离心管、滤器、注射器、枪头、移液枪等)置于超净台紫外辐照30min以上;
(3)实验所用样品灭菌准备,试样正反面进行紫外辐照30min,灭菌完成后置于无菌24孔板中;
(4)将生长状况正常且形态良好的内皮细胞用0.9%的氯化钠清洗三遍,加入胰酶进行消化,时间为1~2min(在光学显微镜下观察,细胞形态皱缩变圆并发亮,加入含有血清的培养基终止消化)。用玻璃吸管将细胞吹打均匀,取出细胞悬浮液并加入离心管中,于离心机中离心5min,转速为1200rpm/min,并进行细胞重悬;
(5)将1mL重悬后的细胞滴加到计数板进行计数,按照本发明使用的接种密度2×104cells/mL进行稀释;
(6)用1mL的移液枪将稀释后的细胞悬浮液接种到装有试样的24孔板中,每孔1mL,并放在孵箱(37℃,5%CO2)培养一天后进行活性检测和免疫荧光染色观察。
3.本发明通过CCK-8检测细胞活性。具体步骤为:每孔中滴加含有10%CCK-8的培养基并孵育3h,用酶标仪测试其吸光度值。
4.利用罗丹明(Rhodamine-phalloidin)进行免疫荧光染色观察其粘附数量和生长形态,具体染色步骤:
(1)将培养板取出,吸出培养基,用0.9%的氯化钠清洗三遍,加入2.5%的戊二醛于室温下固定4h;
(2)吸出戊二醛,用0.9%的氯化钠清洗三遍并吹干,避光条件下加入70μL罗丹明,反应15min。之后用0.9%的氯化钠清洗三遍并吹干,通过荧光显微镜观察。
内皮细胞的培养结果如图4所示,经过一天培养,不锈钢表面粘附了部分状态良好的细胞,镍钛金属片和镍钛密网支架表面也粘附了少量细胞,形态铺展良好。在改性后的镍钛金属片和镍钛密网支架表面粘附了大量的细胞,并且细胞形态良好,铺展状态正常,说明支架键接阿魏酸涂层能够显著的促进内皮细胞的粘附和铺展。
内皮细胞的活性检测如图5所示,可以看出,改性后的镍钛合金密网支架的吸光度高于不锈钢和未改性的镍钛合金密网支架,与荧光染色结果一致。上述结果充分说明了该小分子涂层能够促进内皮细胞的粘附增殖和铺展。
考察实施例1制得的促内皮化的密网支架的体外促内皮细胞迁移的能力。
1.内皮细胞的接种步骤与上述一致,本实验所用的内皮细胞接种于样品表面,种植密度均为5×104cells/ml,培养至铺满样品表面后,用200μL的枪头轻划样品表面,并用氯化钠清洗三遍,加入新的培养基。在孵箱(37℃,5%CO2)中培养12h,24。在对应的时间点用罗丹明进行染色(染色步骤与前述一致),利用荧光显微镜观察拍照。
内皮细胞迁移结果如图6所示,从荧光照片可以看出,在培养12h后改性后的镍钛合金密网支架表面的内皮细胞迁移距离大于不锈钢和镍钛金属;在24h培养后,不锈钢和镍钛金属表面的内皮细胞迁移尚未完全闭合,而改性后的镍钛合金密网支架表面的迁移已经完全将划痕覆盖。表明改性后的样品能够显著的促进内皮细胞的迁移,有利于快速内皮化。
虽然结合实施例对本发明的具体实施方式进行了详细地描述,但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内,本领域技术人员不经创造性劳动即可作出的各种修改和变形仍属本专利的保护范围。
Claims (1)
1.一种促内皮化的密网支架,其特征在于:包括镍钛合金密网支架基底以及键接于镍钛合金密网支架基底表面的阿魏酸涂层,阿魏酸涂层的厚度为400nm;所述促内皮化的密网支架经过以下步骤制得:
S1:将密网支架基底置于去离子水中,超声清洗三次,每次5min;然后置于无水乙醇中,超声清洗三次,每次5min;再置于丙酮中,超声清洗三次,每次5min;最后真空干燥箱干燥;
S2:将经过S1处理后的镍钛合金密网支架基底浸没于温度为50℃的处理液中,并用氢氟酸将处理液的pH调节至3.88,浸泡3h后取出密网支架基底;处理液由氟钛酸铵溶液和硼酸溶液混合而成,其中氟钛酸铵的浓度为0.1M,硼酸的浓度为0.3M;
S3:将经过S2处理后的密网支架基底置于紫外下照射6h;
S4:将经过紫外照射后的密网支架基底浸没于浓度为1mg/mL阿魏酸溶液中,于40℃下反应1h,取出,清洗并干燥,即得。
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