Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (30): 4836-4843.doi: 10.12307/2023.558
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Zhao Zheng1, Ding Wenfei1, Shao Shuxin2, Wang Jinyan1, Jian Xigao1
Received:
2022-08-17
Accepted:
2022-10-26
Online:
2023-10-28
Published:
2023-04-03
Contact:
Wang Jinyan, Professor, Doctoral supervisor, Department of Polymer Science, Dalian University of Technology, Dalian 116024, Liaoning Province, China
About author:
Zhao Zheng, MD, Department of Polymer Science, Dalian University of Technology, Dalian 116024, Liaoning Province, China
Supported by:
CLC Number:
Zhao Zheng, Ding Wenfei, Shao Shuxin, Wang Jinyan, Jian Xigao. Degradation and drug release behavior of sirolimus-poly(trimethylene carbonate) modified magnesium alloy[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(30): 4836-4843.
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2.1 不同涂层镁合金样品的表征结果 图1A为聚三亚甲基碳酸酯涂层及各载药涂层的微观表面形貌。所制备的聚三亚甲基碳酸酯涂层和各载药涂层表面平整,没有观察到西罗莫司的聚集现象,说明西罗莫司在涂层中分散性较好。同时,通过接触角测试对各样品的表面亲疏水性进行表征,如图1B,聚三亚甲基碳酸酯修饰后的接触角为(74.9±0.4)°。加入西罗莫司后,样品接触角有所增大,且随着涂层中药物占比的升高接触角增大更明显,说明西罗莫司的加入可使涂层的疏水性提高。在载药涂层中加入聚乙二醇400后,样品的接触角有所下降,并且随着聚乙二醇400含量的增加,接触角下降更明显,说明聚乙二醇400的加入可使载药涂层的亲水性有所增强。"
2.2 载药涂层对镁合金降解速率的影响 电化学阻抗谱低频区的阻抗模量大小,通常意味着涂层抗腐蚀能力的强弱[25],裸镁合金、聚三亚甲基碳酸酯涂层镁合金及S1、S2、S3、S2-1、S2-2样品电化学阻抗谱中0.1 Hz处的阻抗模量分别为4.0×102,1.3×108,2.6×108,1.6×108,1.4×108,1.3×108,8.8×107 Ω?cm2。 可以看出,随着西罗莫司占比的增加,样品低频区的阻抗模量有所升高,说明样品在浸泡初期的腐蚀抵抗能力有所提高。随着聚乙二醇400的加入,样品的阻抗模量有所减小,腐蚀抵抗能力有所降低。阻抗模量的变化趋势和接触角的变化趋势一致,说明涂层的亲疏水性影响样品最初的腐蚀抵抗能力,疏水性越强,最初的腐蚀抵抗能力也就越强。但是所制备的这6种涂层的阻抗模量在一个数量级,腐蚀抵抗能力区别并不显著。 2.3 载药涂层对镁合金降解速率的影响 通过对浸泡过程中溶液pH值和Mg2+浓度的检测,可以直观地反映浸泡过程中腐蚀速率的变化过程。如图2A,B所示,在所有样品中,裸镁合金组展现出较快的腐蚀速率,在最初的浸泡阶段(第1天)即展现出较高的pH值和较高的Mg2+析出量。裸镁合金组在第5天观察到短暂的下降而后出现进一步的上升,解释为腐蚀产物在镁合金表面的积累抑制了镁合金的腐蚀速率。然而腐蚀产物并不能对镁合金产生持续保护,第5天后pH值和Mg2+浓度进一步上升,腐蚀程度进一步加剧。经过聚三亚甲基碳酸酯修饰之后,样品pH值和Mg2+浓度相比裸镁合金来说在整个浸泡阶段显著下降,说明聚三亚甲基碳酸酯涂层显著抑制了镁合金的溶解。对于S1、S2和S3修饰的样品来说,在前期浸泡过程中,pH值和Mg2+浓度与聚三亚甲基碳酸酯组并无显著差异,但随着浸泡时间的延长,差异逐渐显著(pH值和Mg2+浓度的大小趋势:S3 > S2 > S1)。 对浸泡28 d的样品表面形貌进行了扫描电镜观察,见图2C,发现载药涂层出现了一些孔洞,推测这些孔洞是在药物溶出后留下的。这些孔洞增加了涂层中的水通路,使溶液更容易渗透到镁合金基底,从而削弱了涂层后期的防护能力。对于这些样品来说,药物占比越高,浸泡后期药物溶出造成的残留孔洞就越多,使得pH值和Mg2+浓度的增加就更明显。对于S2-1和S2-2样品来说,pH值和Mg2+浓度升高更明显一些,且聚乙二醇含量越高升高更明显(即S2-2 > S2-1 > S2)。28 d后的涂层表面展现出较多的孔洞,这与亲水性的提高及聚乙二醇的溶出有关。"
2.5 基底对样品药物释放的影响及机制研究 图4A展示了载药涂层修饰铁合金和镁合金的药物释放曲线,与铁合金表面的药物释放相比,镁合金表面在后期药物释放速率有略微加快,推测是由于镁合金在降解过程中产生的碱性环境,造成溶液pH值改变引起的。为了验证这个可能的原因,实验将溶液pH值调整到不同梯度的值(7,8,9,10;其中pH=10约为镁溶解饱和溶液的pH最大值),并测试了在这些不同pH值的溶液中铁合金表面药物涂层的药物释放情况。 由图4B可知,随着pH值的上升,药物释放在整体时间范围上出现了加速的现象,pH值越大,药物释放速率也就越快。通过观察28 d浸泡后表面膜的扫描电镜发现(图4C),随着pH值的增大,其表面孔洞变大变多,这可能是由于在碱性条件下,涂层聚三亚甲基碳酸酯降解加速造成的。强碱性条件造成聚三亚甲基碳酸酯的降解速度的加快,直接或间接地造成了药物释放的速率加快。这就意味着在镁合金基底上,由于镁合金的腐蚀行为造成了局部的碱性微环境,从而轻微改变了药物释放动力学,使得药物释放速率产生增加,这种现象在后期尤为明显。这种现象会改善药物释放在后期动力不足造成溶出速率过慢的问题,对药物释放是有利的,有利于改善血管内皮化延迟的现象。"
2.6 镁合金的细胞相容性 细胞增殖检测结果:由于所搭载的药物具有细胞增殖抑制作用,因此使用样品浸提液进行了细胞增殖抑制率测试。由图5A,B可以看出,无载药聚三亚甲基碳酸酯组的细胞增殖率和阴性对照组无显著差异,说明聚三亚甲基碳酸酯不会对两种细胞产生增殖抑制作用;与不载药涂层组相比,载药涂层组细胞增殖率明显下降,说明样品释放到浸提液中的西罗莫司有效抑制了细胞增殖。两种细胞展现出相似的规律,即西罗莫司展现出对两种细胞很弱的选择性。各个载药涂层样品组的细胞存活率展现出略微不同,其中随着涂层中药物占比的升高,细胞增殖率下降;随着聚乙二醇含量的增加,细胞增殖率下降,这些不同可能和最初的西罗莫司释放速率差异有关。西罗莫司释放速率越快,其浸提液中的药物含量也就越高,细胞受到抑制的效果也就越强。图5C展示了活/死细胞测试的荧光图片,无载药聚三亚甲基碳酸酯的细胞染色显示绿色荧光,说明细胞处于正常的细胞周期当中;载药涂层组的细胞状态则有部分细胞变为亮黄色,随着浸提液析出西罗莫司的含量越高,亮黄色的细胞也就越多,这是细胞进入早期凋亡阶段的象征[26]。西罗莫司作为一种细胞增殖抑制剂,阻滞了细胞从G0期向G1期的发展,部分细胞在阻滞阶段进入了细胞凋亡周期当中。"
2.7 不同涂层镁合金样品的血液相容性 血液相容性主要评价不同涂层镁合金样品对凝血和溶血系统的影响。图7A为血小板在样品表面的黏附,其通常被用来评价材料表面的血栓形成活性,通过观察样品表面黏附的血小板形态和数量可以对材料的血栓形成活性进行对比评价[28]。由图7A可知,在无载药的聚三亚甲基碳酸酯样品表面观察到一定数量的血小板黏附,血小板形态展现出圆形,无聚集和伪足产生,说明血小板并未出现明显的激活现象;而对于载药涂层来说,血小板在其表面的黏附数量显著下降,且血小板形态正常,说明血栓形成的风险减小。样品表面的血小板黏附数量和西罗莫司的释放速率显示出一定的关系,西罗莫司释放速率越快,表面黏附的血小板数量也就越少,见图7B,说明西罗莫司的释放抑制了血小板在其表面的黏附。样品对内外源凝血机制的影响通过活化部分促凝血酶原激酶时间、凝血酶原时间和凝血酶时间实验结果展现。由图7C可以看出,各样品组的各项凝血时间没有表现出显著差异,证明样品的修饰并不会对内外源凝血机制产生影响。由图7D可知,所有载药样品组的溶血率均在极低的水平,远低于ISO 10993-4标准中5%的限制,说明载药涂层修饰样品不会诱发溶血。以上结果表明载药涂层改善了样品的抗血栓能力,并展现出良好的血液相容性。"
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