CN107715179B - 复合型人工小血管支架及其制备方法 - Google Patents

复合型人工小血管支架及其制备方法 Download PDF

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CN107715179B
CN107715179B CN201711191617.6A CN201711191617A CN107715179B CN 107715179 B CN107715179 B CN 107715179B CN 201711191617 A CN201711191617 A CN 201711191617A CN 107715179 B CN107715179 B CN 107715179B
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游庆军
王�锋
黄朝晖
茆勇
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Affiliated Hospital of Jiangnan University
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Abstract

本发明涉及复合型人工小血管支架及其制备方法，包括内层和外层，内层为去细胞心内膜，外层为主动脉弹力层微米颗粒多孔支架，外层和内层间采用胶原连接。制备时首先将心内膜去细胞后采用戊二醛处理，主动脉弹力层组织研磨成颗粒后制成溶液在模具中干燥制成多孔支架，将内层材料连同轴心套入多孔支架中，向内层和外层间的空隙中注入液态胶原粘合。本发明制备方法简单，步骤易于操作，分层仿生构建小口径组织工程血管，制备出具有与正常血管相似的理化、生物力学性能并可与自身血管组织再生相匹配的可降解性小口径血管支架。

Description

复合型人工小血管支架及其制备方法
技术领域
本发明涉及一种复合型人工小血管支架及其制备方法，属于医疗血管支架技术领域。
背景技术
冠状动脉旁路移植术是目前治疗冠心病行之有效的手段之一，移植血管常用材料有大隐静脉、乳内动脉以及桡动脉等。静脉来源相对丰富，但管壁薄、弹性差，存在内膜增生、易堵塞等缺点。从通畅率考虑，应尽可能多地用动脉材料，但自体动脉来源有限。目前临床上应用较广泛的两种人工血管涤纶(dacron)和膨体聚四氟乙烯(expanded polytet—rafluoroethylene，ePTFE），由于弹性差、顺应性差以及易形成血栓（缺乏内膜）等缺点，作为血管移植物，尤其是小口径血管移植物（＜5mm），尚不能令人满意。
组织工程学血管(tissue engineering blood vessel，TEBV)作为组织工程学领域的产物之一，其发展使心血管病的治疗充满希望，标志着组织工程学由单一组织再生向复合组织的预制迈出了重大的一步。它的核心是种子细胞、可供种子细胞进行生命活动的血管支架材料以及细胞与血管支架的相互作用，其主要研究内容是血管支架材料的制备、种子细胞的筛选和培养。近年来，高分子生物材料和生物工程技术的迅速发展为人类寻找并研制理想的组织工程化血管支架提供了新的途径。关于人工合成可降解高分子血管支架材料，国内外研究最多的是聚乙酸(polyglycolic acid，PGA)、聚氨酯、聚乳酸(polylacticacid，PLA)及其共聚物。现有的人造血管材料均有不尽人意之处，特别是用于替代直径<5mm的小血管时，常常由于血栓形成和新生内膜增厚导致血管闭塞而使移植失败。目前面临着以下几个困难：（1）机械强度和弹性：目前植入体内的TEBV可承受一定的肺循环压力，但对于体循环压力的承受力仍需进一步提高。如PGA、PLA材料降解时间为6~8周，降解时间过快，导致组织工程血管松软、脆弱，机械支撑力较弱；（2）远期通畅率：目前大口径TEBV可保持一定的远期通畅率，但是小口径TEBV的远期通畅率仍不理想；（3）材料亲水性差致细胞吸附力较弱。如聚氨酯材料，其顺应性非常好，力学性能也很好，在组织工程血管的研究方面文献也较多。但这种材料降解率较差，细胞在其上粘附能力和增殖活性较差，目前很多实验的重点放在促进聚氨酯表面内皮化的研究上。理想的人造血管应能在愈合过程中随着支架的降解而重塑，形成一条与宿主动脉具有相同机械和生物性能的新血管。
发明内容
本发明的目的是为了解决上述问题，提供了一种复合型人工小血管支架及其制备方法，在支架上种植种子细胞可完成体外成血管，将此血管移植动物体内，通过降解和再生过程，最终自体血管组织再生代替移植血管，达到移植血管完全自体化的目的，得到与宿主动脉具有相似的机械和生物性能的新生小血管。
本发明采用如下技术方案：一种复合型人工小血管支架，所述小血管支架包括内层和外层，所述内层为去细胞心内膜，所述外层为主动脉弹力层微米颗粒多孔支架，所述外层和内层间采用蛋白质交联剂连接。
进一步的，所述小血管支架的外径为4~5mm。
进一步的，所述小血管支架的内径为3~3.2mm。
复合型人工小血管支架的制备方法，包括如下步骤：
（1）内层材料的制备：取新鲜猪心脏，用生理盐水冲洗干净，以心内膜为原料采用去细胞液进行去细胞处理；将去细胞的心内膜浸入含有2-3 %戊二醛液体作用3-4h，用双蒸水反复冲洗去残余戊二醛，环氧乙烷消毒备用得到内层去细胞心内膜；
（2）外层材料的制备：取猪的主动脉弹力层组织，并在匀浆器中打碎至小颗粒，在液氮中进行反复研磨后取直径5-30μm的颗粒；用离心机离心加入1%tritonx-100 12-24小时后，加入去细胞液48-72 h，用双蒸水反复悬浮、离心去除去细胞液；
（3）取预先制备的直径为3mm 的不锈钢轴，将去细胞心内膜缝制于轴上，长度为3-4cm；
（4）取预先制备的管状模具，将颗粒制成30-50%体积比的溶液，然后依次在-20℃、-80℃下冷冻1~1.5小时，去除模具形成多孔支架，用蛋白质交联剂交联；
（5）内层和外层的复合：将内层材料连同轴心套入多孔支架中，向内层和外层间的空隙中注入液态胶原粘合；
（6）细胞种植：将Brdu 标好的骨髓干细胞诱导后的平滑肌样细胞种植于外层的表面，在体外生物反应器中培养2-3 周，拔出轴心，将转染绿色荧光蛋白的慢病毒载的CD34+诱导后的内皮样细胞种植于内表面，体外继续培养1-2 周。
进一步的，所述蛋白质交联剂为质量分数为0.45-0.55%的京尼平。
进一步的，所述步骤（1）中的去细胞液为0.5% tritonx-100+0.5% SDS+ 150 IU/mL DNase I+100μg/mL RNase A。
进一步的，所述多孔支架的孔径为25~100μm，孔隙度为70%。
去细胞心内膜与动脉内膜在结构和力学性能上极为相似，均为内皮细胞生长的天然场所，可作为构建小口径组织工程血管很好的内膜材料，有利于提高细胞粘附并实现有效内皮化。
主动脉弹力层的主要成分为断裂的弹力纤维，该种结构能够用于提高小口径组织工程血管收缩弹性有效的方法。
由弹力层微米颗粒制备的多孔支架力学性能较差，脆性较大，采用京尼平交联后明显提高其力学性能，细胞接种实验发现，血管平滑肌样细胞很容易接种并长入多孔支架内，经体外培养后，材料的力学强度得到显著提高，并具有较好的收缩弹性。
本发明制备方法简单，步骤易于操作，以去细胞心内膜为内层，以主动脉弹力层微颗粒多孔支架为外层，分层仿生构建小口径组织工程血管，制备出的小血管支架具有与正常血管相似的理化、生物力学性能，口径小、并能够与自身血管组织再生相匹配、可降解。
附图说明
图1为本发明中小血管支架的外层原料主动脉弹力层微米颗粒。
图2为本发明中经冷冻干燥后管状支架的示意图。
图3为本发明中多孔支架扫描电镜观察。
图4为本发明中小血管支架的内层原料去细胞心内膜。
图5为本发明中管状去细胞心内膜内层缝制的实图。
图6为本发明中去细胞心内膜胶原染色和弹力纤维染色的对照图。
图7为本发明的人工小血管支架的结构示意图。
附图标记：内层1、外层2。
具体实施方式
下面将结合附图对本发明作进一步的描述。
一种复合型人工小血管支架，小血管支架包括内层1和外层2，内层1为去细胞心内膜，外层2为主动脉弹力层微米颗粒多孔支架，外层2和内层1间采用胶原连接，小血管支架的外径为4~4.5mm，小血管支架的内径为3~3.2mm。
实施例1：
一种复合型人工小血管支架的制备方法，包括如下步骤：
（1）内层材料的制备：取新鲜猪心脏，用生理盐水冲洗干净，以心内膜为原料采用去细胞液进行去细胞处理；将去细胞的心内膜浸入含有2 %戊二醛液体作用4h，用双蒸水反复冲洗去残余戊二醛，环氧乙烷消毒备用得到内层去细胞心内膜，去细胞液为0.5%tritonx-100+0.5% SDS+ 150 IU/mL DNase I+100μg/mL RNase A；
（2）外层材料的制备：取猪的主动脉弹力层组织，并在匀浆器中打碎至小颗粒，在液氮中进行反复研磨后取直径5-30μm的颗粒；用离心机离心加入1%tritonx-100 12小时后，加入去细胞液72 h，用双蒸水反复悬浮、离心去除去细胞液。
（3）取预先制备的直径为3mm 的不锈钢轴，将去细胞心内膜缝制于轴上，长度为3-4cm；
（4）取预先制备的管状模具，将颗粒制成30%体积比的溶液，在-20℃、-80℃下分别冷冻1小时，加入真空干燥机内冻干燥48h，去除模具形成多孔支架，用0.5%京尼平交联；
（5）内层和外层的复合：将内层材料连同轴心套入多孔支架中，向内层和外层间的空隙中注入液态胶原粘合；
（6）细胞种植：将Brdu 标好的骨髓干细胞诱导后的平滑肌样细胞种植于外层的表面，在体外生物反应器中培养3 周，拔出轴心，将转染绿色荧光蛋白的慢病毒载的CD34+诱导后的内皮样细胞种植于内表面，体外继续培养1 周。
实施例3：
一种复合型人工小血管支架的制备方法，包括如下步骤：
（1）内层材料的制备：取新鲜猪心脏，用生理盐水冲洗干净，以心内膜为原料采用去细胞液进行去细胞处理；将去细胞的心内膜浸入含有3 %戊二醛液体作用3h，用双蒸水反复冲洗去残余戊二醛，环氧乙烷消毒备用得到内层去细胞心内膜，去细胞液为0.5%tritonx-100+0.5% SDS+ 150 IU/mL DNase I+100 μg/mL RNase A；
（2）外层材料的制备：取猪的主动脉弹力层组织，并在匀浆器中打碎至小颗粒，在液氮中进行反复研磨后取直径5-30μm的颗粒；用离心机离心加入1%tritonx-100 24小时后，加入去细胞液48 h，用双蒸水反复悬浮、离心去除去细胞液。
（3）取预先制备的直径为3mm 的不锈钢轴，将去细胞心内膜缝制于轴上，长度为3-4cm；
（4）取预先制备的管状模具，将颗粒制成50%体积比的溶液，在-80℃下进行冷冻干燥，去除模具形成多孔支架，用0.5%京尼平交联；
（5）内层和外层的复合：将内层材料连同轴心套入多孔支架中，向内层和外层间的空隙中注入液态胶原粘合；
（6）细胞种植：将Brdu 标好的骨髓干细胞诱导后的平滑肌样细胞种植于外层的表面，在体外生物反应器中培养2 周，拔出轴心，将转染绿色荧光蛋白的慢病毒载的CD34+诱导后的内皮样细胞种植于内表面，体外继续培养2 周。

Claims (7)

1.一种复合型人工小血管支架，其特征在于：所述小血管支架包括内层和外层，所述内层为去细胞心内膜，所述外层为主动脉弹力层微米颗粒多孔支架，所述外层和内层间采用胶原连接：
其制备方法，包括如下步骤：
（1）内层材料的制备：取新鲜猪心脏，用生理盐水冲洗干净，以心内膜为原料采用去细胞液进行去细胞处理；将去细胞的心内膜浸入含有2-3 %戊二醛液体作用3-4h，用双蒸水反复冲洗去残余戊二醛，环氧乙烷消毒备用得到内层去细胞心内膜；
（2）外层材料的制备：取猪的主动脉弹力层组织，并在匀浆器中打碎至小颗粒，在液氮中进行反复研磨后取直径5-30μm的颗粒；用离心机离心加入质量百分比为1%的tritonx-100 处理 12-24小时后，加入去细胞液48-72 h，用双蒸水反复悬浮、离心去除去细胞液；
（3）取预先制备的直径为3mm 的不锈钢轴，将去细胞心内膜缝制于轴上，长度为3-4cm；
（4）取预先制备的管状模具，将颗粒制成30-50%体积比的溶液，将溶液置于管状模具中，依次在-20℃、-80℃下冷冻1~1.5小时，真空干燥36-48小时，去除模具形成多孔支架，用蛋白质交联剂交联；
（5）内层和外层的复合：将内层材料连同轴心套入多孔支架中，向内层和外层间的空隙中注入液态胶原粘合，依次在-20℃、-80℃下冷冻1-1.5小时后真空干燥36-48小时；
（6）细胞种植：将Brdu 标好的骨髓干细胞诱导后的平滑肌样细胞种植于外层的表面，在体外生物反应器中培养2-3 周，拔出轴心，将转染绿色荧光蛋白的慢病毒载的CD34+诱导后的内皮样细胞种植于内表面，体外继续培养1-2 周。
2.如权利要求1所述的复合型人工小血管支架，其特征在于：所述小血管支架的外径为4~4.5mm。
3.如权利要求1所述的复合型人工小血管支架，其特征在于：所述小血管支架的内径为3~3.2mm。
4.如权利要求1述的复合型人工小血管支架，其特征在于：所述蛋白质交联剂为质量分数为0.45-0.55%的京尼平。
5.如权利要求1所述的复合型人工小血管支架，其特征在于：所述步骤（1）中的去细胞液为0.5% tritonx-100+0.5% SDS+ 150 IU/mL DNase I+100 μg/mL RNase A。
6.如权利要求1所述的复合型人工小血管支架，其特征在于：所述液态胶原的浓度为30-60mg/ml。
7.如权利要求1所述的复合型人工小血管支架，其特征在于：所述多孔支架的孔径为25~100μm，孔隙度为70%。
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