CN107376023A - 一种适用于尿道修复重建的支架材料的制备方法 - Google Patents

一种适用于尿道修复重建的支架材料的制备方法 Download PDF

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CN107376023A
CN107376023A CN201710774380.8A CN201710774380A CN107376023A CN 107376023 A CN107376023 A CN 107376023A CN 201710774380 A CN201710774380 A CN 201710774380A CN 107376023 A CN107376023 A CN 107376023A
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彭绪峰
汪继洪
张心如
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Shanghai Sixth Peoples Hospital
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Abstract

本发明提供了一种适用于尿道修复重建的支架材料的制备方法，包括以下步骤：(1)制备大孔径高孔隙率的***脱细胞基质；(2)制备左氧氟沙星PLGA共聚物纳米微囊，将制备的微囊进行重悬，大孔径高孔隙率***脱细胞基质浸入微囊溶液中，静置后干燥处理。本发明将***环切术后废弃的***作为原材料，利用过氧乙酸的氧化性制备了高孔隙率和大孔径的***脱细胞基质，更有利于营养物质的输送和代谢废物的排出以及细胞的长入。制备了能缓释左氧氟沙星的PLGA纳米微囊，并将纳米微囊复合在支架材料上，赋予了支架持续释放左氧氟沙星的抗菌特性，该特性可降低组织工程尿道修复重建术后的感染率，从而降低因***导致的尿道修复重建的失败率。

Description

一种适用于尿道修复重建的支架材料的制备方法
技术领域
本发明属于生物医用材料技术领域，具体涉及一种适用于尿道修复重建的支架材料的制备方法。
背景技术
各种原因导致的复杂性尿道疾病历来是泌尿外科临床中的一个难题。临床上，多种自体组织如：舌黏膜、颊黏膜、结肠黏膜以及膀胱黏膜等已被成功的应用于尿道狭窄的修复重建。然而供体材料的有限性以及供体部位的并发症(如疼痛、唾液腺损伤、麻木等)限制了这些方法的应用。组织工程技术的发展有望成为一种新型且有效的修复方法。目前，国内外文献报道中各种组织工程材料在尿道修复重建中的研究已经逐步从实验室走向临床；其中不乏有小范围实际尿道狭窄临床治疗的案例，并且获得比较令人满意的初步疗效。
相对于种子细胞研究的日趋完善，在尿道组织工程修复重建中选择何种支架材料最为理想却始终未能达成共识。目前在尿道组织工程中主要采用两大类支架：人工合成材料和脱细胞基质材料。前者虽然适合规模化生产，但缺乏细胞识别信号，不利于细胞的识别与粘附，而且降解产物可引起周围组织的无菌性炎症反应；后者虽然制备方便，但由于多取材于异种组织，因而在生物安全性方面一直受到部分学者质疑，很难被患者所接受，而且其孔隙率、孔径大小并不完全符合尿道修复重建的要求。另一方面，尿道重建修复术后需常规放置导尿管2到4周以引流尿液及支撑修复处的组织；根据文献报道，导尿管的放置极大的增加了***的风险，而***可导致修复重建的失败。既往报道使用的支架材料都无抗菌特性。基于上述背景，目前在该研究领域，制备高孔隙率、大孔径且拥有抗菌特点的材料日益受到关注。现有研究中，已有利用尸体真皮做成脱细胞基质材料应用于尿道组织工程修复重建。但同其他传统脱细胞基质材料一样其较小的孔径及较低的孔隙率影响了微血管的长入、营养物质的补给以及代谢废物的运出；而这一问题也是导致利用组织工程材料进行尿道重建术后出现狭窄复发的主要原因之一。同时由于其缺乏抗菌特性，尿道重建术后需长时间放置导尿管，所以***的高发生率也降低了尿道修复重建的成功率；此外,仅将传统剂型的左氧氟沙星(为***首选抗生素)加入移植物环境中并不能达到有效持久的抗菌目的。
发明内容
为了克服上述现有技术的不足，本发明提供了一种可用于尿道组织工程的具有抗菌特性和高孔隙率以及大孔径***脱细胞基质支架的制备方法。
为达到上述目的，具体技术方案如下：
一种适用于尿道修复重建的支架材料的制备方法，包括以下步骤：
步骤1、制备大孔径高孔隙率的***脱细胞基质：
a.收集经***环切术后废弃的***，清水洗净，蒸馏水浸泡并置于恒温振荡器内振荡48小时，振荡频率为125r/min，温度为25℃；
b.将清洗振荡后的***置于5％的过氧乙酸中4-6个小时，利用其氧化性去除有免疫原性的细胞成分，并氧化打断胶原从而增加孔隙率和增大孔径；
c.将氧化后的***用蒸馏水洗净，加入500mL含1％Triton X-100和0.1％氨水中14天进一步脱去细胞成分；同时置于恒温振荡器内振荡，振荡频率为125r/min，温度为25℃；后用蒸馏水洗净，冷冻干燥机将上述***脱细胞基质冻干处理并封装，CO60γ射线辐射灭菌，4℃保存；
步骤2、制备左氧氟沙星PLGA共聚物纳米微囊，将制备的微囊进行重悬，大孔径高孔隙率***脱细胞基质浸入微囊溶液中，静置后干燥处理；
a.秤取左氧氟沙星8mg，PLGA 80mg，将两者溶解在2mL的二氯甲烷中；
b.待上述溶剂充分溶解后，倒入n mL的含1％的聚乙烯醇水溶液中，在冰水浴溶液中涡旋1min，静置5min，再在200W功率条件下超声乳化1min，之后加入m mL的的含1％聚乙烯醇的水溶液室温下磁力低速300r/min搅拌过夜，通过挥发去除二氯甲烷，其中n：m＝3:5～12，通过调整n、m的比例可满足左氧氟沙星载药量达到较高的同时包封率较高且缓释性能达到最佳；
c.将上述含纳米微囊的悬液于11000r/min下高速离心30min收集纳米粒，以蒸馏水反复洗涤、冻干、CO60γ射线辐射灭菌，制得左氧氟沙星PLGA纳米微囊；
d.重悬步骤2中步骤c得到的左氧氟沙星PLGA纳米微囊，将制得的大孔径高孔隙率***脱细胞基质浸入纳米微囊溶液中10h，静置后干燥处理。
一种本发明所述的制备方法制备的适用于尿道修复重建的支架材料。
有益效果：本发明提供了一种适用于尿道修复重建的支架材料的制备方法，1、本发明将***环切术后废弃的***作为原材料，利用过氧乙酸的氧化性制备了高孔隙率和大孔径的***脱细胞基质，相较于其它异种真皮脱细胞基质，这种同种异体真皮脱细胞基质免疫原性更低，生物安全性更高，其大孔径及高孔隙率更有利于营养物质的输送和代谢废物的排出以及细胞的长入。2、本发明制备了能缓释左氧氟沙星的PLGA纳米微囊，并将纳米微囊复合在支架材料上，赋予了支架持续释放左氧氟沙星的抗菌特性，该特性可降低组织工程尿道修复重建术后的感染率，从而降低了因***导致的尿道修复重建的失败率。
附图说明
图1本发明的制备的***脱细胞基质实物图的疏松面。
图2本发明的可缓释左氧氟沙星的PLGA纳米微囊的扫描电镜图。
图3本发明的复合左氧氟沙星PLGA纳米微囊的***脱细胞基质的扫描电镜图。
图4正常兔子尿道的HE染色图片(×200)。
图5纯***脱细胞基质材料修复兔尿道感染模型的HE染色图片(×200)。
图6复合左氧氟沙星PLGA纳米微囊后的***脱细胞基质修复兔尿道感染模型的HE染色图片(×200)。
具体实施方式
为使本领域技术人员更全面地理解本发明，以下结合实施例和附图对本发明的技术方案进行说明，但不以任何方式限制本发明。
一种适用于尿道修复重建的支架材料的制备方法，包括以下步骤：
步骤1、制备大孔径高孔隙率的***脱细胞基质：
a.泌尿外科门诊收集经***环切术后废弃的***，清水洗净，蒸馏水浸泡并置于恒温振荡器内振荡48小时，振荡频率为125r/min，温度为25℃；
b.将清洗振荡后的***置于5％的过氧乙酸中4个小时，利用其氧化性去除有免疫原性的细胞成分，并氧化打断胶原从而增加孔隙率和增大孔径；
c.将氧化后的***用蒸馏水洗净，加入500mL含1％Triton X-100和0.1％氨水中14天进一步脱去细胞成分；同时置于恒温振荡器内振荡，振荡频率为125r/min，温度为25℃。后用蒸馏水洗净，冷冻干燥机将上述真皮脱细胞基质冻干处理并封装，CO60γ射线辐射灭菌，4℃保存，如图1所示，***脱细胞基质表面疏松，可见许多大孔径的孔隙。
步骤2、制备左氧氟沙星PLGA(聚乳酸-羟基乙酸共聚物)纳米微囊，将制备的微囊进行重悬，大孔径高孔隙率***脱细胞基质浸入微囊溶液中，静置后干燥处理。
a.秤取左氧氟沙星8mg，PLGA 80mg，将两者溶解在2mL的二氯甲烷中；
b.待上述溶剂充分溶解后，倒入3mL的含1％的聚乙烯醇水溶液中，在冰水浴溶液中涡旋1min，静置5min，再超声(200W)乳化1min，之后加入5mL的的含1％聚乙烯醇的水溶液室温下磁力低速(300r/min)搅拌过夜，通过挥发去除二氯甲烷；
c.将上述含纳米微囊的悬液于11000r/min下高速离心30min收集纳米粒，以蒸馏水反复洗涤、冻干、CO60γ射线辐射灭菌，制得左氧氟沙星PLGA纳米微囊，如图2所示；
d.通过高效液相色谱法测得其其包封率为88.9±0.5％，载药量为13.2±0.6％；可持续释放左氧氟沙星达14天；
e.重悬最优条件下的左氧氟沙星PLGA纳米微囊，将制得的大孔径高孔隙率***脱细胞基质浸入纳米微囊溶液中10h，静置后干燥处理得到复合后的支架材料，如图3所示。
f.依据文献报道建立兔***模型，去除部分尿道组织，将复合有左氧氟沙星PLGA纳米微囊的***脱细胞基质和单纯的材料分别缝合在缺损组织处，7日后处死兔子，并将其尿道回收行HE染色，正常尿道未见单核巨噬细胞(见图4)；单纯***脱细胞基质材料的尿道有大量炎症细胞浸润(单核巨噬细胞)，炎症反应较重(见图5)；而复合有左氧氟沙星PLGA纳米微囊的***脱细胞基质只有极少的炎症细胞浸润(见图6)，说明复合左氧氟沙星PLGA纳米微囊后的***脱细胞基质因能持续释放左氧氟沙星，故而拥有良好的抗菌性能。

Claims (2)

1.一种适用于尿道修复重建的支架材料的制备方法，其特征在于，包括以下步骤：
步骤1、制备大孔径高孔隙率的***脱细胞基质：
a.收集经***环切术后废弃的***，清水洗净，蒸馏水浸泡并置于恒温振荡器内振荡48小时，振荡频率为125r/min，温度为25℃；
b.将清洗振荡后的***置于5％的过氧乙酸中4-6小时进行氧化；
c.将氧化后的***用蒸馏水洗净，加入500mL含1％Triton X-100和0.1％氨水中14天进一步脱去细胞成分；同时置于恒温振荡器内振荡，振荡频率为125r/min，温度为25℃；后用蒸馏水洗净，冷冻干燥机将上述***脱细胞基质冻干处理并封装，CO60γ射线辐射灭菌，4℃保存；
步骤2、制备左氧氟沙星PLGA共聚物纳米微囊，将制备的微囊进行重悬，大孔径高孔隙率***脱细胞基质浸入微囊溶液中，静置后干燥处理；
a.秤取左氧氟沙星8mg，PLGA 80mg，将两者溶解在2mL的二氯甲烷中；
b.待上述溶剂充分溶解后，倒入n mL的含1％的聚乙烯醇水溶液中，在冰水浴溶液中涡旋1min，静置5min，再在200W功率条件下超声乳化1min，之后加入m mL的的含1％聚乙烯醇的水溶液室温下磁力低速300r/min搅拌过夜，通过挥发去除二氯甲烷，其中n：m＝3:5～12，通过调整n、m的比例可满足左氧氟沙星载药量达到较高的同时包封率较高且缓释性能达到最佳；
c.将上述含纳米微囊的悬液于11000r/min下高速离心30min收集纳米粒，以蒸馏水反复洗涤、冻干、CO60γ射线辐射灭菌，制得左氧氟沙星PLGA纳米微囊；
d.重悬步骤2中步骤c得到的左氧氟沙星PLGA纳米微囊，将步骤1制得的大孔径高孔隙率***脱细胞基质浸入纳米微囊溶液中10h，静置后干燥处理。
2.一种权利要求1所述的制备方法制备的适用于尿道修复重建的支架材料。
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