CN116459393A - 一种负载鹿茸多肽的阵列微管仿生神经支架的制备及应用 - Google Patents
一种负载鹿茸多肽的阵列微管仿生神经支架的制备及应用 Download PDFInfo
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Abstract
本发明公开了一种负载鹿茸多肽的阵列微管仿生神经支架的制备及应用。所述负载鹿茸多肽的阵列微管仿生神经支架是以PLGA、Ⅰ型牛腱胶原、Ⅲ型人源重组胶原和鹿茸多肽为原料制备而成,结构包括内含负载鹿茸多肽的PLGA微球的神经支架填充层和神经支架外层。本发明制备的负载鹿茸多肽的阵列微管仿生神经支架综合了材料仿生、结构仿生和外源性生长因子三个方面,具有生物相容性良好、可降解、免疫原性低等特点,结合支架内部阵列微管能为神经细胞粘附和增殖提供适宜微环境,并通过持续释放生长因子,加快了神经修复总进程,对周围神经缺损大鼠模型有很好的修复用,是一种符合临床需求的神经修复支架材料。
Description
技术领域
本发明属于生物医用技术领域,具体涉及一种负载鹿茸肽的阵列微管仿生神经支架制备及其应用方法。
背景技术
周围神经遍布人体的各个部位,主要起控制人体运动、感觉等功能。周围神经损伤是指周围神经干或分支受到了外界直接或间接的伤害而导致躯干和肢体的运动和感觉以及自主神经功能障碍的病症。外伤、压迫、感染、缺血、肿瘤和营养代谢障碍等诸多因素均会导致周围神经损伤。此外,随着社会经济的发展,运动损伤、建筑事故、交通事故、战争以及自然灾害等亦增加了神经损伤发生的几率。因此,寻求有效治疗周围神经损伤的方法具有重大的临床意义。目前,通过组织工程治疗周围神经损伤成为国内外研究的热点,因而,制备组织工程人工神经支架来修复周围神经损伤具有较大的临床意义。
胶原蛋白是哺乳动物中最丰富的蛋白质,作为结构蛋白为所有组织提供机械稳定性。同时胶原蛋白也是外周神经细胞外基质(ECM)的主要成分,在细胞分化、附着、迁移、存活和增殖中发挥作用。型和Ш型胶原都存在于神经外膜、神经束膜和神经内膜中,型胶原主要分布于神经外膜,Ш型胶原主要分布于神经束膜和神经内膜。有研究表明/Ш型胶原神经导管可作为潜在的神经引导基质,通过扩大神经间隙来保证神经再生。
PLGA由聚乳酸(PLA)和聚乙醇酸(PGA)随机聚合而成,是一种可降解的功能高分子有机化合物。它具有良好的生物相容性、无毒、良好的成囊和成膜性能。因此被广泛应用于组织工程支架材料、手术缝合线、药物缓释载体、医疗缝合补强材料和骨折内固定材料等,并已被美国食品药品监督管理局批准临床应用。同时可通过调整LA与GA比例来调节PLGA降解时间、机械强度和疏水性,这相比与PGA、PLA等组织工程中常用的高分子有机化合物具有很大的优势,使得PLGA成为周围神经修复领域中极具吸引力的材料。
鹿茸多肽是鹿茸再生发育过程中表达的功能短肽,是鹿茸中主要生物活性成分。鹿茸多肽中富含胰岛素样生长因子(IGF)、表皮生长因子(EGF)和神经生长因子(NGF)等各种细胞生长因子,其中IGF和EGF属于神经营养因子,能在神经修复过程中提供营养,与NGF协同促进轴突生长。研究表明,鹿茸多肽能从抗氧化应激、抑制细胞凋亡、提供多种生长因子和提供修复所需的微环境等多方面保护神经细胞和促神经细胞增殖分化。同时其提取技术成熟,来源丰富,因此在促进周围神经修复方面具有良好的发展前景。
天然神经结构具备三层膜,能够支持和保护神经纤维,分离神经纤维成束且使神经纤维沿轴突方向定向排列,因此制备的人工神经导管应能起到引导再生神经方向、将再生的轴突与瘢痕组织分离和保护再生神经免受周围压迫作用。填充式结构可增大导管内比表面积,模拟细胞外基质的微环境,利于纤维方向性生长,也可增加细胞黏附位点,利于细胞迁移和增殖,更能有效增强神经导管机械性能;定向阵列微管结构可模拟神经束膜结构,有助于雪旺细胞有序排列从而引导神经定向生长,并明显降低再生纤维的分散性。结合填充式结构和阵列微管通道结构的优势,从不同的角度模拟神经的自然室间结构有望提高神经支架的理化性能和促神经修复效率。
在前期工作中(王营营,负载鹿茸多肽的/Ш胶原基复合支架调控软骨内源性修复的研究[D],福州大学,2019),研究发现负载鹿茸多肽的/Ш胶原基复合支架具有良好的生物相容性,对软骨损伤具有极好的修复作用,并能有效引导内源性骨髓间充质干细胞参与修复。在此基础上,结合上述“Ⅰ型胶原主要分布于神经外膜,Ш型胶原主要分布于神经束膜和神经内膜”,我们认为如果进一步以Ⅰ型和Ш胶原为基质材料,改变其3D网络化孔隙结构为平行阵列微管结构,并复合鹿茸多肽中多种活性因子尤其NGF作用,结合外覆PLGA制备的神经外膜,将会得到一种能有效修复周围神经缺损的神经支架材料。
因此,本发明综合了材料仿生、结构仿生和外源性生长因子三个方面的优势,制备了一种负载鹿茸多肽的阵列微管仿生神经支架。
发明内容
本发明的目的在于提供一种负载鹿茸多肽的阵列微管仿生神经支架的制备及应用,所述负载鹿茸多肽的阵列微管仿生神经支架具有良好的生物相容性、机械性能强、免疫原性低、生物可降解等优点,能显著提升周围神经修复的速度与质量,是一种较好的适用于周围神经损伤修复的生物医用材料。
为实现上述目的,本发明采用以下技术方案:
一种负载鹿茸多肽的阵列微管仿生神经支架的制备方法,其包括如下步骤:
(1)将Ⅰ型牛腱胶原蛋白溶于乙酸溶液中得到COLⅠ溶液,将Ⅲ型人源重组胶原蛋白溶于乙酸溶液中得到COLⅢ溶液;
(2)将步骤(1)得到的COLⅠ溶液和COLⅢ溶液混合,得到COLⅠ/Ⅲ溶液;
(3)制备负载鹿茸多肽的PLGA微球;
(4)将(3)中负载鹿茸多肽的PLGA微球以物理混合的方式加入到步骤(2)中的COLⅠ/Ⅲ溶液中,磁力搅拌过夜后灌注至软管中,再以1cm/min的速度将软管垂直浸入液氮中进行冷冻,而后取出,剖除外部软管,冷冻干燥,得到支架填充层;
(5)将步骤(4)中的支架填充层浸入交联剂中进行交联反应,交联后用去离子水清洗,冷冻干燥,得到交联后的支架填充层;
(6)将步骤(5)交联后的支架填充层装填至PLGA导管中,得到负载鹿茸多肽的阵列微管仿生神经支架。
所述步骤(1)中,COLⅠ溶液中Ⅰ型牛腱胶原蛋白的质量分数为0.8%,COLⅢ溶液中Ⅲ型人源重组胶原蛋白的质量分数为0.8%。
所述步骤(2)中,COLⅠ溶液和COLⅢ溶液的质量比为7:3、6:4、5:5、3:7、4:6中的任意一种。
所述步骤(3)具体如下:
1)将0.12g分子量为5万、乳酸和羟基乙酸比例为75:25的PLGA固体溶于2.4mL二氯甲烷中,得到PLGA溶液;
2)将1g醇解度为99%、聚合度为1700的PVA固体加入至100mL去离子水中,于95℃水浴锅中搅拌加热至充分溶解,得到PVA溶液;
3)将0.5g鹿茸多肽粉末溶于100mLPBS缓冲液中,在磁力搅拌器上充分搅拌,得到VAPs溶液;
4)向2.4mLPLGA溶液中加入1.2mLVAPs溶液,于细胞破碎仪中以超声功率200W、探头振幅40%在冰浴条件下超声3min使溶液乳化,得到初乳;
5)将3.6mL初乳快速倒入18mLPVA溶液中,于细胞破碎仪中以超声功率200W、探头振幅40%在冰浴条件下超声3min,得到复乳;
6)将得到的复乳在磁力搅拌器上300r/min搅拌6h,再于4℃、5000rpm离心6min,固相用去离子水洗涤,而后冷冻干燥,得到负载鹿茸多肽的PLGA微球。
所述步骤(4)中,负载鹿茸多肽的PLGA微球与COLⅠ/Ⅲ溶液的比例为1.5mg/cm3。
所述步骤(5)中,交联剂为将NHS和EDC溶于90vol%乙醇溶液中制得,所述交联剂中NHS和EDC的含量分别为50mmol/L、20mmol/L。
一种如上述制备方法制得的负载鹿茸多肽的阵列微管仿生神经支架。
上述一种负载鹿茸多肽的阵列微管仿生神经支架在制备修复周围神经损伤的产品中的应用。
本发明的显著优点在于:
(1)本发明中制备的负载鹿茸多肽的阵列微管神经支架具备定向阵列微管通道结构,通道直径较适合神经细胞的生长,同时具备孔隙率高、吸水性强、溶失率低、生物相容性良好、生物可降解和免疫原性低等优点。
(2)本发明的制备方法简便易操作,原材料价廉易得。
(3)本发明所制备的修复支架可用于周围神经损伤的修复,在动物实验中已经证明了其具有良好的周围神经缺损修复效果。
附图说明
图1:负载鹿茸多肽的阵列微管神经支架填充层扫描电镜图。A~E分别代表实施例1~5。
图2:负载鹿茸多阵列微管仿生神经支架的孔隙率测试。
图3:负载鹿茸多阵列微管仿生神经支架的溶胀倍数测试。
图4:负载鹿茸多阵列微管仿生神经支架的热溶失率测试。
图5:负载鹿茸多肽的阵列微管仿生神经支架的体外细胞试验。
图6:负载鹿茸多肽的阵列微管仿生神经支架修复周围神经缺损动物模型试验。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是下述的实例仅仅是本发明其中的例子而已,并不代表本发明所限定的权利保护范围,本发明的权利保护范围以权利要求书为准。
实施例1
一种负载鹿茸多肽的阵列微管仿生神经支架的制备方法,具体步骤如下:
(1)取80g质量分数为1.6%的Ⅰ型牛腱胶原蛋白(COLⅠ,购买于福建省博特生物科技有限公司,货号C20200106),加入48mL去离子水和32mL0.02moL/L冰醋酸溶液,在磁力搅拌器上充分搅拌4h,得到COLⅠ溶液;将0.25gШ型人重组胶原蛋白(COLШ,购买于赛尔瑞成(北京)生命科学技术有限公司,货号PMB011)干粉溶于31.25mL0.02moL/L的醋酸溶液中,在磁力搅拌器上充分搅拌4h,得到COLШ溶液。
(2)将步骤(1)中得到的COLⅠ溶液和COLШ溶液按照5:5的质量比混合,在磁力搅拌器上充分搅拌5h,得到COLⅠ/Ш溶液。
(3)将0.12g分子量为5万、乳酸和羟基乙酸比例为75:25的PLGA固体溶于2.4mL二氯甲烷中,得到PLGA溶液;将1g醇解度为99%、聚合度为1700的PVA固体加入至100mL去离子水中,于95℃水浴锅中搅拌加热至充分溶解,得到PVA溶液;将0.5g鹿茸多肽(VAPs,购买于浙江海宁市紫金港生物科技有限公司,货号:ZJG20200103)粉末溶于100mL0.01M、pH7.4的PBS缓冲液中,在磁力搅拌器上充分搅拌,得到VAPs溶液;向2.4mLPLGA溶液中加入1.2mLVAPs溶液,于细胞破碎仪中以超声功率200W、探头振幅40%在冰浴条件下超声3min使溶液乳化,得到初乳;将3.6mL初乳快速倒入18mL PVA溶液中,于细胞破碎仪中以超声功率200W、探头振幅40%在冰浴条件下超声3min,得到复乳;将得到的复乳在磁力搅拌器上300r/min搅拌6h,再于4℃、5000rpm离心6min,固相用去离子水洗涤5~6次、每次5min,而后冷冻干燥,得到包载VAPs的PLGA微球。
(4)将步骤(3)中包载VAPs的PLGA微球按1.5mg/cm3的比例加入到步骤(2)中的COLⅠ/Ш溶液中,4℃搅拌过夜,得到混合PLGA微球的COLⅠ/Ш溶液。
(5)将步骤(4)中混合PLGA微球的COLⅠ/Ш溶液灌注至直径为5mm、长为5cm的聚苯乙烯材质软管中,再以1cm/min的速度将软管垂直浸入-196℃液氮中,冷冻24h后取出,剖除外部软管,冷冻干燥,得到支架填充层。
(6)将9.585g1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC)、2.301gN-羟基硫代琥珀酰亚胺(NHS)粉末溶于100mL体积分数为90%的乙醇溶液中,得到交联剂,再将步骤(5)中的支架填充层浸没于交联剂中,避光交联9h,而后用去离子水清洗6次、每次30min,冷冻干燥,得到交联后的支架填充层。
(7)将1g分子量为5万、乳酸和羟基乙酸比例为50:50的PLGA固体溶于10mL二氯甲烷中,均匀滴加在规格为2cm×5cm的洁净的玻璃板上保持水平让其自然展开铺满,然后置于通风橱中让溶剂自然挥发过夜,脱膜,得到PLGA膜状材料。
(8)将PLGA膜状材料卷成管状,用少许二氯甲烷粘合,制备成PLGA导管,PLGA导管的直径为5mm、长为50mm、管壁厚度为1mm。
(9)将步骤(6)交联后的支架填充层装填到步骤(8)中的PLGA导管中,得到负载鹿茸多肽的阵列微管仿生神经支架(记为神经支架A)。
实施例2
一种负载鹿茸多肽的阵列微管仿生神经支架的制备方法,具体步骤与实施例1基本相同,不同之处在于:步骤(2)中COLⅠ溶液和COLШ溶液按照7:3的质量比混合。将得到的阵列微管仿生神经支架记为神经支架B。
实施例3
一种负载鹿茸多肽的阵列微管仿生神经支架的制备方法,具体步骤与实施例1基本相同,不同之处在于:步骤(2)中COLⅠ溶液和COLШ溶液按照6:4的质量比混合。将得到的阵列微管仿生神经支架记为神经支架C。
实施例4
一种负载鹿茸多肽的阵列微管仿生神经支架的制备方法,具体步骤与实施例1基本相同,不同之处在于:步骤(2)中COLⅠ溶液和COLШ溶液按照3:7的质量比混合。将得到的阵列微管仿生神经支架记为神经支架D。
实施例5
一种负载鹿茸多肽的阵列微管仿生神经支架的制备方法,具体步骤与实施例1基本相同,不同之处在于:步骤(2)中COLⅠ溶液和COLШ溶液按照4:6的质量比混合。将得到的阵列微管仿生神经支架记为神经支架E。
实施例6
一种阵列微管仿生神经支架的制备方法,具体步骤如下:
(1)取80g质量分数为1.6%的Ⅰ型牛腱胶原蛋白(COLⅠ),加入48mL去离子水和32mL0.02moL/L冰醋酸溶液,在磁力搅拌器上充分搅拌4h,得到COLⅠ溶液;将0.25gШ型人重组胶原蛋白(COLШ)干粉溶于31.25mL0.02moL/L的醋酸溶液中,在磁力搅拌器上充分搅拌4h,得到COLШ溶液。
(2)将步骤(1)中得到的COLⅠ溶液和COLШ溶液按照5:5的质量比混合,在磁力搅拌器上充分搅拌5h,得到COLⅠ/Ш溶液。
(3)将步骤(2)中的COLⅠ/Ш溶液灌注至直径为5mm、长为5cm的聚苯乙烯材质软管中,再以1cm/min的速度将软管垂直浸入-196℃液氮中,冷冻24h后取出,剖除外部软管,冷冻干燥,得到支架填充层。
(4)将9.585g1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC)、2.301gN-羟基硫代琥珀酰亚胺(NHS)粉末溶于100mL体积分数为90%的乙醇溶液中,得到交联剂,再将步骤(3)中的支架填充层浸没于交联剂中,避光交联9h,而后用去离子水清洗6次、每次30min,冷冻干燥,得到交联后的支架填充层。
(5)将1g分子量为5万、乳酸和羟基乙酸比例为50:50的PLGA固体溶于10mL二氯甲烷中,均匀滴加在规格为2cm×5cm的洁净的玻璃板上保持水平让其自然展开铺满,然后置于通风橱中让溶剂自然挥发过夜,脱膜,得到PLGA膜状材料。
(6)将PLGA膜状材料卷成管状,用少许二氯甲烷粘合,制备成PLGA导管,PLGA导管的直径为5mm、长为50mm、管壁厚度为1mm。
(7)将步骤(4)交联后的支架填充层装填到步骤(6)中的PLGA导管中,得到阵列微管仿生神经支架(记为神经支架F)。
实施例7
一种阵列微管仿生神经支架的制备方法,具体步骤如下:
(1)取80g质量分数为1.6%的Ⅰ型牛腱胶原蛋白(COLⅠ),加入48mL去离子水和32mL0.02moL/L冰醋酸溶液,在磁力搅拌器上充分搅拌4h,得到COLⅠ溶液;将0.25gШ型人重组胶原蛋白(COLШ)干粉溶于31.25mL0.02moL/L的醋酸溶液中,在磁力搅拌器上充分搅拌4h,得到COLШ溶液。
(2)将步骤(1)中得到的COLⅠ溶液和COLШ溶液按照5:5的质量比混合,在磁力搅拌器上充分搅拌5h,得到COLⅠ/Ш溶液。
(3)将0.12g分子量为5万、乳酸和羟基乙酸比例为75:25的PLGA固体溶于2.4mL二氯甲烷中,得到PLGA溶液;将1g醇解度为99%、聚合度为1700的PVA固体加入至100mL去离子水中,于95℃水浴锅中搅拌加热至充分溶解,得到PVA溶液;向2.4mL PLGA溶液中加入1.2mL1ng/mL的神经生长因子(NGF)溶液,于细胞破碎仪中以超声功率200W、探头振幅40%在冰浴条件下超声3min使溶液乳化,得到初乳;将3.6mL初乳快速倒入18mLPVA溶液中,于细胞破碎仪中以超声功率200W、探头振幅40%在冰浴条件下超声3min,得到复乳;将得到的复乳在磁力搅拌器上300r/min搅拌6h,再于4℃、5000rpm离心6min,固相用去离子水洗涤5~6次、每次5min,而后冷冻干燥,得到包载NGF的PLGA微球。
(4)将步骤(3)中包载NGF的PLGA微球按1.5mg/cm3的比例加入到步骤(2)中的COLⅠ/Ш溶液中,4℃搅拌过夜,得到混合PLGA微球的COLⅠ/Ш溶液。
(5)将步骤(4)中混合PLGA微球的COLⅠ/Ш溶液灌注至直径为5mm、长为5cm的聚苯乙烯材质软管中,再以1cm/min的速度将软管垂直浸入-196℃液氮中,冷冻24h后取出,剖除外部软管,冷冻干燥,得到支架填充层。
(6)将9.585g1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC)、2.301gN-羟基硫代琥珀酰亚胺(NHS)粉末溶于100mL体积分数为90%的乙醇溶液中,得到交联剂,再将步骤(5)中的支架填充层浸没于交联剂中,避光交联9h,而后用去离子水清洗6次、每次30min,冷冻干燥,得到交联后的支架填充层。
(7)将1g分子量为5万、乳酸和羟基乙酸比例为50:50的PLGA固体溶于10mL二氯甲烷中,均匀滴加在规格为2cm×5cm的洁净的玻璃板上保持水平让其自然展开铺满,然后置于通风橱中让溶剂自然挥发过夜,脱膜,得到PLGA膜状材料。
(8)将PLGA膜状材料卷成管状,用少许二氯甲烷粘合,制备成PLGA导管,PLGA导管的直径为5mm、长为50mm、管壁厚度为1mm。
(9)将步骤(6)交联后的支架填充层装填到步骤(8)中的PLGA导管中,得到阵列微管仿生神经支架(记为神经支架G)。
实施例8
实施例1~5中阵列微管神经支架的填充层的扫描电镜图见图1。由图中可以看出,5种神经支架填充层均具有定向阵列微管结构;COLⅠ与COLШ的比例对神经支架填充层微观结构存在一定的影响,随COLШ比例不断增加,神经支架填充层内部结构越来越紧凑,阵列微管直径越来越小,微管数量随之提升,这可能与COLШ结构有关,COLШ为细长型纤维;在5种神经支架填充层中,B组支架填充层的微管孔径约在15~20μm范围内,这很适合雪旺细胞(schwann,又名施万细胞)沿着微管通道定向排列,从而引导神经纤维从断裂近端向远端伸长。
实施例9
将实施例1~5中制备的阵列微管神经支架分别称重记为W,然后浸没于5mL无水乙醇中,37℃温水浴24h,而后将支架取出用滤纸吸去其表面的多余水分,再称其重量记为W1。按如下公式计算其孔隙率:
孔隙率(%)=4(W1-W)/ρπd2h×100%,
式中W为支架干重(mg),W1为吸乙醇后支架湿重(mg),ρ为乙醇的密度,d为支架直径,h为支架高度。
结果如图2所示,实施例1~5中制备的阵列微管神经支架的孔隙率都很高,其中以实施例4制备的阵列微管神经支架孔隙率最高、其孔隙率高达76.99±5.84%。
实施例10
将实施例1~5中制备的阵列微管神经支架分别称重记为W1,然后浸入5mL0.01M、pH7.4的PBS溶液中,37℃温水浴24h,而后将支架取出用滤纸吸去其表面的多余水分,再称其重量记为W。按如下公式计算其溶胀倍数:
吸水溶胀率(%)=(W-W1)/W1×100%,
式中W1为材料干重(mg),W为溶胀后材料湿重(mg)。
结果如图3所示,实施例1~5中制备的阵列微管神经支架的溶胀倍数均超过50倍,表明其吸水能力均很强。
实施例11
将实施例1~5中制备的阵列微管神经支架分别称重记为M1,然后浸入装有4mLPBS溶液中的离心管中,用封口膜封口后置于恒温摇床(37℃,120r/min)振荡48h,而后将支架取出用超纯水清洗3~5次,冷冻干燥,再称其质量记为M2。按如下公式计算其热溶失率:
热溶失率(%)=(M1-M2)/M2×100%,
式中M1为材料干重(mg),M2为溶失后材料干重(mg)。
结果如图4所示,实施例1~5中制备的阵列微管神经支架在37℃条件下均有一定的质量损失,其中实施例4制备的阵列微管神经支架的热水溶失率最高,达到3.28±0.28%,且与其他四组均有显著性差异(P<0.5)。
实施例12
将实施案例3中神经支架C和实施案例7中神经支架G分别置于48孔板内。将对数期NIH/737细胞制备成细胞悬液并计数,使用1mL一次性注射器将细胞接种到神经支架内部(103个/孔),并补充适量的培养基和血清,于恒温培养箱中培养。分别在培养1,3,5天后吸弃培养基,并使用PBS清洗3遍,尽可能洗去支架内残留培养基。再使用4%多聚甲醛于4℃固定24h,固定细胞形态。采用免疫荧光染色和扫描电镜观察细胞在支架内增殖和粘附状况。
结果如图5所示,神经支架G和神经支架C均能促进NIH/737细胞的增殖和粘附。
实施例13
手术操作:将45只SD大鼠随机分为5组,每组9只。分组后按0.1mL/100g剂量腹腔注射戊巴比妥钠溶液,使大鼠麻醉。大鼠剃毛后将其四肢固定在木板上,使大鼠在手术中维持俯卧姿势。在大鼠左侧用手指感受腹股沟位置,70%酒精消毒,切开皮肤,钝性分离,分开后可见一条白色较粗的神经,即为坐骨神经。用剪刀将暴露出来的坐骨神经去除0.8cm,构建坐骨神经缺损模型。空白对照组(A组):不做任何处理;模型对照组(B组):缺损处不做任何处理;实验组(C组):缺损处移植实施案例6中神经支架F;实验组(D组):缺损处移植实施案例7中神经支架G组;实验组(E组):缺损处移植实施案例3中神经支架C。移植时,将神经支架使用γ射线灭菌后,使用11-0可吸收手术缝线将其缝合在神经断端处,然后逐层缝合肌肉和皮肤(分别使用5-0可吸收手术缝线和2-0不可吸收缝线),手术结束后每天注射一次青霉素防止伤口感染(5万U/只),共注射7天。分别于术后第4、8、12周将大鼠处死,取其坐骨神经进行组织观察、甲苯胺蓝(TB)染色、劳克坚牢蓝(LFB)染色。
TB染色结果如图6所示。神经中尼龙氏小体能被TB染成蓝色,同时SCs细胞被染成深蓝色,轴突不着色或浅蓝色。从图中可以看出,与B组相比,C、D、E组对坐骨神经的修复效果均较优异,但D、E两组较C组的修复效果要更好;同时与A组相比,至12周时,C、D两组的组织状况与A组组织状况相似,表明C、D两组均能有效提高坐骨神经修复的速度与质量。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (8)
1.一种负载鹿茸多肽的阵列微管仿生神经支架的制备方法,其特征在于:包括如下步骤:
(1)将Ⅰ型牛腱胶原蛋白溶于乙酸溶液中得到COL Ⅰ溶液,将Ⅲ型人源重组胶原蛋白溶于乙酸溶液中得到COL Ⅲ溶液;
(2)将步骤(1)得到的COL Ⅰ溶液和COL Ⅲ溶液混合,得到COL Ⅰ/Ⅲ溶液;
(3)制备负载鹿茸多肽的PLGA微球;
(4)将步骤(3)中负载鹿茸多肽的PLGA微球以物理混合的方式加入到步骤(2)中的COLⅠ/Ⅲ溶液中,磁力搅拌过夜后灌注至软管中,再以1 cm/min的速度将软管垂直浸入液氮中进行冷冻,而后取出,剖除外部软管,冷冻干燥,得到支架填充层;
(5)将步骤(4)中的支架填充层浸入交联剂中进行交联反应,交联后用去离子水清洗,冷冻干燥,得到交联后的支架填充层;
(6)将步骤(5)交联后的支架填充层装填至PLGA导管中,得到负载鹿茸多肽的阵列微管仿生神经支架。
2.根据权利要求1所述的制备方法,其特征在于:所述步骤(1)中,COL Ⅰ溶液中Ⅰ型牛腱胶原蛋白的质量分数为0.8%,COL Ⅲ溶液中Ⅲ型人源重组胶原蛋白的质量分数为0.8%。
3.根据权利要求1所述的制备方法,其特征在于:所述步骤(2)中,COL Ⅰ溶液和COL Ⅲ溶液的质量比为7:3、6:4、5:5、3:7、4:6中的任意一种。
4.根据权利要求1所述的制备方法,其特征在于:所述步骤(3)具体如下:
1)将0.12 g分子量为5万、乳酸和羟基乙酸比例为75:25的PLGA固体溶于2.4 mL二氯甲烷中,得到PLGA溶液;
2)将1 g醇解度为99%、聚合度为17000的PVA固体加入至100 mL去离子水中,于95℃水浴锅中搅拌加热至充分溶解,得到PVA溶液;
3)将0.5 g鹿茸多肽粉末溶于100 mL PBS缓冲液中,在磁力搅拌器上充分搅拌,得到VAPs溶液;
4)向2.4 mL PLGA溶液中加入1.2 mL VAPs溶液,于细胞破碎仪中以超声功率200W、探头振幅40%在冰浴条件下超声3 min使溶液乳化,得到初乳;
5)将3.6 mL初乳快速倒入18 mL PVA溶液中,于细胞破碎仪中以超声功率200W、探头振幅40%在冰浴条件下超声3 min,得到复乳;
6)将得到的复乳在磁力搅拌器上300 r/min搅拌6 h,再于4℃、5000 rpm离心6 min,固相用去离子水洗涤,而后冷冻干燥,得到负载鹿茸多肽的PLGA微球。
5.根据权利要求1所述的制备方法,其特征在于:所述步骤(4)中,负载鹿茸多肽的PLGA微球与COL Ⅰ/Ⅲ溶液的比例为1.5 mg/cm3。
6.根据权利要求1所述的制备方法,其特征在于:所述步骤(5)中,交联剂为将NHS和EDC溶于90 vol%乙醇溶液中制得,所述交联剂中NHS和EDC的含量分别为50 mmol/L、20 mmol/L。
7.一种如权利要求1所述制备方法制得的负载鹿茸多肽的阵列微管仿生神经支架。
8.如权利要求7所述的负载鹿茸多肽的阵列微管仿生神经支架在制备修复周围神经损伤的产品中的应用。
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