CN105525385B - 一种多层核壳纳米纤维支架及其与黑素细胞构建组织工程材料的方法 - Google Patents

一种多层核壳纳米纤维支架及其与黑素细胞构建组织工程材料的方法 Download PDF

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CN105525385B
CN105525385B CN201510983527.5A CN201510983527A CN105525385B CN 105525385 B CN105525385 B CN 105525385B CN 201510983527 A CN201510983527 A CN 201510983527A CN 105525385 B CN105525385 B CN 105525385B
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layer
solution
core
nano fiber
shell
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CN105525385A (zh
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许爱娥
王文俊
吴非凡
尉晓冬
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No3 People's Hospital Hangzhou City
Zhejiang University ZJU
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No3 People's Hospital Hangzhou City
Zhejiang University ZJU
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Abstract

本发明公开了一种多层核壳结构的纳米纤维支架及其与黑素细胞构建组织工程材料的方法。其中所述多层核壳纳米纤维通过静电纺丝制备，外层为生物相容性材料，中层为高分子阻隔层，核层为可负载药物的芯材。利用该多层纳米纤维支架与黑素细胞或其与成纤维细胞、角质形成细胞构建组织工程材料，可用于色素脱失症（如白癜风等）的治疗。本发明的多层核壳结构的纳米纤维支架具有良好的生物相容性和机械性能，并能实现药物的长久可控释放，用其构建的组织工程支架能有效支持黑素细胞及共培养细胞的生长、增殖和移植。

Description

一种多层核壳纳米纤维支架及其与黑素细胞构建组织工程材 料的方法
技术领域
本发明属生物医学领域，尤其涉及一种具有药物缓释功能的多层核壳纳米纤维支架及其与黑素细胞构建组织工程材料的方法。
发明背景
白癜风是一种常见多发的色素脱失性皮肤病,临床表现主要为局限性或局发性白色斑片，边界清楚，无自觉症状，组织学及免疫细胞化学显示其皮损表皮黑素细胞消失。白癜风在人群中发病率达到0.5-2％，白斑处因缺乏黑色素，容易被阳光灼伤甚至引发癌变，同时由于影响人体外观形象，往往给患者造成严重的心理障碍，降低其生活质量。传统的治疗手段主要有药物治疗和光疗法。但药物治疗和紫外线照射的治愈率有限。外科手术治疗方法主要包括自体表皮移植、黑素细胞悬液移植等，这些治疗方法证实是有效的。但表皮移植需大面积取皮，不适合大面积白癜风治疗，而黑素细胞悬液移植能以较小的皮肤取材，治疗较大范围的皮损，具有良好的应用前景。目前该疗法的主要问题是，细胞悬液在活动部位难附着，另外移植操作会对细胞造成损伤，影响疗效。同时，黑色素细胞培养和移植过程中，生长因子等生物药物及抗氧化剂等药物起到很好的提高黑色素细胞活性和移植疗效的作用。因此，构建合适的黑素细胞载体，实现黑素细胞的培养和转移一体化，同时赋予载体具有药物缓释的功能，能有效提高悬液移植疗法的成功率。
利用组织工程技术、通过细胞负载与转移和药物缓释，为治疗白癜风提供新的途径。CN103668485A公开了一种同轴三层高活性包埋生物酶的静电纺丝纳米纤维膜，以实现生物酶的高活性和稳定性。文献ACS Macro Letters 2013,2,466-468.公开了一种同轴三层纳米纤维，提出可以对各层材料进行优化选择，以便同时满足纳米纤维的机械性能、生物相容性以及药物缓释的不同要求。CN103757743A公开了一种包载脂质体的核壳纳米纤维，活性物质装载在核层的脂质体内以实现缓释。上述公开的核壳纳米纤维支架不能实现药物长久缓释，更不能用于人体黑素细胞的培养。
发明内容
本发明的目的在于针对现有技术的不足，提供一种多层核壳纳米纤维支架及其与黑素细胞构建组织工程材料的方法。
本发明的目的是通过以下技术方案来实现的：一种多层核壳结构的纳米纤维支架，所述多层核壳纳米纤维支架由多层核壳纳米纤维构成，所述多层核壳纳米纤维的外层为生物相容性材料，中层为高分子阻隔层，核层为可负载药物的芯材。纤维外径在100nm-10μm。
进一步地，通过以下步骤制备得到：
(1)将生物相容性材料溶解于壳层溶剂中，配成重量百分比为1-10％的溶液，得到壳层溶液；所述生物相容性材料为明胶、胶原蛋白、纤维蛋白、透明质酸、硫酸软骨素中的一种或任意两种按照质量比1:50-50:1混合组成；
(2)将中层材料溶于中层溶剂中，配置成质量分数为1-10％的溶液，得到中层溶液；所述中层材料由乙烯基单体、二乙烯基单体与核层聚合物按10-50:0.1-5:50-90的重量比例混合组成；
(3)在中层溶液中再加入光引发剂，光引发剂的质量分数为0.1-3％；
(4)将核层聚合物溶解在核层溶剂中，配成重量百分比为1-10％的溶液，加入药物，得到核层溶液；其中药物的质量分数为0～0.1％。
(5)将外层溶液、中层溶液、核层溶液分别装入注射器中，在纺丝电压5-30kv，接收距离10-30cm，温度20-60℃，外层溶液推进速度0.1-2ml/h，中层溶液推进速度0.1-2ml/h，核层溶液推进速度0.1-2ml/h，紫外光照下进行同轴电纺，得到多层核壳纳米纤维支架。纺丝过程中，中层溶液在紫外光照下发生聚合，外、中、核层溶剂挥发；
(6)将多层核壳纳米纤维支架在真空、20-50℃下烘2-48小时；然后用交联剂对多层核壳纳米纤维支架在20-50℃交联处理0.5-24小时，交联处理后用纯净水冲洗除净残留的交联剂；
(7)再在鼓风条件下于30-120℃下烘2-48小时，真空条件下于20～40℃下干燥2-24h，去除残余溶剂。
进一步地，通过以下步骤制备得到：
(1)将生物相容性材料溶解于壳层溶剂中，配成重量百分比为1-10％的溶液，得到壳层溶液；所述生物相容性材料为壳聚糖；
(2)将中层材料溶于中层溶剂中，配置成质量分数为1-10％的溶液，得到中层溶液；所述中层材料由乙烯基单体、二乙烯基单体与核层聚合物按10-50:0.1-5:50-90的重量比例混合组成；
(3)在中层溶液中再加入光引发剂，光引发剂的质量分数为0.1-3％；
(4)将核层聚合物溶解在核层溶剂中，配成重量百分比为1-10％的溶液，加入药物，得到核层溶液；其中药物的质量分数为0～0.1％。
(5)将外层溶液、中层溶液、核层溶液分别装入注射器中，在纺丝电压5-30kv，接收距离10-30cm，温度20-60℃，外层溶液推进速度0.1-2ml/h，中层溶液推进速度0.1-2ml/h，核层溶液推进速度0.1-2ml/h，紫外光照下进行同轴电纺，得到多层核壳纳米纤维支架。纺丝过程中，中层溶液在紫外光照下发生聚合，外、中、核层溶剂挥发；
(6)将多层核壳纳米纤维支架在真空、20-50℃下烘2-48小时；然后用交联剂对多层核壳纳米纤维支架在20-50℃交联处理0.5-24小时，交联处理后用纯净水冲洗除净残留的交联剂；
(7)用氢氧化钠-碳酸钠水溶液浸泡多层核壳纳米纤维支架24小时，再用去离子水清洗，所述氢氧化钠-碳酸钠水溶液中，氢氧化钠和碳酸钠的总质量与水的质量配比为1-10:90-99混合组成，且氢氧化钠与碳酸钠的质量配比为1:3-3:1；
(8)再在鼓风条件下于30-120℃下烘2-48小时，真空条件下于20～40℃下干燥2-24h，去除残余溶剂。
进一步地，所述外层溶剂选自水、甲酸、乙酸、三氟乙酸、三氟乙醇、六氟异丙醇、二甲基亚砜、二氯甲烷、氯仿中的一种或者两种按重量配比10:1-1:10混合组成的混合溶剂。所述中层溶剂为乙酸、水、乙醇、甲酸、三氟乙酸、三氟乙醇、六氟异丙醇、二甲基亚砜中的一种或几种两种按重量配比10:1-1:10混合组成的混合溶剂；所述核层溶剂为乙酸、水、乙醇、甲酸、三氟乙酸、三氟乙醇、六氟异丙醇、二甲基亚砜、二氯甲烷、氯仿中的一种或几种两种按重量配比10:1-1:10混合组成的混合溶剂；
进一步地，所述的乙烯基单体选自聚乙二醇(甲基)丙烯酸酯、(甲基)丙烯酸羟乙酯、(甲基)丙烯酸羟丙酯、乙烯基吡咯烷酮、(甲基)丙烯酸。所述的二乙烯基单体选自N,N'-亚甲基双丙烯酰胺、乙二醇二(甲基)丙烯酸酯、一缩二乙二醇二(甲基)丙烯酸酯、二缩三乙二醇二(甲基)丙烯酸酯、三缩四乙二醇二(甲基)丙烯酸酯、聚乙二醇二(甲基)丙烯酸酯，1,4-丁二醇二丙烯酸酯。所述的核层聚合物选自聚环氧乙烷、聚乙烯醇、聚乙烯吡咯烷酮、透明质酸、硫酸软骨素。
进一步地，所述的光引发剂选自2-羟基-1-[4-(羟基)苯基]-2-甲基-1-丙酮、苯基双(2,4,6-三甲基苯甲酰基)氧化膦、苯甲酮、1-[4-(2-羟基乙氧基)苯基]-2-羟基-2甲基-1-丙烷-1-酮、1-羟基环己基苯基甲酮。
进一步地，所述的交联剂选自硫酸钠、柠檬酸钠、三聚磷酸钠、戊二醛、乙二醛、水杨醛、京尼平、香草醛等。
一种多层核壳纳米纤维支架与黑素细胞构建组织工程材料的方法，该方法为，将灭菌处理后的多层核壳纳米纤维支架置于细胞培养板中，向细胞培养板里注入培养液，置于细胞培养箱中于37℃、体积分数为5％的CO2、饱和湿度条件进行孵化；将按1-40:0-200:0-100的细胞个数比例共培养的黑素细胞、成纤维细胞和角质形成细胞以5×103-5×105个细胞/cm2的密度接种在孵化过的多层核壳纳米纤维支架上，同时加入1-10毫升的细胞培养液，置于细胞培养箱于37℃、体积分数为5％的CO2、饱和湿度条件下培养，每两天更换一次细胞培养液，培养时间为1-10天，可获得细胞-多层核壳纳米纤维支架的复合材料。
进一步地，所述培养液由100毫升F12培养基、0.1-100毫升FBS、10-50000纳克CT、10-50000微克IBMX、0.1-100毫升Glutamine和100-500000微克Gentamicin等成分混合组成。
一种由上述多层核壳结构的纳米纤维支架构建的组织工程材料。
本发明的有益效果是：所得的纳米纤维具有多层核壳结构，其外层材料使用天然高分子材料有利于提高生物相容性；其中间层材料使用交联型水凝胶，通过水凝胶交联密度的设计与定制，可有效地控制药物的释放速率；其核层能高效地负载药物并能提高材料强度。多层核壳纳米纤维支架对药物高效负载与可控释放，可很好地提高培养过程中黑素细胞活性和移植过程中的疗效。纳米纤维支架具有多孔结构，适合细胞贴附、生长。同时具有良好的机械性能，在制备、负载、转移和移植过程不发生破碎和断裂，符合转移和移植的要求。细胞-纳米纤维支架复合材料的构建过程中，细胞在生物相容性纳米纤维支架材料上的形态良好，多孔的纳米纤维结构益于培养液的更新，从而促进了细胞的增殖，使细胞保持良好的活性。
附图说明
图1为本发明纳米纤维的结构示意图，图中，外层1、中层2、核层3。
具体实施方式
本发明属生物医学领域，利用组织工程技术构建具有良好生物相容性多层核壳纳米纤维支架材料，药物可负载于其核层，并实现药物的可控释放。该支架材料用于黑素细胞或其与成纤维细胞、角质形成细胞的(共)培养，实现细胞的体外培养及移植。具体涉及具有多层核壳纳米纤维支架材料的制备、细胞-纳米纤维支架复合材料的构建及其应用，满足色素脱失症(如白癜风)和皮肤颜色调节的要求。
本发明选用生物相容性良好的材料，纳米纤维支架材料制备条件温和，多孔的纳米纤维结构模仿人体细胞外基质，能够促进细胞的贴附、生长和增殖，使细胞保持良好的活性。经交联处理的纳米纤维支架材料，具有优越的机械性能，满足可直接从细胞培养板中取出用于细胞转移和移植的要求。纳米纤维支架材料可负载药物并实现药物的可控释放，很好地提高了培养过程中黑素细胞活性和移植过程中的疗效。此外，本发明通过调节细胞的接种密度、培养时间和培养方法，保持细胞活性，实现对不同个体皮肤颜色的调控，使黑素细胞的功效性在移植中得到成功的表达，具有实际的可操作性，为大面积色素脱失症和皮肤颜色的调节提供了极大的便利。
本发明的多层核壳纳米纤维支架，通过以下步骤制备得到：
将生物相容性材料溶解于壳层溶剂中，配成重量百分比为1-10％的溶液，得到壳层溶液；
将中层材料溶于中层溶剂中，配置成质量分数为1-10％的溶液，得到中层溶液；所述中层材料由乙烯基单体、二乙烯基单体与核层聚合物按10-50:0.1-5:50-90的重量比例混合组成；
在中层溶液中再加入光引发剂，光引发剂的质量分数为0.1-3％；
将核层聚合物溶解在核层溶剂中，配成重量百分比为1-10％的溶液，加入药物得到核层溶液；
将外层溶液、中层溶液、核层溶液分别装入注射器中，在纺丝电压5-30kv，接收距离10-30cm，温度20-60℃，外层溶液推进速度0.1-2ml/h，中层溶液推进速度0.1-2ml/h，核层溶液推进速度0.1-2ml/h，紫外光照下进行电纺。纺丝过程中，中层溶液在紫外光照下发生聚合，外、中、内层溶剂挥发，得到多层核壳纳米纤维支架；
将多层核壳纳米纤维支架在真空、20-50℃下烘2-48小时；然后用交联剂对多层核壳纳米纤维支架在20-50℃交联处理0.5-24小时(交联剂与核壳型纳米纤维支架的质量配比以及交联处理的具体实施手段为本领域的公知常识，例如，可以参考Journal ofBiomedical Materials Research Part A 2012,100A,673-683.)，交联处理后用纯净水冲洗除净残留的交联剂；
然后根据需要，用氢氧化钠-碳酸钠水溶液浸泡多层核壳纳米纤维支架24小时，再用去离子水清洗(一般清洗至清洗后的溶液的PH为中性)，所述氢氧化钠-碳酸钠水溶液中，氢氧化钠和碳酸钠的总质量与水的质量配比为1-10:90-99混合组成，且氢氧化钠与碳酸钠的质量配比为1:3-3:1；再在鼓风条件下于30-120℃下烘2-48小时；真空干燥去除残余溶剂2-24h。
所述外层溶剂选自水、甲酸、乙酸、三氟乙酸、三氟乙醇、六氟异丙醇、二甲基亚砜、二氯甲烷、氯仿中的一种或者两种按重量配比10:1-1:10混合组成的混合溶剂。所述中层溶剂为乙酸、水、乙醇、甲酸、三氟乙酸、三氟乙醇、六氟异丙醇、二甲基亚砜中的一种或几种两种按重量配比10:1-1:10混合组成的混合溶剂；所述核层溶剂为乙酸、水、乙醇、甲酸、三氟乙酸、三氟乙醇、六氟异丙醇、二甲基亚砜、二氯甲烷、氯仿中的一种或几种两种按重量配比10:1-1:10混合组成的混合溶剂；
所述的生物相容性材料选自壳聚糖、明胶、胶原蛋白、纤维蛋白、透明质酸、硫酸软骨素中的一种或任意两种按照质量比1:50-50:1混合组成。
所述的乙烯基单体选自聚乙二醇(甲基)丙烯酸酯、(甲基)丙烯酸羟乙酯、(甲基)丙烯酸羟丙酯、乙烯基吡咯烷酮、(甲基)丙烯酸。所述的二乙烯基单体选自N,N'-亚甲基双丙烯酰胺、乙二醇二(甲基)丙烯酸酯、一缩二乙二醇二(甲基)丙烯酸酯、二缩三乙二醇二(甲基)丙烯酸酯、三缩四乙二醇二(甲基)丙烯酸酯、聚乙二醇二(甲基)丙烯酸酯，1,4-丁二醇二丙烯酸酯。所述的核层聚合物选自聚环氧乙烷、聚乙烯醇、聚乙烯吡咯烷酮、透明质酸、硫酸软骨素。
所述的光引发剂选自2-羟基-1-[4-(羟基)苯基]-2-甲基-1-丙酮、苯基双(2,4,6-三甲基苯甲酰基)氧化膦、苯甲酮、1-[4-(2-羟基乙氧基)苯基]-2-羟基-2甲基-1-丙烷-1-酮、1-羟基环己基苯基甲酮。
所述的交联剂选自硫酸钠、柠檬酸钠、三聚磷酸钠、戊二醛、乙二醛、水杨醛、京尼平、香草醛等。
采用上述多层核壳纳米纤维支架构建细胞-多层核壳纳米纤维支架组织工程材料的方法如下：
将多层核壳纳米纤维支架用于构建组织工程材料，具体为，将灭菌处理后的多层核壳纳米纤维支架置于细胞培养板中，向细胞培养板里注入培养液，置于细胞培养箱中于37℃、体积分数为5％的CO2、饱和湿度条件进行孵化；将按1-40:0-200:0-100的细胞个数比例共培养的黑素细胞、成纤维细胞和角质形成细胞以5×103-5×105个细胞/cm2的密度接种在孵化过的多层核壳纳米纤维支架上，同时加入1-10毫升的细胞培养液，置于细胞培养箱于37℃、体积分数为5％的CO2、饱和湿度条件下培养，每两天更换一次细胞培养液，培养时间为1-10天，可获得细胞-多层核壳纳米纤维支架的复合材料。
所述培养液由100毫升F12培养基、0.1-100毫升FBS、10-50000纳克CT、10-50000微克IBMX、0.1-100毫升Glutamine和100-500000微克Gentamicin等成分混合组成。
下面根据实施例详细描述本发明，本发明的目的和效果将变得更加明显。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1.壳聚糖-聚环氧乙烷-聚环氧乙烷多层核壳纳米纤维支架材料的制备
A.将壳聚糖用三氟乙酸/二氯甲烷重量比3:1混合溶剂溶解，配成浓度为5％的壳层溶液；
B.将聚环氧乙烷，聚乙二醇甲基丙烯酸酯，聚乙二醇二甲基丙烯酸酯，按80：20:1重量比溶解在水中，配成浓度为5％的中层溶液，在纺丝前加入1％的光引发剂1-[4-(2-羟基乙氧基)苯基]-2-羟基-2甲基-1-丙烷-1-酮；
C.将聚环氧乙烷溶解在水中，配成浓度为4％的核层溶液；
D.在电压为15KV、三层推进速率均为0.5ml/h、接收距离为15cm、温度为30℃，紫外光照射条件下进行静电纺丝；
E.将多层核壳纳米纤维支架在真空、50℃下烘12小时；
F.20℃下，纳米纤维支架用硫酸钠交联2小时，交联处理后用纯净水冲洗除净残留的交联剂；
G.用氢氧化钠-碳酸钠水溶液(氢氧化钠和碳酸钠的总质量与水的质量配比为1:90混合组成，且氢氧化钠与碳酸钠的质量配比为3:1)浸泡核壳型纳米纤维支架24小时，再用去离子水清洗至清洗后的溶液的PH＝7.2-7.4。
H.将纳米纤维支架置于鼓风烘箱中，50℃条件下干燥24小时；再在真空条件下于20～40℃下干燥2-24h，去除残余溶剂。
I.得到可用于细胞培养的壳聚糖-聚环氧乙烷-聚环氧乙烷多层核壳纳米纤维支架材料。
实施例2.壳聚糖-聚乙烯吡咯烷酮-聚乳酸核壳纳米纤维支架材料的制备
A.将壳聚糖用乙酸/水(重量配比1:1)混合溶剂溶解，配成浓度为4％的壳聚糖/聚环氧乙烷溶液；
B.将乙烯基吡咯烷酮，甲基丙烯酸羟乙酯，N,N'-亚甲基双丙烯酰胺，按70：30:3重量比溶解在水中，配成浓度为6％的核层溶液，在纺丝前加入3％的光引发剂苯基双(2,4,6-三甲基苯甲酰基)氧化膦；
C.将聚乳酸用二氯甲烷溶解，配成浓度为7％的溶液；
D.在电压为20KV、壳层推进速率为0.5ml/h、壳层、中层、核层推进速度分别为0.8、0.4、0.4ml/h，接收距离为18cm、温度为20℃，紫外光照条件下进行静电纺丝；
E.将壳聚糖-聚乙烯吡咯烷酮-聚乳酸多层核壳纳米纤维支架在真空、25℃下烘24小时；
F.20℃下，纳米纤维支架用京尼平交联12小时，交联处理后用纯净水冲洗除净残留的交联剂；
G.用氢氧化钠-碳酸钠水溶液(氢氧化钠和碳酸钠的总质量与水的质量配比为1：9混合组成，且氢氧化钠与碳酸钠的质量配比为1:3)浸泡核壳型纳米纤维支架24小时，再用去离子水清洗至清洗后的溶液的PH＝7.2-7.4。
H.将纳米纤维支架置于鼓风烘箱中，50℃条件下干燥24小时；再在真空条件下于20～40℃下干燥2-24h，去除残余溶剂。
I.得到可用于细胞培养的壳聚糖-聚乙烯吡咯烷酮-聚乳酸核壳纳米纤维支架材料。
实施例3.明胶-聚乙烯醇-聚乙烯吡咯烷酮核壳纳米纤维支架材料的制备
A.将明胶用三氟乙醇/水(质量比8:2)溶剂溶解，配成浓度为10％的明胶溶液；
B.将聚乙烯醇，甲基丙烯酸，二缩乙二醇二甲基丙烯酸酯，按60:40:0.4重量比溶解在乙酸/水(质量比1:1)中，配成浓度为3％的核层溶液，在纺丝前加入0.2％的光引发剂2-羟基-1-[4-(羟基)苯基]-2-甲基-1-丙酮；
C.在电压为25KV、壳层推进速率为1ml/h、核层推进速率为0.5ml/h，接收距离为20cm、温度为40℃，紫外光照条件下进行静电纺丝；
D.将聚乙烯吡咯烷酮用乙醇溶解，配成浓度为8％核层溶液；
E.将明胶-聚乙烯醇-聚乙烯吡咯烷酮多层核壳纳米纤维支架在真空、25℃下烘24小时；
F.50℃下，纳米纤维支架用戊二醛交联12小时，交联处理后用纯净水冲洗除净残留的交联剂；
G.将纳米纤维支架置于鼓风烘箱中，50℃条件下干燥24小时；再在真空条件下于20～40℃下干燥2-24h，去除残余溶剂。
H.得到可用于细胞培养的明胶-聚乙烯醇-聚乙烯吡咯烷酮核壳纳米纤维支架材料。
实施例4.负载生物药物壳聚糖-聚环氧乙烷-聚环氧乙烷多层核壳纳米纤维支架材料的制备
A.将壳聚糖用三氟乙酸/二氯甲烷重量比3:1混合溶剂溶解，配成浓度为5％的壳层溶液；
B.将聚环氧乙烷，聚乙二醇甲基丙烯酸酯，聚乙二醇二甲基丙烯酸酯，按80：20:1重量比溶解在水中，配成浓度为5％的中层溶液，在纺丝前加入1％的光引发剂1-[4-(2-羟基乙氧基)苯基]-2-羟基-2甲基-1-丙烷-1-酮；
C.将聚环氧乙烷溶解在水中，配成浓度为4％的核层溶液，并加入0.1％的肝细胞生长因子；
D.在电压为15KV、三层推进速率均为0.5ml/h、接收距离为15cm、温度为30℃，紫外光照射条件下进行静电纺丝；
E.将多层核壳纳米纤维支架在真空、50℃下烘12小时；
F.20℃下，纳米纤维支架用硫酸钠交联2小时，交联处理后用纯净水冲洗除净残留的交联剂；
G.用氢氧化钠-碳酸钠水溶液(氢氧化钠和碳酸钠的总质量与水的质量配比为1：20混合组成，且氢氧化钠与碳酸钠的质量配比为1:1)浸泡核壳型纳米纤维支架24小时，再用去离子水清洗至清洗后的溶液的PH＝7.2-7.4。
H.将纳米纤维支架置于鼓风烘箱中，50℃条件下干燥24小时；再在真空条件下于20～40℃下干燥2-24h，去除残余溶剂。
I.得到可用于细胞培养的负载有肝细胞生长因子的壳聚糖-聚环氧乙烷-聚环氧乙烷多层核壳纳米纤维支架材料。
实施例5.负载药物壳聚糖-聚环氧乙烷-聚环氧乙烷多层核壳纳米纤维支架材料的制备
A.将壳聚糖用三氟乙酸/二氯甲烷重量比3:1混合溶剂溶解，配成浓度为5％的壳层溶液；
B.将聚环氧乙烷，聚乙二醇甲基丙烯酸酯，聚乙二醇二甲基丙烯酸酯，按80：20:1重量比溶解在水中，配成浓度为5％的中层溶液，在纺丝前加入1％的光引发剂1-[4-(2-羟基乙氧基)苯基]-2-羟基-2甲基-1-丙烷-1-酮；
C.将聚环氧乙烷溶解在水中，配成浓度为4％的核层溶液，并加入5％的槲皮素；
D.在电压为15KV、三层推进速率均为0.5ml/h、接收距离为15cm、温度为30℃，紫外光照射条件下进行静电纺丝；
E.将多层核壳纳米纤维支架在真空、50℃下烘12小时；
F.20℃下，纳米纤维支架用硫酸钠交联2小时，交联处理后用纯净水冲洗除净残留的交联剂；
G.用氢氧化钠-碳酸钠水溶液(氢氧化钠和碳酸钠的总质量与水的质量配比为1：15混合组成，且氢氧化钠与碳酸钠的质量配比为2:3)浸泡核壳型纳米纤维支架24小时，再用去离子水清洗至清洗后的溶液的PH＝7.2-7.4。
H.将纳米纤维支架置于鼓风烘箱中，50℃条件下干燥24小时；再在真空条件下于20～40℃下干燥2-24h，去除残余溶剂。
I.得到可用于细胞培养的负载有槲皮素的壳聚糖-聚环氧乙烷-聚环氧乙烷多层核壳纳米纤维支架材料。
实施例6.细胞-多层核壳纳米纤维支架复合材料的构建
A.将实例1所述的壳聚糖-聚环氧乙烷-聚环氧乙烷多层核壳纳米纤维支架置于体积分数为75％的酒精中浸泡12小时后用PBS冲洗干净；
B.将覆盖纳米纤维支架的细胞培养板置于紫外灯下照射12小时；
C.在细胞培养板里注入3毫升细胞培养液，置于细胞培养箱于37℃、体积分数为5％的CO2、饱和湿度下孵化4小时；
D.将黑素细胞以8×104个细胞/cm2的密度接种在纳米纤维支架上，置于细胞培养箱于37℃、体积分数为5％的CO2、饱和湿度下培养；
E.每两天换一次细胞培养液，培养时间为6天；
F.得到黑素细胞与壳聚糖-聚环氧乙烷-聚环氧乙烷多层核壳纳米纤维支架复合材料，可进一步用于移植。
实施例7.细胞-多层核壳纳米纤维支架复合材料的构建
A.将实例2所述的壳聚糖-聚乙烯吡咯烷酮-聚乳酸多层核壳纳米纤维支架置于体积分数为75％的酒精中浸泡12小时后用PBS冲洗干净；
B.将覆盖多层核壳纳米纤维支架的细胞培养板置于紫外灯下照射12小时；
C.在细胞培养板里注入2毫升细胞培养液，置于细胞培养箱于37℃、体积分数为5％的CO2、饱和湿度下孵化4小时；
D.将黑素细胞和成纤维细胞以比例为1:2，密度为8×104个细胞/cm2接种在纳米纤维支架上，置于细胞培养箱37℃、体积分数为5％的CO2、饱和湿度下培养；
E.每两天换一次细胞培养液，培养时间为1-10天；
F.得到黑素细胞和成纤维细胞与壳聚糖-聚乙烯吡咯烷酮-聚乳酸多层核壳纳米纤维支架复合材料，可进一步用于移植。
实施例8.细胞-多层核壳纳米纤维支架复合材料的构建
A.将实例3所述的明胶-聚乙烯醇-聚乙烯吡咯烷酮多层核壳纳米纤维支架置于体积分数为75％的酒精中浸泡12小时后用PBS冲洗干净；
B.将覆盖纳米纤维支架的细胞培养板置于紫外灯下照射12小时；
C.在细胞培养板里注入2毫升细胞培养液，置于细胞培养箱于37℃、体积分数为5％的CO2、饱和湿度下孵化4小时；
D.将黑素细胞、成纤维细胞、角质形成细胞比例以1:2:1，密度为8×104个细胞/cm2接种在纳米纤维支架上，置于细胞培养箱于37℃、体积分数为5％的CO2、饱和湿度下培养；
E.每两天换一次细胞培养液，培养时间为6天；
F.得到黑素细胞/成纤维细胞/角质形成细胞与明胶-聚乙烯醇-聚乙烯吡咯烷酮多层核壳纳米纤维支架复合材料，可进一步用于移植。
实施例9.细胞-多层核壳纳米纤维支架复合材料的构建
A.将实例4所述的负载有肝细胞生长因子的壳聚糖-聚环氧乙烷-聚环氧乙烷多层核壳纳米纤维支架置于体积分数为75％的酒精中浸泡12小时后用PBS冲洗干净；
B.将覆盖纳米纤维支架的细胞培养板置于紫外灯下照射12小时；
C.在细胞培养板里注入3毫升细胞培养液，置于细胞培养箱于37℃、体积分数为5％的CO2、饱和湿度下孵化4小时；
D.将黑素细胞以8×104个细胞/cm2的密度接种在纳米纤维支架上，置于细胞培养箱于37℃、体积分数为5％的CO2、饱和湿度下培养；
E.每两天换一次细胞培养液，培养时间为6天；
F.得到黑素细胞与壳聚糖-聚环氧乙烷-聚环氧乙烷多层核壳纳米纤维支架复合材料，其黑素细胞增殖和活性明显优于实施例6。该负载细胞的膜复合材料可进一步用于移植。本发明的支架可通过肝细胞因子缓释进一步提高黑素细胞的增殖和活性，有利于移植后病灶区的复色，提高治疗成功率。
本发明利用组织工程技术实现黑素细胞或其与成纤维细胞和角质形成细胞的体外培养、负载及移植。通过将细胞-多层核壳纳米纤维支架复合材料移植到脱色皮肤创口处，使黑素细胞或其与成纤维细胞和角质形成细胞由载体材料迁移到皮肤创口处，为脱色处皮肤提供黑素细胞，从而实现色素脱失症(如白癜风)和皮肤颜色的调节。利用多层核壳纳米纤维支架的药物负载和控释的功能，提高了黑色素细胞活性和移植疗效。

Claims (9)

1.一种多层核壳结构的纳米纤维支架，所述多层核壳结构的纳米纤维支架由多层核壳纳米纤维构成，所述多层核壳纳米纤维的外层为生物相容性材料，中层为高分子阻隔层，核层为可负载药物的芯材；纤维外径在100nm-10μm；其特征在于，通过以下步骤制备得到：
(1)将生物相容性材料溶解于壳层溶剂中，配成重量百分比为1-10％的溶液，得到壳层溶液；所述生物相容性材料为明胶、胶原蛋白、纤维蛋白、透明质酸、硫酸软骨素中的一种或任意两种按照质量比1:50-50:1混合组成；
(2)将中层材料溶于中层溶剂中，配置成质量分数为1-10％的溶液，得到中层溶液；所述中层材料由乙烯基单体、二乙烯基单体与核层聚合物按10-50:0.1-5:50-90的重量比例混合组成；
(3)在中层溶液中再加入光引发剂，光引发剂的质量分数为0.1-3％；
(4)将核层聚合物溶解在核层溶剂中，配成重量百分比为1-10％的溶液，加入药物，得到核层溶液；其中药物的质量分数为0～0.1％；
(5)将外层溶液、中层溶液、核层溶液分别装入注射器中，在纺丝电压5-30kv，接收距离10-30cm，温度20-60℃，外层溶液推进速度0.1-2mL /h，中层溶液推进速度0.1-2mL /h，核层溶液推进速度0.1-2mL /h，紫外光照下进行同轴电纺，得到多层核壳纳米纤维支架；纺丝过程中，中层溶液在紫外光照下发生聚合，外、中、核层溶剂挥发；
(6)将多层核壳纳米纤维支架在真空、20-50℃下烘2-48小时；然后用交联剂对多层核壳纳米纤维支架在20-50℃交联处理0.5-24小时，交联处理后用纯净水冲洗除净残留的交联剂；
(7)再在鼓风条件下于30-120℃下烘2-48小时，真空条件下于20～40℃下干燥2-24h，去除残余溶剂。
2.一种多层核壳结构的纳米纤维支架，所述多层核壳结构的纳米纤维支架由多层核壳纳米纤维构成，所述多层核壳纳米纤维的外层为生物相容性材料，中层为高分子阻隔层，核层为可负载药物的芯材；纤维外径在100nm-10μm；其特征在于，通过以下步骤制备得到：
(1)将生物相容性材料溶解于壳层溶剂中，配成重量百分比为1-10％的溶液，得到壳层溶液；所述生物相容性材料为壳聚糖；
(2)将中层材料溶于中层溶剂中，配置成质量分数为1-10％的溶液，得到中层溶液；所述中层材料由乙烯基单体、二乙烯基单体与核层聚合物按10-50:0.1-5:50-90的重量比例混合组成；
(3)在中层溶液中再加入光引发剂，光引发剂的质量分数为0.1-3％；
(4)将核层聚合物溶解在核层溶剂中，配成重量百分比为1-10％的溶液，加入药物，得到核层溶液；其中药物的质量分数为0～0.1％；
(5)将外层溶液、中层溶液、核层溶液分别装入注射器中，在纺丝电压5-30kv，接收距离10-30cm，温度20-60℃，外层溶液推进速度0.1-2mL /h，中层溶液推进速度0.1-2mL /h，核层溶液推进速度0.1-2mL /h，紫外光照下进行同轴电纺，得到多层核壳纳米纤维支架；纺丝过程中，中层溶液在紫外光照下发生聚合，外、中、核层溶剂挥发；
(6)将多层核壳纳米纤维支架在真空、20-50℃下烘2-48小时；然后用交联剂对多层核壳纳米纤维支架在20-50℃交联处理0.5-24小时，交联处理后用纯净水冲洗除净残留的交联剂；
(7)用氢氧化钠-碳酸钠水溶液浸泡多层核壳纳米纤维支架24小时，再用去离子水清洗，所述氢氧化钠-碳酸钠水溶液中，氢氧化钠和碳酸钠的总质量与水的质量配比为1-10:90-99混合组成，且氢氧化钠与碳酸钠的质量配比为1:3-3:1；
(8)再在鼓风条件下于30-120℃下烘2-48小时，真空条件下于20～40℃下干燥2-24h，去除残余溶剂。
3.根据权利要求1或2所述的多层核壳结构的纳米纤维支架，其特征在于，所述外层溶剂选自水、甲酸、乙酸、三氟乙酸、三氟乙醇、六氟异丙醇、二甲基亚砜、二氯甲烷、氯仿中的一种或者两种按重量配比10:1-1:10混合组成的混合溶剂；所述中层溶剂为乙酸、水、乙醇、甲酸、三氟乙酸、三氟乙醇、六氟异丙醇、二甲基亚砜中的一种或几种两种按重量配比10:1-1:10混合组成的混合溶剂；所述核层溶剂为乙酸、水、乙醇、甲酸、三氟乙酸、三氟乙醇、六氟异丙醇、二甲基亚砜、二氯甲烷、氯仿中的一种或几种两种按重量配比10:1-1:10混合组成的混合溶剂。
4.根据权利要求1或2所述的多层核壳结构的纳米纤维支架，其特征在于，所述的乙烯基单体选自聚乙二醇(甲基)丙烯酸酯、(甲基)丙烯酸羟乙酯、(甲基)丙烯酸羟丙酯、乙烯基吡咯烷酮、(甲基)丙烯酸；所述的二乙烯基单体选自N,N'-亚甲基双丙烯酰胺、乙二醇二(甲基)丙烯酸酯、一缩二乙二醇二(甲基)丙烯酸酯、二缩三乙二醇二(甲基)丙烯酸酯、三缩四乙二醇二(甲基)丙烯酸酯、聚乙二醇二(甲基)丙烯酸酯，1,4-丁二醇二丙烯酸酯；所述的核层聚合物选自聚环氧乙烷、聚乙烯醇、聚乙烯吡咯烷酮、透明质酸、硫酸软骨素。
5.根据权利要求1或2所述的多层核壳结构的纳米纤维支架，其特征在于，所述的光引发剂选自2-羟基-1-[4-(羟基)苯基]-2-甲基-1-丙酮、苯基双(2,4,6-三甲基苯甲酰基)氧化膦、苯甲酮、1-[4-(2-羟基乙氧基)苯基]-2-羟基-2甲基-1-丙烷-1-酮、1-羟基环己基苯基甲酮。
6.根据权利要求1或2所述的多层核壳结构的纳米纤维支架，其特征在于，所述的交联剂选自硫酸钠、柠檬酸钠、三聚磷酸钠、戊二醛、乙二醛、水杨醛、京尼平、香草醛。
7.一种权利要求1或2所述的多层核壳结构的纳米纤维支架与黑素细胞构建组织工程材料的方法，其特征在于，该方法为：将灭菌处理后的多层核壳结构的纳米纤维支架置于细胞培养板中，向细胞培养板里注入培养液，置于细胞培养箱中于37℃、体积分数为5％的CO2、饱和湿度条件进行孵化；将按1-40:0-200:0-100的细胞个数比例共培养的黑素细胞、成纤维细胞和角质形成细胞以5×103-5×105个细胞/cm2的密度接种在孵化过的多层核壳结构的纳米纤维支架上，同时加入1-10毫升的细胞培养液，置于细胞培养箱于37℃、体积分数为5％的CO2、饱和湿度条件下培养，每两天更换一次细胞培养液，培养时间为1-10天，可获得细胞-多层核壳纳米纤维支架的复合材料。
8.根据权利要求7所述的方法，其特征在于，所述培养液由100毫升F12培养基、0.1-100毫升FBS、10-50000纳克CT、10-50000微克IBMX、0.1-100毫升Glutamine和100-500000微克Gentamicin混合组成。
9.一种权利要求7所述方法构建的组织工程材料。
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