CN105611952A - 通过单步过程制备双层支架的方法以及利用由该制备方法获得的双层支架进行组织再生的方法 - Google Patents

通过单步过程制备双层支架的方法以及利用由该制备方法获得的双层支架进行组织再生的方法 Download PDF

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CN105611952A
CN105611952A CN201380079610.9A CN201380079610A CN105611952A CN 105611952 A CN105611952 A CN 105611952A CN 201380079610 A CN201380079610 A CN 201380079610A CN 105611952 A CN105611952 A CN 105611952A
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double
layer scaffold
preparing
cell
layer
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CN105611952B (zh
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韩星洙
崔顺模
迪普·蒂·辛格
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Industry Academic Cooperation Foundation of Yeungnam University
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Industry Academic Cooperation Foundation of Yeungnam University
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Abstract

本发明涉及通过单步过程制备双层支架的方法以及利用由该制备方法获得的双层支架进行组织再生的方法。所述制备双层支架的方法可在没有单独的粘合过程的情况下通过单步过程产生完全粘合型的双层支架，这与传统技术中借助分别制备各聚合物支架随后彼此粘合的两步过程的双层支架制备方法不同。进而，可通过在制备的双层支架中培养细胞，并将获得的双层支架施用至进行组织再生或创伤愈合的皮肤缺陷位点；或者在制备的双层支架中对软骨细胞和骨细胞进行共培养，并将获得的双层支架施用至组织(例如骨或软骨)彼此接触的位点，促进由于创伤、烧伤、肿瘤等损失的皮肤组织的再生。

Description

通过单步过程制备双层支架的方法以及利用由该制备方法获得的双层支架进行组织再生的方法
技术领域
本发明涉及通过单步过程制备双层支架的方法以及利用由该制备方法获得的双层支架进行组织再生的方法。
背景技术
支架是为了对组织细胞进行体外培养和移植而制备的物理支撑和贴附基底；支架用于为人组织再生进行的细胞移植；并且，支架对于细胞的大量培养和增殖十分重要。其原因在于上皮细胞的细胞贴附以及相伴的迁移和增殖发生在细胞和基底之间的接触部分。
在体内或体外与物质相接触时，大部分具有生物活性的细胞首先通过细胞贴附而存活。特别地，在成纤维细胞和组织细胞的存活中，细胞优先贴附至基底，随后细胞质中的细胞器代谢变得活跃，并移向新的位点，从而促进增殖和营养供应。
因此，用于使细胞有效堆积的表面对于细胞的增殖而言是最基本的手段。可借助基底的组分对细胞基底的贴附性能进行人为控制。支架是细胞再生和生长的支撑载体(即，人工基底中作为基础的材料)，近来被用作大量培养和增殖的容器或烧瓶的涂层。
另一方面，韩国专利公开号2004-0016984公开了用于培养细胞和组织的载体及其培养方法，其中，成纤维细胞的过度生长可被阻抑，并且靶组织或器官可有效再生。然而，上述支架存在诸多问题：其贴附性并不足以对细胞进行大量培养，其物理性质并不出众，制备方法也并不简便和符合期望。
此外，传统的双层支架是通过在分别制造各聚合物支架后将其彼此附着来制备的，然而，所述双层支架不能完全附着，存在很大的分离可能，并且，由于聚合物支架的制备为两步法，工艺很复杂。
因此，本发明的发明人通过将阴离子表面活性剂加入至两种聚合物共混的溶液中，在高温高速下搅拌反应以诱导相分离，并确认了由此产生的支架是结构和化学上特异的双层支架，从而完成了本发明。
发明内容
技术问题
本发明的一个目的在于提供通过简单的单步过程制备结构和化学上特异的、不会分离的双层支架的方法。
此外，本发明的另一目的在于提供利用所述双层支架进行组织再生的方法。
技术方案
为了实现本发明的目的，本发明提供了通过简单的单步过程制备双层支架的方法，所述方法包含：制备第一聚合物水性溶液；将第二聚合物加入至所述第一聚合物水性溶液中，并对反应物进行搅拌；将表面活性剂加入至经搅拌的反应物中，并对所述反应物进行高温高速搅拌；对经搅拌的反应物进行冷冻干燥，从而获得海绵状物(sponge)；将所述海绵状物浸渍于交联剂中，使其交联；以及对经交联的反应物进行冷冻干燥。
本发明提供了制备双层支架的方法，所述方法包含：通过在60-70℃的温度下以100-300rpm的速度对水中的明胶进行搅拌以使其完全溶解，制备明胶水性溶液；将聚乙烯醇(PVA)加入至所述明胶水性溶液中，并对溶液进行搅拌；将十二烷基硫酸钠加入至经搅拌的溶液中，并在80-120℃的温度下以800-2000rpm进行搅拌；对经搅拌的溶液进行冷冻干燥，从而获得海绵状物；将所述海绵状物浸渍于交联剂中，使其交联，所述交联剂选自于由戊二醛或乙基二乙基氨基丙基碳化二亚胺(EDC)所组成的组；以及对经交联的反应物进行冷冻干燥。
本发明提供了通过在由上述制备方法制备的双层支架中培养细胞进行组织再生的方法。
此外，本发明提供了用于组织再生和创伤愈合的组合物，所述组合物包含上述双层支架。
有益效果
与通过分别制造各聚合物支架后将其粘合的两步过程而制备的传统双层支架不同，根据本发明的制备方法，可通过单步过程制备完全附着的双层支架，并且，可通过在制备的双层支架中培养细胞，并将其施用至进行组织再生或创伤愈合的皮肤缺陷位点；或者在制备的双层支架中对软骨细胞和骨细胞进行共培养，并将其施用至组织(例如骨和软骨)彼此接触的位点，促进由于创伤、烧伤、肿瘤等损失的皮肤组织的再生。
附图说明
图1描绘了根据本发明的双层支架制备过程的示意流程图。
图2为根据本发明的双层支架的扫描电子显微图像。
图3为根据本发明的双层支架的数字图像(A：含水的双层支架；B：干燥的双层支架；C：交联前的干燥的双层支架)。
图4为根据本发明的双层支架的FT-IR显微图像和曲线图(明胶层)。
图5为根据本发明的双层支架的FT-IR显微图像和曲线图(PVA层)。
图6为根据本发明的双层支架的显微CT图像(A：双层支架；B：明胶层)。
图7为在根据本发明的双层支架中培养1天的细胞的扫描电子显微图像。
图8为在根据本发明的双层支架中培养5天的细胞的扫描电子显微图像。
图9和10展示了利用根据本发明的双层支架处理的创伤组织中的组织再生效果。
图11为利用双层支架处理创伤组织后将其切下进行的RT-PCR分析。
具体实施方式
本发明提供了通过单步过程制备双层支架的方法，所述方法包含：制备第一聚合物水性溶液；将第二聚合物加入至所述第一聚合物水性溶液中，并对反应物进行搅拌；将表面活性剂加入至经搅拌的反应物中，并对所述反应物进行高温高速搅拌；对经搅拌的反应物进行冷冻干燥，从而获得海绵状物；将所述海绵状物浸渍于交联剂中，使其交联；以及对经交联的反应物进行冷冻干燥。
所述第一聚合物和所述第二聚合物可彼此不同，并可选自于由如下聚合物所组成的组：明胶、壳聚糖(chitosan)、弹性蛋白、透明质酸、羟基磷灰石、藻酸盐、胶原、纤维素、聚乙二醇(PEG)、聚环氧乙烷(PEO)、聚己内酯(PCL)、聚乳酸(PLA)、聚乙醇酸(PGA)、聚(乳酸-共-乙醇酸)(PLGA)、聚[(3-羟基丁酸酯)-共-(3-羟基戊酸酯)](PHBV)、聚二噁烷酮(PDO)、聚[(L-丙交酯)-共-(己内酯)]、聚酯型聚氨酯(poly(esterurethane)，PEUU)、聚[(L-丙交酯)-共-(D-丙交酯)]、聚(乙烯-共-乙烯醇)(PVOH)、聚丙烯酸(PAA)、聚乙烯醇(PVA)、聚乙烯吡咯烷酮(PVP)、聚苯乙烯(PS)和聚苯胺(PAN)。优选地，所述第一聚合物可以是明胶，所述第二聚合物可以是聚乙烯醇(PVA)。
所述表面活性剂可以是阴离子表面活性剂，例如可选自于由如下表面活性剂所组成的组：十二烷基硫酸钠(SDS)、月桂基硫酸铵(ALS)、月桂基乙烯硫酸钠(SodiumLaurylEthyleneSulfate，SLES)、直链烷基苯磺酸盐(LAS)、α-烯烃磺酸盐(AOS)、烷基硫酸盐(AS)、烷基醚硫酸盐(AES)和链烷磺酸钠(SodiumAlkaneSulfonate，SAS)；优选地，所述表面活性剂为十二烷基硫酸钠(SDS)。
高温高速搅拌优选在80-120℃的温度下以800-2000rpm进行。
所述交联剂可选自于由如下交联剂所组成的组：戊二醛、乙基二乙基氨基丙基碳化二亚胺(EDC)、京尼平(genepin)和转谷氨酰胺酶(TG)。
此外，本发明提供了制备上述双层支架的方法，其中，所述方法包含：通过在60-70℃的温度下以100-300rpm的速度对水中的明胶进行搅拌以使其完全溶解，制备明胶水性溶液；将聚乙烯醇(PVA)加入至所述明胶水性溶液中，并对溶液进行搅拌；将十二烷基硫酸钠加入至经搅拌的溶液中，并在80-120℃的温度下以800-2000rpm进行搅拌；对经搅拌的溶液进行冷冻干燥，从而获得海绵状物；将所述海绵状物浸渍于交联剂中，使其交联，所述交联剂选自于由戊二醛或乙基二乙基氨基丙基碳化二亚胺(EDC)所组成的组；以及对经交联的反应物进行冷冻干燥。
进而，本发明提供了通过在由上述方法制备的双层支架中培养细胞进行组织再生的方法。
所述细胞可为选自于由如下细胞所组成的组中的任意一种或两种以上：软骨细胞、骨细胞、皮肤细胞。可通过在双层支架的上层和下层分别培养不同的细胞，对所述细胞进行共培养。
此外，本发明提供了用于组织再生和创伤愈合的组合物，所述组合物包含由上述方法制备的双层支架。
将具体描述根据本发明的双层支架制备方法的一个实施方式，其中，所述第一聚合物为明胶，所述第二聚合物为聚乙烯醇(PVA)，所述表面活性剂为十二烷基硫酸钠(SDS)，并且所述交联剂为戊二醛或乙基二乙基氨基丙基碳化二亚胺(EDC)。
根据本发明的一个实施方式，双层支架可通过如下方式制备：通过在60-70℃的温度下以100-300rpm的速度对水中的明胶进行搅拌以使其完全溶解，制备明胶水性溶液；将聚乙烯醇(PVA)加入至所述明胶水性溶液中，并对溶液进行搅拌；将十二烷基硫酸钠加入至经搅拌的溶液中，并在80-120℃的温度下以800-2000rpm进行搅拌；在-80℃下对经搅拌的反应物进行冷冻干燥24-48h，从而获得海绵状物；将所述海绵状物浸渍于交联剂中，使其交联，所述交联剂选自于由戊二醛或乙基二乙基氨基丙基碳化二亚胺(EDC)所组成的组；以及在-80℃下对经交联的反应物进行冷冻干燥。
首先，通过将天然聚合物明胶加入至水中，并在60-70℃的温度下以100-300rpm进行搅拌以使其完全溶解，制备明胶水性溶液。所述明胶水性溶液可为1-99wt％、优选2.5wt％。如果明胶水性溶液中的明胶含量落在上述范围之外，可能会发生无法在视觉上确认分层的问题。
如果未在该条件下进行搅拌，明胶水性溶液不能均匀溶解，或者分层可能出现问题。
通过将聚乙烯醇(PVA)加入至明胶水性溶液中并搅拌，从而制备含有1-99wt％、优选12.5wt％的PVA的聚合物共混溶液。如果PVA含量落在上述范围之外，可能会发生无法在视觉上确认分层的问题。
将作为阴离子表面活性剂的十二烷基硫酸钠(SDS)加入至经搅拌的聚合物共混溶液中，并在80-120℃的高温下以800-2000rpm的高速进行搅拌。SDS的加入使得基于聚合物共混溶液的总量含有0.01-2wt％的SDS。如果SDS含量落在上述范围之外，不能对分层反应进行控制，并且可能发生难以操控的问题。
此外，如果未在所述高温高速的条件下进行搅拌，不会发生均匀的分层，并且不能对分层反应进行控制。
在-80℃下将经搅拌的反应物进行冷冻干燥24-48h，从而获得海绵状物；并将所述海绵状物浸渍于交联剂中，使其交联，所述交联剂选自于戊二醛或EDC(乙基二乙基氨基丙基碳化二亚胺)。此时，使用含有0.1-1wt％的交联剂的水性溶液进行3-24h来产生交联，所述交联剂例如为戊二醛或EDC(乙基二乙基氨基丙基碳化二亚胺)。如果交联剂含量落在上述范围之外，不发生交联反应，导致溶液降解；或者，由于未反应的交联剂残留，可能导致细胞培养有问题。
在-80℃下对经交联的反应物再次进行冷冻干燥，从而制备双层支架。
根据所述实施方式，可简单地通过单步过程制备如图2和图3中示出的未出现层脱离的双层支架，这与通过分别制造各聚合物支架后将其粘合的传统双层支架工艺过程不同。特别地，图2证实了双层支架具有位于左侧的多孔明胶层和位于右侧的高密度PVA层。
进而，图4和图5分别展示了根据本发明的双层支架的FT-IR显微图像中的明胶层和PVA层，并将它们与分别制备的明胶支架对照和PVA支架对照进行了比较。
此外，图6为根据本发明的双层支架的显微CT图像(A：双层支架；B：明胶层)，其中，红色部分和灰色部分代表多孔明胶层和高密度PVA层。
并且，作为利用双层支架培养细胞的结果，如图7所示，在细胞接种后1天细胞开始贴附至支架并增殖；如图8所示，证实了在细胞接种后5天细胞分泌的细胞外基质(ECM)堆积至支架中。
此外，如反映了使用双层支架在创伤组织中的组织再生效果的图9和图10所示的，在利用根据本发明的双层支架处理1天后，组织开始从皮下鼓泡；5天后，组织整合至双层支架中；10天后，组织再生，同时，明胶层借助自发降解(然而不完全降解)开始脱离。15天后，双层支架的明胶层几乎完全降解并自发脱离，创伤周围的组织几乎完全再生。另一方面，即使在5天后，对照也维持了80％的创伤区域。
特别地，本发明在不使得创伤收缩的情况下使组织再生，创伤收缩与组织再生和愈合的概念不同，其通过创伤收缩而使创伤区域变硬且密度变高，从而成为通常的疤痕组织。
此外，图11示出了利用根据本发明的双层支架对损伤区域处理后1天，对具有双层支架的部位的组织进行的RT-PCR分析，结果：第1天表达ITGA3，ITGA3为在与ECM(细胞外基质)蛋白反应的整合蛋白的形成过程中表达的；未显示ITGA3在第5天的表达，IL-8在第5天不表达，证实根据本发明的双层支架不引起免疫应答，并且，CD55的表达升高，其抑制与形成ECM的主要成分胶原的原纤维(fibril)的COL16A1、血管内皮生长因子VEGF和血液凝固相关的炎性物质C5a的形成。
因此，根据本发明的双层支架在组织工程学、医学、药学和材料科学领域中广泛地用作药物、细胞、蛋白和生长因子的递送材料或人工皮肤等，特别是有利地用作用于组织再生和创伤愈合的组合物。
也就是说，通过在由上述制备方法制备的双层支架中培养细胞，可将所述双层支架用于组织再生和创伤愈合。所述细胞可为选自于由如下细胞所组成的组中的任意一种或两种以上：软骨细胞、骨细胞、皮肤细胞；并且，可通过在双层支架的上层和下层分别培养不同的细胞，对所述细胞进行共培养。
特别地，可将根据本发明的双层支架施用于皮肤缺陷。换句话说，在由天然聚合物制得的下层支架中对细胞进行培养，将其用作组织再生层；并将药物加载至由合成聚合物制得的上层支架，从而在组织再生中阻断细菌感染。所述天然聚合物支架在组织再生过程中通过生物降解而被去除，所述合成聚合物支架则在组织再生完成时去除。
此外，根据本发明的双层支架可通过对两种细胞进行共培养并将其施用至组织(例如骨和软骨)彼此接触的位点来帮助再生。也就是说，在由天然聚合物制得的下层支架中培养软骨细胞，并在由合成聚合物制得的上层支架中培养骨细胞，将该双层支架施用至组织彼此接触的位点，从而帮助组织再生。
下文将根据以下实施例对本发明进行更为详细的说明。然而，这些实施例对本发明并不是限制性的。
实施例
<实施例1>制备双层支架
1)制备双层支架
制备2.5wt％的明胶水性溶液，在80℃下以300rpm进行搅拌。明胶完全溶解后，加入聚乙烯醇(PVA)以使其为10wt％，并搅拌。加入十二烷基硫酸钠以使其为0.2wt％，并在95℃的高温下以900rpm的高速进行搅拌。将经搅拌的溶液倾倒入培养皿，并在-80℃冷冻干燥至少24小时，获得海绵状物。将该海绵状物浸渍于含有戊二醛(0.5wt％)的水性溶液中至少12小时，使其交联。在-80℃再次冷冻干燥，产生双层支架。
2)双层支架的SEM和数字图像
借助场致发射型扫描电子显微镜(FE-SEM)(S-4100，HITACHI，LTD)对如上制备的双层支架进行观测，获得的SEM图像示于图2中；借助场致发射型扫描电子显微镜(FE-SEM)(S-4100，HITACHI，LTD)获得的数字图像示于图3中。换句话说，证实了该双层支架以一体化的形式完全附着，成为没有脱离可能的安全的双层结构支架。
3)双层支架的FT-IR图像分析
借助显微FTIR分光光度计(Spectrum100，PerkinElmer，USA)/成像系统(Spotlight200，PerkinElmer，USA)，在含有液氮的区室内通过扫描对如上制备的双层支架进行分析，并使用分别制备的Latin支架或PVA支架作为对照。
作为结果，如图4和图5所示，证实了根据本发明的双层支架中的明胶层和PVA层。
4)双层支架的显微CT图像分析
借助SKYSCAN1172(Skyscan，German)对如上制备的双层支架的显微CT图像进行分析，其中，单色光束为40kV，样品和检测器之间的距离为45.14mm，曝光时间为147ms，像素大小固定为4.84362μm。
作为结果，图6示出了根据本发明的双层支架的显微CT图像(A：双层支架；B：明胶层)，其中，红色部分和灰色部分分别表示多孔明胶层和高密度PVA层。
<实验实施例1>双层支架的体外性能
将实施例中制备的双层支架制为直径1cm后，将2×104个CRL-2310(ATCC编号；角化细胞，经人乳头瘤病毒16(HPV-16)E6/E7转化)分配在该双层支架上。细胞分配后，作为SEM观测的结果，如图7所示，细胞分配1天后细胞开始贴附至支架并再生；如图8所示，证实了细胞分配5天后细胞分泌的细胞外基质(ECM)堆积至支架中。
<实验实施例2>双层支架的体内创伤再生效果
使用活检穿孔器制造7mm的创伤，并使具有与创伤(7mm)相同尺寸的双层支架弯曲，从而明胶层与创伤位点相接触。在此情况下，分别将支架浸没在20％、40％、60％、80％和100％的乙醇中1小时以用于灭菌，最后在70％的乙醇中浸没24小时，并在室温下浸没在PBS缓冲液中3小时以供实验使用。在对照组中保持创伤开放；而在测试组中用根据本发明的支架将所制造的创伤覆盖。
作为结果，如图9和图10所示，从利用双层支架处理后1天的照片看来，组织开始从皮下鼓泡，而在对照中至少维持90％的创伤，测试组创伤的大小减小至80％。从其后5天的照片看来，组织整合入根据本发明的双层支架中，对照组创伤的大小减小至80％，而测试组创伤的大小减小至最初大小的40％以下。从其后10天的照片看来，组织已经再生，即便组织再生仍不完全，组织鼓泡的程度足以完全覆盖创伤，同时明胶层降解，支架自动脱离，然而该脱离不完全。从其后15天的照片看来，双层支架的明胶层几乎完全降解并自发脱离，创伤周围的组织几乎完全再生。
<实验实施例3>RT-PCR分析
以与实验实施例2相同的方式利用根据本发明的双层支架对创伤进行处理并在其后1天将双层支架周围的组织切下以进行RT-PCR分析。RT-PCR的条件如下：94℃5min(预变性)；进行30个循环的94℃30秒(变性)、根据引物的熔点在55-60℃30秒(退火)和72℃1分钟(延伸)；最后72℃10分钟(最后延伸)。通过凝胶电泳对由此获得的PCR产物进行分析。在此情况之下，使用的引物组如下所示。
[表1]
作为结果，如图11所示，第1天表达了ITGA3，ITGA3为在与ECM(细胞外基质)蛋白反应的整合蛋白的形成过程中表达的；未显示ITGA3在第5天的表达，IL-8在第5天不表达，证实根据本发明的双层支架不引起免疫应答。此外，CD55的表达升高，其抑制与形成ECM的主要成分胶原的原纤维的COL16A1、血管内皮生长因子VEGF和血液凝固相关的炎性物质C5a的形成。
虽然已参考具体特征对本发明进行了详细说明，本领域的普通技术人员将理解的是，所进行的说明仅是为了描述优选的实施方式，而并不旨在限制本发明的范围。本领域技术人员能够在所附权利要求书中记载的本发明的精神和范围的等同范围内作出各种修改及变化。

Claims (11)

1.一种通过单步过程制备双层支架的方法，所述方法包含：
制备第一聚合物水性溶液；
将第二聚合物加入至所述第一聚合物水性溶液中，并对反应物进行搅拌；
将表面活性剂加入至经搅拌的反应物中，并对所述反应物进行高温高速搅拌；
对经搅拌的反应物进行冷冻干燥，从而获得海绵状物；
将所述海绵状物浸渍于交联剂中，使其交联；以及
对经交联的反应物进行冷冻干燥。
2.根据权利要求1所述的制备双层支架的方法，其中，所述第一聚合物和所述第二聚合物彼此不同，并且所述第一聚合物和所述第二聚合物选自于由如下聚合物所组成的组：
明胶、壳聚糖、弹性蛋白、透明质酸、羟基磷灰石、藻酸盐、胶原、纤维素、聚乙二醇(PEG)、聚环氧乙烷(PEO)、聚己内酯(PCL)、聚乳酸(PLA)、聚乙醇酸(PGA)、聚(乳酸-共-乙醇酸)(PLGA)、聚[(3-羟基丁酸酯)-共-(3-羟基戊酸酯)](PHBV)、聚二噁烷酮(PDO)、聚[(L-丙交酯)-共-(己内酯)]、聚酯型聚氨酯(PEUU)、聚[(L-丙交酯)-共-(D-丙交酯)]、聚(乙烯-共-乙烯醇)(PVOH)、聚丙烯酸(PAA)、聚乙烯醇(PVA)、聚乙烯吡咯烷酮(PVP)、聚苯乙烯(PS)和聚苯胺(PAN)。
3.根据权利要求1所述的制备双层支架的方法，其中，所述第一聚合物是明胶，所述第二聚合物是聚乙烯醇(PVA)。
4.根据权利要求1所述的制备双层支架的方法，其中，所述表面活性剂选自于由如下表面活性剂所组成的组：
十二烷基硫酸钠(SDS)、月桂基硫酸铵(ALS)、月桂基乙烯硫酸钠(SLES)、直链烷基苯磺酸盐(LAS)、α-烯烃磺酸盐(AOS)、烷基硫酸盐(AS)、烷基醚硫酸盐(AES)和链烷磺酸钠(SAS)。
5.根据权利要求1所述的制备双层支架的方法，所述高温高速搅拌在80-120℃的温度下以800-2000rpm进行。
6.根据权利要求1所述的制备双层支架的方法，其中，所述交联剂选自于由如下交联剂所组成的组：
戊二醛、乙基二乙基氨基丙基碳化二亚胺(EDC)、京尼平和转谷氨酰胺酶(TG)。
7.根据权利要求1所述的制备双层支架的方法，其中，所述方法包含：
通过在60-70℃的温度下以100-300rpm的速度对水中的明胶进行搅拌以使其完全溶解，制备明胶水性溶液；
将聚乙烯醇(PVA)加入至所述明胶水性溶液中，并对溶液进行搅拌；
将十二烷基硫酸钠加入至经搅拌的溶液中，并在80-120℃的温度下以800-2000rpm进行搅拌；
对经搅拌的溶液进行冷冻干燥，从而获得海绵状物；
将所述海绵状物浸渍于交联剂中，使其交联，所述交联剂选自于由戊二醛或乙基二乙基氨基丙基碳化二亚胺(EDC)所组成的组；以及
对经交联的反应物进行冷冻干燥。
8.通过在根据权利要求1-7中任一项所述的方法制备的双层支架中培养细胞进行组织再生的方法。
9.根据权利要求8所述的进行组织再生的方法，其中，所述细胞为选自于由如下细胞所组成的组中的任意一种或两种以上：
软骨细胞、骨细胞、皮肤细胞。
10.根据权利要求8所述的进行组织再生的方法，其中，通过在所述双层支架的上层和下层分别培养不同的细胞，对所述细胞进行共培养。
11.一种用于组织再生和创伤愈合的组合物，所述组合物包含根据权利要求1-7中任一项所述的方法制备的双层支架。
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