CN112999425B - 一种双层水凝胶管状组织工程支架及其制备方法 - Google Patents
一种双层水凝胶管状组织工程支架及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种双层水凝胶管状组织工程支架及其制备方法,包括管状的内层水凝胶和外层水凝胶,所述的内层水凝胶由可紫外光交联水凝胶和细胞或导电水凝胶复合而成;所述的外层水凝胶由两种或多种可紫外光交联水凝胶复合而成。本发明的制备方法,制造成本低、速度快,工艺灵活性大,制得的工程支架内层能够提供多功能的组织微环境,具有较好的生物相容性;外层提供机械支撑,且改变不同水凝胶的浓度及配比可以定制其机械性能。
Description
技术领域
本发明属于生物制造领域,尤其是涉及一种具有复合功能的双层水凝胶管状组织工程支架及其制备方法。
背景技术
管状结构的组织,如血管、胆管、气管和肠道,在高等动物的身体和器官系统中普遍存在。一旦这些管状组织发生疾病或功能障碍,通常需要用自体植入物、供体组织或人工假体替代它们。然而,由于缺乏合适的自体组织和供体移植物来源,限制了组织移植的发展。人工假体往往难以满足机械性能和生物性能的要求,易造成术后功能障碍和再生不良。管状组织的特殊管状形态和不同类型的细胞及其分布是管状组织的两个重要特征。
组织工程的发展为体外构建结构和功能与天然管状组织类似的移植物提供了新的途径,对指导管状及非管状组织结构的修复和再生具有重要意义。理想的导管应具有一定的机械强度,以承受外科手术,并应具用有特定的功能性,例如含有可再生的活细胞、对应生长因子或赋予导电功能特性等。
近几十年来,已有许多病例报道使用开发的生物材料和制备的人工种植体对各种受损组织进行良好修复。各种天然或合成材料制成的生物材料包括透明质酸、明胶、壳聚糖、海藻酸钠、聚己内酯(PCL)、聚乳酸乙醇酸(PLGA)等,已被用于气管修复、胆管或血管移植、神经再生等场合。同时,电纺丝、浸涂、溶液铸造、薄板轧制、3D打印等生产工艺得到了全面应用。水凝胶因其良好的生物相容性、组织弹性、保水性和给药性能,在组织工程和器官再生中得到了广泛的应用。
有学者(Journal of Biomedical Materials Research Part B:AppliedBiomaterials,2018,106(2),2817-2829)制备了填充有辛伐他汀/普朗尼克F127水凝胶的壳聚糖导管,并证实其对周围神经损伤的修复有一定的促进作用。也有学者(Biofabrication,2016,9(1),015001)在传统浸涂法的基础上,加入电机系统实现浸涂棒的旋转和移动,从而实现了组织工程小直径血管多层结构的自动制造,并证明该系统可以简单、快速地制作结构类似于天然血管的复杂细胞化多层血管移植。
然而,上述管状支架的力学性能尚未见报道。另外,类似静电纺丝或3D打印等制造方法不仅需要复杂的设备,对材料的流变性能也有一定的要求,为制备诸如含有活细胞等功能的导管或支架带来困难。
发明内容
本发明提供了一种具有复合功能的双层水凝胶管状组织工程支架及其制备方法,制造成本低、速度快,工艺灵活性大。
一种具有复合功能的双层水凝胶管状组织工程支架,包括管状的内层水凝胶和外层水凝胶,所述的内层水凝胶由可紫外光交联水凝胶和细胞或导电水凝胶复合而成;所述的外层水凝胶由两种或多种可紫外光交联水凝胶复合而成。
本发明中,支架由两层不同孔隙结构不同功能的水凝胶层组成;内层由低浓度可紫外光交联的大分子水凝胶和细胞或导电水凝胶复合而成,外层由两种或多种可紫外光交联的大分子水凝胶复合而成。支架内层能够提供多功能的组织微环境,具有较好的生物相容性;支架外层提供机械支撑,且改变不同水凝胶的浓度及配比可以定制其机械性能。
优选地,组成内层水凝胶的可紫外光交联水凝胶为甲基丙烯酸酐化明胶GelMA,细胞为间充质干细胞,导电水凝胶为PEDOT:PSS。
优选地,所述的外层水凝胶由两种可紫外光交联水凝胶复合而成,分别为甲基丙烯酸酐化明胶GelMA和聚乙二醇二丙烯酸酯PEGDA。
上述优选方案中,内外层主成分中采用了相同具有热、光双交联特性的材料——GelMA,从而有效保证内外两层之间的结合力,功能特性外层提供机械支撑,内层提供对应功能属性的特定配方种类。
本发明还提供了一种具有复合功能的双层水凝胶管状组织工程支架制备方法,包括以下步骤:
(1)模具制备:
模具包括底座、一根中空透明管和两根直径不同的中央芯柱;底座上同轴设置有与中空透明管匹配的定位槽以及与两根中央芯柱匹配的定位阶梯孔;内层水凝胶的厚度对应大小两根中央芯柱的半径差;
(2)水凝胶原液制备:
(2-1)外层水凝胶原液:将两种或多种可紫外光交联水凝胶按照一定配比溶解在含有光引发剂的去离子水中或者PBS中,搅拌过滤除菌后置于特定环境中备用;
(2-2)内层水凝胶原液:将可紫外光交联水凝胶溶解于含有光引发剂的PBS或者培养基中,添加功能特性材料并混合均匀,过滤除菌后置于特定环境中备用;所述的功能特性材料为细胞或者导电水凝胶;
上述的特定环境指的是环境温度在25~37℃的无菌环境。
(3)组织工程支架制备:
(3-1)将中空透明管和大直径的中央芯柱分别插入模具底座上的定位槽和定位阶梯孔并固定;
(3-2)将外层水凝胶原液注入中空透明管与大直径中央芯柱之间的空隙中,将模具静置直到溶液物理交联为止;
(3-3)在保持温度同步骤(3-2)的环境下,取出大直径的中央芯柱,插入小直径的中央芯柱并固定,然后将内层水凝胶原液注入至外层材料与小直径中央芯柱之间的空隙;
(3-4)将整个模具放置于紫外光下照射一定时间直到内、外两层材料全部光交联固化为止;
(3-5)拆卸模具,取出双层水凝胶管状组织工程支架。
步骤(2-1)中,外层水凝胶原液配置的具体过程为:将5~30%(w/v)GelMA与0~30%(w/v)PEGDA的粉末材料溶解于含有0.3~0.5%(w/v)光引发剂的去离子水或PBS中,在37~45℃搅拌配置成均匀溶液并过滤除菌后备用。
步骤(2-2)中,当功能特性材料为细胞时,内层水凝胶原液的配置过程为:
将5~10%(w/v)GelMA粉末材料溶解于含有0.3~0.5%(w/v)光引发剂的PBS中形成均匀溶液后过滤除菌;
取适量培养于培养基中的间充质干细胞,用0.25%的胰蛋白酶分离,离心去掉上清液后,添加到除菌后的5~10%的GelMA溶液中,搅拌均匀后备用。
当功能特性材料为导电水凝胶时,内层水凝胶原液的配置过程为:
将5~10%(w/v)GelMA粉末材料溶解于含有0.3~0.5%(w/v)光引发剂的PBS中形成均匀溶液,过滤除菌备用;
取适量PEDOT:PSS水溶液,超声波处理并通过0.75微米过滤筛过滤去除聚集大颗粒后,过滤除菌并添加到除菌后的5~10%的GelMA溶液中,搅拌形成0.1~0.4%(w/v)浓度分散均匀的悬浮液备用。
优选地,步骤(3-2)中,将模具在温度12~18℃的环境下静置8~12min,直到溶液物理交联。
进一步地,步骤(3-4)中,紫外光的波长365nm,紫外光照射密度为2W/cm2。
步骤(3-1)至(3-5)均需要在无菌环境下进行。
与现有技术相比,本发明具有以下有益效果:
1、本发明中,双层结构的设计,赋予管状支架复合功能,外层发挥可定制的机械性能,内层引入活细胞、导电凝胶等功能特性材料修饰提供多功能的微环境,使得支架机械性能与功能性(如生物相容性、导电性等)均能发挥到最优;
2、本发明的制备方法操作简单、材料选择范围广泛、支架结构稳定、管径调节灵活、方便进行各种功能性修饰(细胞或导电等)。此外,方法可直接获得包封活细胞的支架,更利于发挥相应的生理和生物学功能;
3、本发明制备方法制备的双层支架两层间结合紧密,不会脱离。未使用生物粘合剂,有效避免了因其降解导致的分层和层间脱离现象。
附图说明
图1为本发明制备方法采用的模具示意图;
图2为本发明制备方法的流程示意图;
图3为本发明制备方法制备的双层管状支架产品图。
具体实施方式
下面结合附图和实施例对本发明做进一步详细描述,需要指出的是,以下所述实施例旨在便于对本发明的理解,而对其不起任何限定作用。
实施例1
本实施例中,双层水凝胶管状组织工程支架的内层水凝胶为GelMA/细胞复合材料,外层水凝胶为GelMA/PEGDA复合材料。
具体的,内层结构由低浓度GelMA水凝胶和细胞复合而成,能够提供多功能的组织微环境,具有较好的生物相容性。外层结构由可增强力学性能的PEGDA和具有热、光双交联特性的大分子GelMA水凝胶复合而成,实现机械强度支撑。两层结构自成孔隙,外层比内层更致密。
两层之间的结合由两层结构中相同的主成分GelMA水凝胶的双交联特性实现,可逆的物理交联定型后不可逆的光交联定性,保证两层结构的紧密结合。
模具方案:
如图1所示,模具包括底座1、中空透明管2、大直径中央芯柱3、小直径中央芯柱4以及对应的模具盖5和模具盖6。底座1上同轴设置有与中空透明管2匹配的定位槽11以及与两根中央芯柱匹配的定位阶梯孔12,模具盖上也对应设有定位阶梯孔和定位槽。
模具材料的透明度根据内外层水凝胶材料中光引发剂含量、所需紫外光波段(常规波段在330-380nm,365是常见UV光波段)及照射时间决定。相应中空透明管的透过率优选为>80%。中央芯柱具有直径差,用来保证管状支架内层结构的厚度,中央芯柱的外径公差直线度与模具底座的阶梯孔配合公差是保证管状支架内外层结构管子壁厚均匀性和同轴度的关键。优选使用参数是H7f7的配合和的同轴度。
内外层水凝胶原液制备:
外层水凝胶原液:将20%(w/v)GelMA与20%(w/v)PEGDA粉末材料溶解于含有0.5%(w/v)光引发剂(LAP)的去离子水中,在37℃搅拌配置成均匀溶液,除菌过滤备用。
内层水凝胶原液:将无菌10%(w/v)GelMA粉末材料溶解于含有0.5%(w/v)光引发剂(LAP)的无菌磷酸缓冲液(PBS)中形成均匀均匀溶液;取适量培养于培养基(DMEM)中的骨髓间充质干细胞(BMSC),用0.25%的胰蛋白酶分离,1000转每分钟的速度离心5分钟后,去掉上清液后,添加到配置的10%的GelMA溶液中并搅拌,得到细胞悬液,整个过程保证无菌操作。
如图2所示,具体制备步骤如下:
步骤1,将最外层的中空透明管及大直径中央芯柱插入模具底座并固定。
步骤2,将外层溶液注入模具中空透明管于大直径中央芯柱之间,将模具置于12~18℃保持10min直到溶液物理交联。
步骤3,在保持12~18℃环境下取出大直径中央芯柱,插入小直径中央芯柱并固定,然后在外层材料于小直径中央芯柱之间空隙中注入内层细胞悬液。
步骤4,将模具至于紫外光(365nm,2W/cm2)下照射30s使两层材料光交联固化。
步骤5,拆模具,取出双层水凝胶管状支架,浸泡于培养基中并于37℃,5%CO2的培养箱中培养备用。
上述整个过程确保无菌操作,最终制备得到的双层水凝胶管状组织工程支架如图3所示,图中,A为横截面的数码照片,B-F为轴向的数码照片,G为横截面的显微图片,H为横截面的SEM图片,I为横截面细胞活死染色下的显微图片。从A-F中可以看出,制备的管状支架结构稳定,壁厚均匀,弹性显著,变形能力强,被弯曲成90°、180°和拉长至两倍后没有裂缝产生,并且外力撤除后能快速恢复。G-I均显示出制备的管状支架为两层结构,两层紧密结合自成一体。从H中可以看出,管状水凝胶支架的两层存在不同的孔隙结构,其中外层相对致密提供机械支撑,而内层的孔径更大可以为细胞的生长提供更好的微环境。细胞的活死染色图I进一步说明包封在内层的细胞具有较高的活性,证明所述制备方法对细胞友好,可以直接获得含活细胞的支架。
实施例2
本实施例中,内层水凝胶为GelMA/PEDOT:PSS复合材料,外层水凝胶为GelMA/PEGDA复合材料。
与实施例1的区别仅在于内层水凝胶原液的配制,其余步骤与实施例一相同。
内层水凝胶原液配制:将无菌10%(w/v)GelMA粉末材料溶解于含有0.5%(w/v)光引发剂(LAP)的无菌磷酸缓冲液(PBS)中形成均匀溶液;取适量PEDOT:PSS水溶液,超声波处理并通过0.75微米过滤筛过滤去除聚集大颗粒后,添加到配置的10%的GelMA溶液中并搅拌形成0.1~0.4%(w/v)浓度分散均匀的悬浮液。
以上所述的实施例对本发明的技术方案和有益效果进行了详细说明,应理解的是以上所述仅为本发明的具体实施例,并不用于限制本发明,凡在本发明的原则范围内所做的任何修改、补充和等同替换,均应包含在本发明的保护范围之内。
Claims (1)
1.一种双层水凝胶管状组织工程支架,其特征在于,包括管状的内层水凝胶和外层水凝胶,所述的内层水凝胶由可紫外光交联水凝胶和细胞或导电水凝胶复合而成;所述的外层水凝胶由两种或多种可紫外光交联水凝胶复合而成;
组成内层水凝胶的可紫外光交联水凝胶为甲基丙烯酸酐化明胶GelMA,细胞为间充质干细胞,导电水凝胶为PEDOT:PSS;外层水凝胶由两种可紫外光交联水凝胶复合而成,分别为甲基丙烯酸酐化明胶GelMA和聚乙二醇二丙烯酸酯PEGDA;
所述双层水凝胶管状组织工程支架的制备方法包括以下步骤:
(1)模具制备:
模具包括底座、一根中空透明管和两根直径不同的中央芯柱;底座上同轴设置有与中空透明管匹配的定位槽以及与两根中央芯柱匹配的定位阶梯孔;内层水凝胶的厚度对应大小两根中央芯柱的半径差;
(2)水凝胶原液制备:
(2-1)外层水凝胶原液:将两种或多种可紫外光交联水凝胶按照一定配比溶解在含有光引发剂的去离子水中或者PBS中,搅拌后过滤除菌备用;具体过程为:
将5~30%(w/v)GelMA与0~30%(w/v)PEGDA的粉末材料溶解于含有0.3~0.5%(w/v)光引发剂的去离子水或PBS中,在37~45℃搅拌配置成均匀溶液后,用0.22微米微孔滤膜滤器过滤除菌备用;
(2-2)内层水凝胶原液:将可紫外光交联水凝胶溶解于含有光引发剂的PBS或者培养基中,添加功能特性材料并混合均匀后过滤除菌备用;所述的功能特性材料为细胞或者导电水凝胶;
所述的功能特性材料为细胞时,内层水凝胶原液的配置过程为:将5~10%(w/v)GelMA粉末材料溶解于含有0.3~0.5%(w/v)光引发剂的PBS中形成均匀溶液,用0.22微米微孔滤膜滤器过滤除菌备用;取适量培养于培养基中的间充质干细胞,用0.25%的胰蛋白酶分离,离心去掉上清液后,添加到除菌后的5~10%的GelMA溶液中,搅拌均匀后备用;
所述的功能特性材料为导电水凝胶时,内层水凝胶原液的配置过程为:将5~10%(w/v)GelMA粉末材料溶解于含有0.3~0.5%(w/v)光引发剂的PBS中形成均匀溶液;取适量PEDOT:PSS水溶液,超声波处理并通过0.75微米微孔滤膜滤器过滤去除聚集大颗粒后,添加到配置的5~10%的GelMA溶液中,搅拌形成0.1~0.4%(w/v)浓度分散均匀的悬浮液备用;
(3)组织工程支架制备:
(3-1)将中空透明管和大直径的中央芯柱分别插入模具底座上的定位槽和定位阶梯孔并固定;
(3-2)将外层水凝胶原液注入中空透明管与大直径中央芯柱之间的空隙中,将模具在温度12~18℃的环境下静置8~12min直到溶液物理交联为止;
(3-3)在保持温度同步骤(3-2)的环境下,取出大直径的中央芯柱,插入小直径的中央芯柱并固定,然后将内层水凝胶原液注入至外层材料与小直径中央芯柱之间的空隙;
(3-4)将整个模具放置于紫外光下照射一定时间直到内、外两层材料全部光交联固化为止;紫外光的波长365nm,紫外光照射密度为2W/cm2;
(3-5)拆卸模具,取出双层水凝胶管状组织工程支架;
步骤(3-1)至(3-5)均需要在无菌环境下进行。
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CN107898533B (zh) * | 2017-11-22 | 2019-11-19 | 上海大学 | 人工载药同轴再生血管支架及其复合工艺制备方法 |
CN110665061A (zh) * | 2018-09-05 | 2020-01-10 | 浙江大学 | 一种脱细胞支架溶液-GelMA水凝胶复合材料及制备方法 |
CN110743040B (zh) * | 2019-10-25 | 2021-08-20 | 福建省安悦莱生物科技有限公司 | 一种多通道挤出3d生物打印制备仿生骨骼肌复合组织 |
CN111481320B (zh) * | 2020-04-30 | 2022-12-09 | 中国医科大学 | 用于制备复杂器官的专用组合模具制备肝脏前体的方法 |
CN111773435B (zh) * | 2020-06-18 | 2021-04-23 | 大连大学附属中山医院 | 一种用于关节软骨修复的双交联一体化无缝复合水凝胶支架 |
CN111921016B (zh) * | 2020-07-20 | 2022-04-26 | 中国医科大学 | 一种人工器官制造模具及人工器官制造方法 |
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