CN114042191A - 一种细胞打印的成骨功能化支架及其制备方法和应用 - Google Patents
一种细胞打印的成骨功能化支架及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及生物骨组织工程支架技术领域，提供了一种细胞打印的成骨功能化支架及其制备方法和应用。本发明公开了将hBMSCs、HUVECs细胞与GelMA水凝胶混合，通过挤出式3D打印技术打印得到具有生物活性的网格状方块水凝胶构建物，然后在光引发剂下采用蓝光照射得到成骨功能化支架。利用3D打印技术打印骨支架满足了不规则骨缺损的修复，该支架不仅具有良好的机械性能和结构维持性，且实现了不同细胞的精确定位个性化打印，细胞能够在支架内部生长与分化，具有良好的生物相容性和促成骨分化、血管形成能力，在骨缺损修复材料领域发挥重要的作用。
Description
技术领域
本发明涉及生物骨组织工程支架技术领域，具体地，涉及一种细胞打印的成骨功能化支架及其制备方法和应用。
背景技术
外伤、感染、肿瘤切除和先天畸形等均可导致严重骨缺损，影响患者外形和功能，“金标准”自体骨移植修复需开辟第二术区，引起新的骨缺损，给患者的生理、心理和经济带来巨大负担，且修复效果也并不理想。临床上大多数骨缺损均为不规则形，传统的骨修复支架难以满足大量的个性化病例。而3D打印技术能够很好地解决个性化病例问题。3D打印技术制备的骨组织工程支架在支架精确性、个性化、机械强度、孔隙调节、空间结构复杂性方面有独特优势。
甲基丙烯酸化水凝胶(GelMA)是由甲基丙烯酸酐(MA)与明胶(Gelatin)制备得到的光敏性水凝胶，其可通过紫外及可见光在光引发剂作用下交联固化成胶，是一种3D打印技术领域中常见的基体材料。GelMA兼具天然和合成生物材料的特征，具有优良的生物相容性和细胞反应特性，如：提供合适的细胞粘附位点及蛋白水解降解性等，它可以取代人工基底膜或其他天然胶原蛋白水凝胶。例如公布号为CN107663377A的中国专利公开了一种具有温敏和光敏特性的混合水凝胶及其3D打印方法，通过添加明胶来改善低浓度单一成分的GelMA水凝胶的力学性能和结构维持性，提高凝胶的可打印性。然而，在现有的骨组织工程技术中，通常采用把细胞植入到支架表面后进行诱导培养，这种方式容易导致细胞脱落率较高、2D黏附的细胞生长环境不能模拟体内细胞外基质的“3D水环境”以及多细胞培养不能精确定位进行个性化打印，而且当骨缺损越大，用于修复的组织工程骨体积也就越大，由于缺乏氧气和营养的供应，越靠近支架中心的细胞越难存活。因此，如何同时改善GelMA水凝胶的物理性能和生物功能仍是3D打印GelMA水凝胶领域所面临的挑战，开发一种兼具良好的机械性能与成骨分化、血管化能力的骨组织支架具有重要的意义。
发明内容
本发明的目的在于克服现有技术中存在的上述缺陷和不足，提供一种细胞打印的成骨功能化支架。
本发明的第二个目的在于提供上述成骨功能化支架的制备方法。
本发明的第三个目的在于提供上述成骨功能化支架在作为或制备骨缺损修复材料中的应用。
本发明的上述目的是通过以下技术方案给予实现的：
本发明提供了成骨功能化支架的制备方法，且对其进行细胞活性检测及成骨成血管功能检测。通过细胞活死检测和碱性磷酸酶染色实验，结果表明，成骨功能化支架不仅具有良好的细胞相容性，细胞能够在水凝胶支架中良好的增殖，而且具有促进成骨分化和血管生成的作用。
一种细胞打印的成骨功能化支架，将甲基丙烯酸酯化水凝胶、人骨髓间充质干细胞与人脐静脉内皮细胞混合，通过挤出式3D打印技术逐层堆叠打印，在光引发剂下采用蓝光照射快速固化成型。
本发明提供的成骨功能化支架，通过将GelMA水凝胶与人骨髓间充质干细胞(hBMSCs)与人脐静脉内皮细胞(HUVECs)混合打印，更有利于细胞在支架的内部生长，促进骨缺损部位成骨化与血管化。
上述成骨功能化支架的制备方法，包含以下步骤：
S1.将光引发剂苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入GelMA水溶液中，混合均匀，经0.22μm滤膜挤出除菌，得到水凝胶混合液，放置备用；
S2.将培养了3～8代的人骨髓间充质干细胞与人脐静脉内皮细胞分别消化重悬，混合，离心，置于培养基重悬；
S3.将步骤S2所述混合细胞液加入步骤S1所述水凝胶混合液中，置于医用注射器中，预冷；
S4.将注射器置于机械臂挤压成型生物打印机中，打印得到网格状方块水凝胶构建物；用蓝光照射，垂直距离2～3cm，即得。
优选地，步骤S1所述GelMA水溶液的制备方法为：将明胶溶于PBS，60℃下搅拌至完全溶解，然后将甲基丙烯酸酐以0.5mL/min的速度加入到明胶溶液中，反应2～4h，用PBS稀释，透析，冷冻干燥即得。
更优选地，步骤S1所述明胶与甲基丙烯酸酐的质量体积比为(1～2)g：(10～20)mL。
优选地，步骤S1所述GelMA水溶液中的GelMA的质量分数为5％～20％；所述苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐与GelMA水溶液的质量体积比为1.5～2mg/mL；所述放置条件为37℃下避光环境。
优选地，步骤S2所述人骨髓间充质干细胞与人脐静脉内皮细胞的重量比为(1～4)：(0～3)。
更优选地，人骨髓间充质干细胞与人脐静脉内皮细胞的重量比为(3～4)：(0～2)。
优选地，步骤S3所述混合细胞在水凝胶中的密度为1×106～3×106个/mL；所述预冷的温度为4℃。
优选地，步骤S4所述打印的条件为打印速度8mm/s，填充率30～40％，接收平台温控4～10℃，料筒温度26～27℃，正转速度2～5％，层高2～4mm。
优选地，步骤S4所述方块水凝胶构建物的孔径为300～500μm。
优选地，步骤S4所述蓝光的波长为波长405nm；所述照射时间为10～30s。
本发明还提供上述制备方法得到的成骨功能化支架在作为或制备人体组织修复材料中的应用。
优选地，上述制备方法得到的成骨功能化支架在作为或制备骨缺损修复材料中的应用。
与现有技术相比，本发明具有以下有益效果：
(1)在光引发剂的作用下，GelMA水凝胶实现快速固化成型，提高了GelMA水凝胶的机械性能，从而提高3D打印成骨功能化支架的力学性能和结构维持性。
(2)本发明提供了将不同的细胞与GelMA水凝胶混合，通过3D细胞打印技术得到成骨功能化支架，满足不规则骨缺损的修复。本发明构建的成骨功能化支架是一个具有生物活性的三维“细胞/材料”体系，其不仅能够实现不同细胞的精确定位个性化打印，且细胞能够在支架内部生长与分化，具有良好的生物相容性以及促成骨分化和血管形成能力。
(3)本发明采用的构建物模型为格栅状网络模型，将支架空隙的大小调节成了适合血管和硬骨组织生长的尺寸。
附图说明
图1为成骨功能化支架的整体形貌图。
图2为成骨功能化支架的光学显微镜图(40×)。
图3为不含细胞的GelMA水凝胶的扫描电镜图。
图4为成骨功能化支架的细胞活死染色图。
图5为成骨功能化支架的碱性磷酸酶染色图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1成骨功能化支架的制备
(1)将2g明胶溶于20mL PBS，60℃下搅拌至完全溶解，然后将15mL甲基丙烯酸酐以0.5mL/min的速度加入到明胶溶液中，反应2～4h，用PBS稀释，透析，冷冻干燥，得到GelMA水溶液；
(2)以2mg/mL的浓度比例将苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入到20mLGelMA水溶液中，混合均匀，经0.22μm滤膜挤出除菌，得到水凝胶混合液，避光放置在37℃水浴备用，防止凝胶化；
(3)将培养至3代的hBMSCs和HUVECs消化重悬，分别按照4:1的比例混合，离心，放入70μL培养基(1:1＝OM：EM)重悬，得到混合细胞液；
(4)将步骤(3)的混合细胞液与步骤(2)的水凝胶混合液以1×106个/mL的细胞密度混合于1mL医用注射器中，随即放入4℃冰箱预冷3分钟，使水凝胶能更好的被挤出打印；
(5)将预冷后的注射器置于机械臂挤压成型生物打印机中，设置打印速度8mm/s、填充率30％、接收平台温控5℃、料筒温度26℃、正转速度2％，层高2mm，打印得到孔径为300μm的网格状方块水凝胶构建物，然后用波长405nm蓝光照射水凝胶10s，垂直距离2cm，得到成骨功能化支架。
本发明提供的成骨功能化支架的整体形貌图如图1所示，由图可知打印得到的成骨功能化支架的结构维持性良好；光学显微镜下的成骨功能化支架图(40×)如图2所示；不含细胞的GelMA水凝胶的扫描电镜图如图3所示，可以看出该支架的表面形貌呈格栅状网络结构。
实施例2成骨功能化支架的制备
(1)GelMA水溶液的制备与实施例1中的步骤(1)一致；
(2)以1.8mg/mL的浓度比例将苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入到30mLGelMA水溶液中，混合均匀得到混合液，经0.22μm滤膜挤出除菌，得到水凝胶混合液，避光放置在37℃水浴备用，防止凝胶化；
(3)将培养至5代的hBMSCs和HUVECs消化重悬，分别按照3:1的比例混合，离心，放入75μL培养基(1:1＝OM：EM)重悬，得到混合细胞液；
(4)将步骤(3)的混合细胞液与步骤(2)的水凝胶混合液以2×106个/mL的细胞密度混合于1mL医用注射器中，随即放入4℃冰箱预冷3分钟，使水凝胶能更好的被挤出打印；
(5)将预冷后的注射器置于机械臂挤压成型生物打印机中，设置打印速度8mm/s、填充率40％、接收平台温控4℃、料筒温度26℃、正转速度3％，层高3mm，打印得到孔径为400μm的网格状方块水凝胶构建物，然后用波长405nm蓝光照射构建物15s，垂直距离3cm，得到成骨功能化支架。
实施例3成骨功能化支架的制备
(1)GelMA水溶液的制备与实施例1中的步骤(1)一致。
(2)以1.5mg/mL的浓度比例将苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入到20mLGelMA水溶液中，混合均匀得到混合液，经0.22μm滤膜挤出除菌，得到水凝胶混合液，避光放置在37℃水浴备用，防止凝胶化；
(3)将培养至8代的hBMSCs和HUVECs消化重悬，分别按照1:1的比例混合，离心，放入70μL培养基(1:1＝OM：EM)重悬，得到混合细胞液；
(4)将步骤(3)的混合细胞液与步骤(2)的水凝胶混合液以3×106个/mL的细胞密度混合于1mL医用注射器中，随即放入4℃冰箱预冷3分钟，使水凝胶能更好的被挤出打印；
(5)将预冷后的注射器置于机械臂挤压成型生物打印机中，设置打印速度8mm/s、填充率35％、接收平台温控10℃、料筒温度26℃、正转速度5％，层高4mm，打印得到孔径为500μm的网格状方块水凝胶，然后用波长405nm蓝光照射水凝胶30s，垂直距离3cm，得到成骨功能化支架。
实施例4成骨功能化支架的制备
仅将实施例3中hBMSCs和HUVECs的比例替换为1:3，制备成骨功能化支架。
测试例1成骨功能化支架的细胞活性检测
将实施例2制备得到成骨功能化支架培养至第7天，用PBS冲洗2～3次，利用活死染色试剂盒对其细胞活力进行检测，染色完毕后用倒置荧光显微镜观察细胞的活死情况。
结果分析：第七天成骨功能化支架的活死染色结果如图4所示，结果显示hBMSCs和HUVECs能够在支架中均匀地分布，且成活率良好。
测试例2成骨功能化支架的功能性检测
通过碱性磷酸酶染色来检测成骨功能化支架的成骨情况。将实施例2制备得到成骨功能化支架培养至第7天后，PBS清洗2～3次，用4％多聚甲醛固定20～30min，再次用PBS清洗2～3次；滴加配置好的碱性磷酸酶工作液，放入湿盒中，避光孵育5～40min，直至显色至预期染色，PBS清洗，干燥，用倒置显微镜观察。
结果分析：在倒置显微镜下观察到的成骨功能化支架的碱性磷酸酶染色图如图5所示，从图中可以看出GelMA水凝胶中有蓝灰色的碱性磷酸酶颗粒，表明该成骨功能化支架具有良好的成骨分化和血管化能力。
Claims (10)
1.一种细胞打印的成骨功能化支架，其特征在于，将甲基丙烯酸酯化水凝胶、人骨髓间充质干细胞与人脐静脉内皮细胞混合，通过挤出式3D打印技术逐层堆叠打印，在光引发剂下采用蓝光照射快速固化成型。
2.权利要求1所述成骨功能化支架的制备方法，其特征在于，包含以下步骤：
S1.将光引发剂苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐加入GelMA水溶液中，混合均匀，除菌，得到水凝胶混合液，放置备用；
S2.将培养了3～8代的人骨髓间充质干细胞与人脐静脉内皮细胞分别消化重悬，混合，离心，置于培养基重悬，得到混合细胞液；
S3.将步骤S2所述混合细胞液加入步骤S1所述水凝胶混合液中，置于医用注射器中，预冷；
S4.将注射器置于机械臂挤压成型生物打印机中，打印得到网格状方块水凝胶构建物；用蓝光照射，垂直距离2～3cm，即得。
3.根据权利要求2所述制备方法，其特征在于，步骤S1所述GelMA水溶液中的GelMA的质量分数为5％～20％；所述苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐与GelMA水溶液的质量比为1.5～2mg/mL；所述放置条件为37℃下避光环境。
4.根据权利要求2所述制备方法，其特征在于，步骤S2所述人骨髓间充质干细胞与人脐静脉内皮细胞的重量比为(1～4)：(0～3)。
5.根据权利要求2所述制备方法，其特征在于，步骤S3所述混合细胞在水凝胶中的密度为1×106～3×106个/mL；所述预冷的温度为4℃。
6.根据权利要求2所述制备方法，其特征在于，步骤S4所述打印的条件为打印速度8mm/s，填充率30～40％，接收平台温控4～10℃，料筒温度26～27℃，正转速度2～5％，层高2～4mm。
7.根据权利要求2所述制备方法，其特征在于，步骤S4所述方块水凝胶构建物的孔径为300～500μm。
8.根据权利要求2所述制备方法，其特征在于，步骤S4所述蓝光的波长为波长405nm；所述照射时间为10～30s。
9.权利要求2～8所述制备方法得到的成骨功能化支架在作为或制备人体组织修复材料中的应用。
10.权利要求2～8所述制备方法得到的成骨功能化支架在作为或制备人体骨缺损修复材料中的应用。
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| CN115212002A (zh) * | 2022-06-15 | 2022-10-21 | 苏州大学 | 一种用于修复软骨缺损的3d生物打印支架及其制备 |
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| CN115212002A (zh) * | 2022-06-15 | 2022-10-21 | 苏州大学 | 一种用于修复软骨缺损的3d生物打印支架及其制备 |
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