CN117618653B - 一种用于面神经缺损修复的3d打印神经再生导管及其制备方法 - Google Patents
一种用于面神经缺损修复的3d打印神经再生导管及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于面神经缺损修复的3D打印神经再生导管及其制备方法,属于神经再生导管技术领域。该神经再生导管包括PCL壳结构和水凝胶核结构。本发明创新性负载诱导为类施万细胞的脱落人乳牙牙髓干细胞scSHED,以起到模仿自体神经移植中崩解后神经纤维的情景,并可规避复杂的缓释系统。本发明创新性构建负载相关药物的PCL膜,在模拟自体神经移植中神经外膜提供足够的机械性能保护和屏障的情况下,亦保持了足够的渗透性,同时负载的药物亦可促使巨噬细胞向M2型极化,促进组织的修复效果。
Description
技术领域
本发明属于神经再生导管技术领域,特别是涉及一种用于面神经缺损修复的3D打印神经再生导管及其制备方法。
背景技术
面神经是第七对脑神经,是混合神经,其运动支司职面部诸多表情肌的运动,损伤可导致患者出现面瘫,干扰患者的社会生活和身心健康。口腔颌面外科常见的周围性面神经损伤可能由贝尔面瘫、外伤、炎症、腮腺区肿瘤及医源性因素等导致。严重的外伤或恶性腮腺肿瘤需要扩大切除时,往往导致神经缺损超过5mm,直接缝合困难,此时外科治疗的“金标准”为自体神经移植术,需要牺牲患者健康的神经,一般是耳大神经或腓肠神经。该修复方式可以达到相对理想的修复效果,但难以完全恢复表情功能,且可能引起连带运动等并发症,依然影响患者的表情功能,供区也必然出现神经功能损伤,如感觉麻木等。而躯体的其他周围神经收到如车祸等而出现损伤时,患者的肢端瘫痪情况较面神经可能更甚,其修复效果亦相对有限,供应神经的选择更为受限。
因此,为神经移植选择合适的供体一直是相关领域的研究热点,除了自体神经移植以外,既往包括同种异体脱细胞导管、脱细胞血管、不同生物材料的人工导管等均被提及和应用,但尚未发现某种材料可以理想的满足临床应用。现有可供患者选择的人工神经移植物如“神桥”,即为脱细胞的细胞外基质,其优势为组份对神经再生具有较好的促进作用,但修复效果仍欠理想,且来源为同种异体或异种供应,一定程度上存在供应有限、免疫排斥等问题。
3D生物打印技术是一种新兴的组织工程支架制造技术,能够在三维空间上控制功能组件放置而生成复杂的三维结构,通过计算机辅助设计和打印材料的调配,产生可控的孔隙率和机械性能,从而成个性化导管。多喷头协同打印技术也可解决传统导管组份单一、没有精细结构的问题,通过预先设计内、外层或高、低层使用不同喷头,可以构建具有个性化和多层次的仿生结构。
现有的传统生产工艺如单纯静电纺丝、模具压制、自组装等,流程复杂且精度有限,难以实现患者的个性化和对微观结构的调控,同时受其生产流程的限制,难以方便负载如细胞、生物因子等可提高神经再生效果的成分,限制了修复的效果。.现有的构造方法中,负载物或为干细胞,或为神经生长因子等单一成分。干细胞在体内的免疫特性及分化情况无法预测,而单一成分的生物因子,其效果相对有限,且需要复杂的缓释系统适配机体自身的修复时间。现有的神经再生导管其机械性能一般弹性差,且单一成分者易存在渗透性过高或过低的情况,对周围毛细血管的长入和营养物质交换存在障碍,植入物亦可引起不同程度的免疫炎症反应。
发明内容
针对现有技术的不足,本发明使用3D打印技术,同时结合静电纺丝技术,可以实现方便负载细胞、生物因子,并分别构建不同组件,降低制造难度。本发明通过负载诱导为类施万细胞的脱落人乳牙牙髓干细胞(scSHED),其可能提供更多种类的促神经恢复相关细胞因子,且可达到缓释的效果。本发明构建同时具有理想的机械性能、渗透性和药物负载能力的模拟神经外膜结构的组件。
本发明的第一方面在于公开一种用于面神经缺损修复的3D打印神经再生导管,包括PCL壳结构和水凝胶核结构。
在本发明的一些实施方式中,所述PCL壳结构包括PCL膜及负载与所述PCL膜的细胞因子。
在本发明的一些实施方式中,所述细胞因子为白细胞介素,优选为IL-4和IL13。
在本发明的一些优选的实施方式中,IL-4和IL13的重量比例为1:(2-5),优选为1:(3-3.5)。
在本发明的一些优选的实施方式中,IL-4在PCL壳结构中的重量含量为0.01-0.06%,IL13在PCL壳结构中的重量含量为0.05-0.15%;优选为,IL-4在PCL壳结构中的重量含量为0.02-0.06%,IL13在PCL壳结构中的重量含量为0.10-0.15%。
在本发明的一些实施方式中,所述水凝胶核结构中含有经诱导的类施万细胞样脱落人乳牙牙髓干细胞。
在本发明的一些优选的实施方式中,所述核结构的水凝胶中的scSHED的含量为2×104到1×107个/ml,优选为1×106到1×107个/ml。
在本发明的一些实施方式中,所述水凝胶核结构为以CaCl2交联的RGD接枝海藻酸钠和甲基丙烯酸明胶制成的平面网状结构。
本发明的第二方面在于公开第一方面所述的用于面神经缺损修复的3D打印神经再生导管的制备方法,包括以下步骤:
S01,制备PCL壳结构;
S02,制备水凝胶核结构;
S03,将所述PCL壳结构包裹所述水凝胶核结构,得到神经再生导管。
在本发明的一些实施方式中,S01中,将PCL制成PCL静电纺丝溶液,纺丝,构建PCL膜,负载细胞因子。
在本发明的一些实施方式中,S01中,所述PCL在PCL静电纺丝溶液中的浓度为10-20%w/v;
和/或,S01中,所述PCL静电纺丝溶液的溶剂为氯仿和甲醇。
在本发明的一些实施方式中,S02中,将所述RGD接枝海藻酸钠、甲基丙烯酸明胶和脱落人乳牙牙髓干细胞制成水凝胶溶液,制成平面网状结构,使用CaCl2溶液交联;优选,将所述水凝胶溶液3D打印制成所述平面网状结构。
在本发明的一些实施方式中,S02中,RGD接枝海藻酸钠的含量为2-6%w/v,GelMA的含量为2-8%w/v;优选为,RGD接枝海藻酸钠的含量为3-7%w/v,GelMA的含量为3-7%w/v。
有益效果:
本发明使用3D打印技术,同时结合静电纺丝技术,可以实现针对不同患者的个性化设计,并方便负载细胞、生物因子,亦针对周围神经的结构进行仿生设计,分别构建不同组件,降低制造难度。
本发明通过负载诱导为类施万细胞的脱落人乳牙牙髓干细胞(scSHED),借助其较低的免疫原性,降低体内排异情况,以延长其在体内存留时间。scSHED可模拟施万细胞,在神经纤维再生中起到诱导、分泌促神经生长因子等多种作用,相较于负载未经诱导的干细胞具有更集中于神经再生的功能,相较于负载生物因子等单一药物,其可能提供更多种类的促神经恢复相关细胞因子,且可达到缓释的效果。
本发明通过静电纺丝技术构建聚己内酯(Polycaprolactone,PCL)膜,在模拟自体神经移植中神经外膜提供足够的机械性能保护和屏障的情况下,亦保持了足够的渗透性,同时负载的药物亦可促使巨噬细胞向M2型极化,促进组织的修复效果。
本发明的3D打印神经再生导管中,IL-4和IL13的浓度和二者之间的比例、scSHED细胞数目以及构建核结构的水凝胶的组分含量共同促进神经再生,显著提高再生神经纤维数目和纤维直径。
附图说明
图1为本发明的一种实施方式的用于面神经缺损修复的3D打印神经再生导管的制备方法流程图。
图2为本发明的一种实施方式的人乳牙牙髓干细胞(SHED)诱导为类施万细胞的脱落人乳牙牙髓干细胞后的因子表达;其中,纵坐标为倍数,以SHED为基准1。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
若非特别指出,实施例和对比例为组分、组分含量、制备步骤、制备参数相同的平行试验。所述CaCl2溶液交联中,CaCl2溶液的浓度为25mM,用量为为浸泡住所打印的支架结构即可,约1ml,浸泡5min。所述平面网状结构,横轴较粗起支持加固作用,纵轴较细起诱导神经纤维再生方向的作用。
若非特别指出,实施例和对比例中脱落人乳牙牙髓干细胞(scSHED)为诱导为类施万细胞的脱落人乳牙牙髓干细胞。
SHED提取方法(胶原酶消化法):自愿捐赠的儿童滞留乳牙拔除后样本,无菌条件下劈开牙齿,获取牙髓,用含双抗的PBS反复冲洗并充分剪碎,加入等体积的一型胶原酶和中性蛋白酶混合物,消化离心,重悬,将细胞均匀接种于培养瓶底,常规培养。待细胞生长至80%融合时,胰酶消化后传代培养。选用流式细胞仪对SHED进行鉴定。收集对数期生长的4-7代细胞用于后续操作。
SHED来源于神经嵴,具有良好的神经分化潜能,将SHED诱导为类施万细胞,其可表达相应的神经再生相关因子,可以更好的模拟周围神经再生环境,从而提高修复效果。并且避免了直接放置未经诱导的干细胞时,细胞在体内具体分化方向不明的情况。
测定细胞诱导前后S-100β蛋白,MPZ蛋白和GFAP蛋白的表达情况,用免疫荧光检查。
以及对NGF,BDNF,GDNF等蛋白基因表达量的测定,用qrtPCR技术。具体包括:
提RNA:拿出细胞版,PBS洗一次;若直接从瓶子里提细胞出来,则每个样取2*10^6个细胞。每个6孔板的孔加入500ul的Trizol液,静置3min;分别吹打至稀薄状液体,分别移至大EP管,做好标记。每管加入100ul氯仿,颠倒混匀,静置10min,4℃12000转15min离心。将每个大EP管的上清液移至新的大EP管。往每个大EP管加入500ul的异丙醇,振荡器上混匀,静置10min,4℃12000转10min离心。用枪头吸去上清,只留管底的白色固体,敞开管口晾干5~10min(注意环境要防尘)。每管加入1ml的75%乙醇,颠倒混匀,4℃7500转5min,用枪头吸去上清,只留管底的白色固体。每管加入1ml无水乙醇,颠倒混匀,4℃7500转5min。用枪头吸去上清,只留管底的白色固体,敞开管口晾干5~10min晾至透明。根据白色固体的大小,加入30~50ul的DEPC水。使用Nanodrop软件测RNA浓度:每次吸取1ul;先用DEPC水洗检测头三次,再进行RNA测定。
qPCR方法(全程冰上操作):
1.逆转录:在小EP管中使用20ul体系
(1)按照每孔6ul水、4ul mix先全部配好在大EP管,混匀在每个小EP管加入10ul。
(2)将每种RNA溶液取少许出来,加入DEPC水稀释至100ng/ul,加10ul至小EP管。
将装有20ul体系的小EP管在瞬离机上瞬离20s,在逆转录PCR仪上设立程序37℃15min+85℃5s+4℃forever,获得cDNA,可放至-20℃保存3个月。
2.荧光定量PCR:
(1)配引物:根据每小管引物的nmol数,加入10x的ul的DEPC水,可放-20℃长期保存6个月。
(2)每孔需要10ul syber+7ul DEPC水+1ul R+F引物,先配好到大EP管,振荡器震荡后瞬离10s,每个PCR板孔加18ul到孔底。分别加稀释后的cDNA溶液2ul到孔壁。贴膜离心,2步法40个循环。
结果见图2,表明诱导后细胞NGF、BDNF、GDNF和PDGF的表达量均有所提高。
实施例1
一种用于面神经缺损修复的3D打印神经再生导管的制备方法,如图1所示:
1.将PCL原料(聚已内酯)溶解在氯仿和甲醇的混合物(5:1v/v)中,制备浓度为15%w/v的PCL静电纺丝溶液,使用静电纺丝机并用可编程注射泵(Cole Parmer,USA)以4ml/h的速度流动。注射器针头连接的正高压电源设置为16kV,接收器收集纺丝,构建6mm×10mm×0.5mm的PCL膜,使用乙二胺法将IL-4和IL13负载于PCL膜上制备壳结构,IL-4的含量为0.04%,IL13含量为0.12%。
2.将RGD(精氨酸-甘氨酸-天冬氨酸,Arg-Gly-Asp)接枝海藻酸钠和甲基丙烯酸明胶(GelMA)、scSHED(脱落人乳牙牙髓干细胞)溶解在PBS中,制备水凝胶溶液;RGD接枝海藻酸钠的含量为6%w/v,GelMA的含量为5%w/v,scSHED的含量2×106个/ml。将水凝胶置入3D生物打印机喷头料筒中,计算机辅助设计控制下打印为10mm×10mm的平面网状结构,使用CaCl2溶液交联,并使用无菌器械将其卷起,构建水凝胶核结构。
3.将PCL壳包裹水凝胶核,制备神经再生导管。
实施例2
一种用于面神经缺损修复的3D打印神经再生导管的制备方法,如图1所示:
1.将PCL原料(聚已内酯)溶解在氯仿和甲醇的混合物(5:1v/v)中,制备浓度为15%w/v的PCL静电纺丝溶液,使用静电纺丝机并用可编程注射泵(Cole Parmer,USA)以4ml/h的速度流动。注射器针头连接的正高压电源设置为16kV,接收器收集纺丝,构建6mm×10mm×0.5mm的PCL膜,使用乙二胺法将IL-4和IL13负载于PCL膜上制备壳结构,IL-4的含量为0.02%,IL13含量为0.10%。
2.将RGD(精氨酸-甘氨酸-天冬氨酸,Arg-Gly-Asp)接枝海藻酸钠和甲基丙烯酸明胶(GelMA)、scSHED(脱落人乳牙牙髓干细胞)溶解在PBS中,制备水凝胶溶液;RGD接枝海藻酸钠的含量为6%w/v,GelMA的含量为5%w/v,scSHED的含量1×106个/ml。将水凝胶置入3D生物打印机喷头料筒中,计算机辅助设计控制下打印为10mm×10mm的平面网状结构,使用CaCl2溶液交联,并使用无菌器械将其卷起,构建水凝胶核结构。
3.将PCL壳包裹水凝胶核,制备神经再生导管。
实施例3
一种用于面神经缺损修复的3D打印神经再生导管的制备方法,如图1所示:
1.将PCL原料(聚已内酯)溶解在氯仿和甲醇的混合物(5:1v/v)中,制备浓度为15%w/v的PCL静电纺丝溶液,使用静电纺丝机并用可编程注射泵(Cole Parmer,USA)以4ml/h的速度流动。注射器针头连接的正高压电源设置为16kV,接收器收集纺丝,构建6mm×10mm×0.5mm的PCL膜,使用乙二胺法将IL-4和IL13负载于PCL膜上制备壳结构,IL-4的含量为0.02%,IL13含量为0.10%。
2.将RGD(精氨酸-甘氨酸-天冬氨酸,Arg-Gly-Asp)接枝海藻酸钠和甲基丙烯酸明胶(GelMA)、scSHED(脱落人乳牙牙髓干细胞)溶解在PBS中,制备水凝胶溶液;RGD接枝海藻酸钠的含量为6%w/v,GelMA的含量为5%w/v,scSHED的含量2×106个/ml。将水凝胶置入3D生物打印机喷头料筒中,计算机辅助设计控制下打印为10mm×10mm的平面网状结构,使用CaCl2溶液交联,并使用无菌器械将其卷起,构建水凝胶核结构。
3.将PCL壳包裹水凝胶核,制备神经再生导管。
实施例4
一种用于面神经缺损修复的3D打印神经再生导管的制备方法,如图1所示:
1.将PCL原料(聚已内酯)溶解在氯仿和甲醇的混合物(5:1v/v)中,制备浓度为15%w/v的PCL静电纺丝溶液,使用静电纺丝机并用可编程注射泵(Cole Parmer,USA)以4ml/h的速度流动。注射器针头连接的正高压电源设置为16kV,接收器收集纺丝,构建6mm×10mm×0.5mm的PCL膜,使用乙二胺法将IL-4和IL13负载于PCL膜上制备壳结构,IL-4的含量为0.06%,IL13含量为0.15%。
2.将RGD(精氨酸-甘氨酸-天冬氨酸,Arg-Gly-Asp)接枝海藻酸钠和甲基丙烯酸明胶(GelMA)、scSHED(脱落人乳牙牙髓干细胞)溶解在PBS中,制备水凝胶溶液;RGD接枝海藻酸钠的含量为6%w/v,GelMA的含量为5%w/v,scSHED的含量1×107个/ml。将水凝胶置入3D生物打印机喷头料筒中,计算机辅助设计控制下打印为10mm×10mm的平面网状结构,使用CaCl2溶液交联,并使用无菌器械将其卷起,构建水凝胶核结构。
3.将PCL壳包裹水凝胶核,制备神经再生导管。
实施例5
一种用于面神经缺损修复的3D打印神经再生导管的制备方法,如图1所示:
1.将PCL原料(聚已内酯)溶解在氯仿和甲醇的混合物(5:1v/v)中,制备浓度为15%w/v的PCL静电纺丝溶液,使用静电纺丝机并用可编程注射泵(Cole Parmer,USA)以4ml/h的速度流动。注射器针头连接的正高压电源设置为16kV,接收器收集纺丝,构建6mm×10mm×0.5mm的PCL膜,使用乙二胺法将IL-4和IL13负载于PCL膜上制备壳结构,IL-4的含量为0.06%,IL13含量为0.15%。
2.将RGD(精氨酸-甘氨酸-天冬氨酸,Arg-Gly-Asp)接枝海藻酸钠和甲基丙烯酸明胶(GelMA)、scSHED(脱落人乳牙牙髓干细胞)溶解在PBS中,制备水凝胶溶液;RGD接枝海藻酸钠的含量为6%w/v,GelMA的含量为5%w/v,scSHED的含量2×106个/ml。将水凝胶置入3D生物打印机喷头料筒中,计算机辅助设计控制下打印为10mm×10mm的平面网状结构,使用CaCl2溶液交联,并使用无菌器械将其卷起,构建水凝胶核结构。
3.将PCL壳包裹水凝胶核,制备神经再生导管。
实施例6
一种用于面神经缺损修复的3D打印神经再生导管的制备方法,如图1所示:
1.将PCL原料(聚已内酯)溶解在氯仿和甲醇的混合物(5:1v/v)中,制备浓度为15%w/v的PCL静电纺丝溶液,使用静电纺丝机并用可编程注射泵(Cole Parmer,USA)以4ml/h的速度流动。注射器针头连接的正高压电源设置为16kV,接收器收集纺丝,构建6mm×10mm×0.5mm的PCL膜,使用乙二胺法将IL-4和IL13负载于PCL膜上制备壳结构,IL-4的含量为0.01%,IL13含量为0.05%。
2.将RGD(精氨酸-甘氨酸-天冬氨酸,Arg-Gly-Asp)接枝海藻酸钠和甲基丙烯酸明胶(GelMA)、scSHED(脱落人乳牙牙髓干细胞)溶解在PBS中,制备水凝胶溶液;RGD接枝海藻酸钠的含量为6%w/v,GelMA的含量为5%w/v,scSHED的含量2×104个/ml。将水凝胶置入3D生物打印机喷头料筒中,计算机辅助设计控制下打印为10mm×10mm的平面网状结构,使用CaCl2溶液交联,并使用无菌器械将其卷起,构建水凝胶核结构。
3.将PCL壳包裹水凝胶核,制备神经再生导管。
对比例1
一种用于面神经缺损修复的3D打印神经再生导管的制备方法,如图1所示:
1.将PCL原料(聚已内酯)溶解在氯仿和甲醇的混合物(5:1v/v)中,制备浓度为15%w/v的PCL静电纺丝溶液,使用静电纺丝机并用可编程注射泵(Cole Parmer,USA)以4ml/h的速度流动。注射器针头连接的正高压电源设置为16kV,接收器收集纺丝,构建6mm×10mm×0.5mm的PCL膜,使用乙二胺法将IL-4和IL13负载于PCL膜上制备壳结构,IL-4的含量为0.04%,IL13含量为0.12%。
2.将RGD(精氨酸-甘氨酸-天冬氨酸,Arg-Gly-Asp)接枝海藻酸钠和甲基丙烯酸明胶(GelMA)、scSHED(脱落人乳牙牙髓干细胞)溶解在PBS中,制备水凝胶溶液;RGD接枝海藻酸钠的含量为8%w/v,GelMA的含量为2%w/v,scSHED的含量2×106个/ml。将水凝胶置入3D生物打印机喷头料筒中,计算机辅助设计控制下打印为10mm×10mm的平面网状结构,使用CaCl2溶液交联,并使用无菌器械将其卷起,构建水凝胶核结构。
3.将PCL壳包裹水凝胶核,制备神经再生导管。
对比例2
一种用于面神经缺损修复的3D打印神经再生导管的制备方法,如图1所示:
1.将PCL原料(聚已内酯)溶解在氯仿和甲醇的混合物(5:1v/v)中,制备浓度为15%w/v的PCL静电纺丝溶液,使用静电纺丝机并用可编程注射泵(Cole Parmer,USA)以4ml/h的速度流动。注射器针头连接的正高压电源设置为16kV,接收器收集纺丝,构建6mm×10mm×0.5mm的PCL膜,使用乙二胺法将IL-4和IL13负载于PCL膜上制备壳结构,IL-4的含量为0.04%,IL13含量为0.12%。
2.将RGD(精氨酸-甘氨酸-天冬氨酸,Arg-Gly-Asp)接枝海藻酸钠和甲基丙烯酸明胶(GelMA)、scSHED(脱落人乳牙牙髓干细胞)溶解在PBS中,制备水凝胶溶液;RGD接枝海藻酸钠的含量为2%w/v,GelMA的含量为8%w/v,scSHED的含量2×106个/ml。将水凝胶置入3D生物打印机喷头料筒中,计算机辅助设计控制下打印为10mm×10mm的平面网状结构,使用CaCl2溶液交联,并使用无菌器械将其卷起,构建水凝胶核结构。
3.将PCL壳包裹水凝胶核,制备神经再生导管。
对比例3
与实施例6的区别在于,采用不是诱导为类施万细胞的脱落人乳牙牙髓干细胞(scSHED),而是未经诱导的SHED。
实验例
取实施例和对比例得到的神经再生导管,无菌处理后,进行兔子的体内试验。
兔沿嘴角至耳前处做长约3cm的切口,离断面神经上颊支,用制备的神经再生导管套接吻合缺损,两断端嵌入1.5mm,缝合。以不植入神经再生导管为阴性对照。8周后电镜观察神经组织横切片中再生神经纤维数目和纤维直径,结果见表1。
表1 3D打印神经再生导管的再生效果
再生神经纤维数目 | 纤维直径μm | |
实施例1 | 1775.5 | 13.24 |
实施例2 | 1619.9 | 11.71 |
实施例3 | 1632.4 | 11.49 |
实施例4 | 1527.3 | 10.75 |
实施例5 | 1512.5 | 10.64 |
实施例6 | 1494.6 | 9.82 |
对比例1 | 1280.6 | 8.35 |
对比例2 | 1257.1 | 8.23 |
对比例3 | 1345.5 | 8.67 |
阴性对照 | 858.1 | 2.95 |
结果显示,实施例1-6的再生神经纤维数目和纤维直径均优于对比例1和对比例2,表明了水凝胶核结构中的RGD和GelMA会显著影响scSHED与IL-4、IL13对面部神经在再生作用。这可能与核结构的水凝胶粘度和渗透性或者与影响scSHED表达蛋白及影响蛋白活性有关。实施例1-5的再生神经纤维数目和纤维直径均优于实施例6,表明了对面部神经在再生作用需要足够数目的scSHED的细胞数目和足够浓度的IL-4和IL13。实施例1的再生神经纤维数目和纤维直径与实施例2、3及实施例4、5之间有显著的差异(P<0.05)。这些差异表明了IL-4和IL13的浓度和二者之间的比例对于与scSHED共同促进神经再生的影响。
以上对本发明优选的具体实施方式和实施例作了详细说明,但是本发明并不限于上述实施方式和实施例,在本领域技术人员所具备的知识范围内,还可以在不脱离本发明构思的前提下作出各种变化。
Claims (5)
1.一种用于面神经缺损修复的3D打印神经再生导管,其特征在于,包括PCL壳结构和水凝胶核结构;
所述PCL壳结构包括PCL膜及负载于所述PCL膜的细胞因子;所述细胞因子为白细胞介素IL-4和IL13;
所述水凝胶核结构中含有经诱导的类施万细胞样脱落人乳牙牙髓干细胞;
所述水凝胶核结构为以CaCl2交联的RGD接枝海藻酸钠和甲基丙烯酸明胶制成的平面网状结构;
所述水凝胶核结构的制备方法为将所述RGD接枝海藻酸钠、甲基丙烯酸明胶和脱落人乳牙牙髓干细胞制成水凝胶溶液,制成平面网状结构,使用CaCl2溶液交联;所述水凝胶溶液中,所述RGD接枝海藻酸钠的浓度为3-7% w/v,所述甲基丙烯酸明胶的浓度为3-7% w/v。
2.一种根据权利要求1所述的用于面神经缺损修复的3D打印神经再生导管的制备方法,其特征在于,包括以下步骤:
S01,制备PCL壳结构;
S02,制备水凝胶核结构;
S03,将所述PCL壳结构包裹所述水凝胶核结构,得到神经再生导管。
3.根据权利要求2所述的用于面神经缺损修复的3D打印神经再生导管的制备方法,其特征在于,S01中,将PCL制成PCL静电纺丝溶液,纺丝,构建PCL膜,负载细胞因子。
4.根据权利要求3所述的用于面神经缺损修复的3D打印神经再生导管的制备方法,其特征在于,S01中,所述PCL在PCL静电纺丝溶液中的浓度为10-20%w/v;
和/或,S01中,所述PCL静电纺丝溶液的溶剂为氯仿和甲醇。
5.根据权利要求2所述的用于面神经缺损修复的3D打印神经再生导管的制备方法,其特征在于,S02中,将水凝胶溶液3D打印制成所述平面网状结构,得到水凝胶核结构。
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