CN116099051B - 一种脱细胞基质构建组织工程血管化牙髓的方法及应用 - Google Patents
一种脱细胞基质构建组织工程血管化牙髓的方法及应用 Download PDFInfo
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Abstract
本发明涉及干细胞培养技术领域，提出了一种脱细胞基质构建组织工程血管化牙髓的方法及应用。所述一种脱细胞基质构建组织工程血管化牙髓的方法，包括以下步骤：S1、大鼠颌下腺脱细胞处理得到脱细胞颌下腺基质；S2、将所述脱细胞颌下腺基质冻干得到冻干支架；S3、将所述冻干支架体外接种牙髓干细胞得到牙髓干细胞‑支架复合体；S4、将所述牙髓干细胞‑支架复合体与牙根切片构建组织工程复合体。通过上述技术方案，解决了相关技术中的牙髓组织获取来源少、组织获取及操作复杂问题。
Description
技术领域
本发明涉及干细胞培养技术领域，具体指，涉及一种脱细胞基质构建组织工程血管化牙髓的方法及应用。
背景技术
牙髓是一种复杂的、包含有多种细胞类型的、高度血管化和受神经支配的结构高度有序组织。牙髓功能与其微环境生态位密切相关，天然牙髓细胞外基质维持牙髓组织的结构完整性，并在牙髓发育、修复和再生中发挥重要作用。
牙髓干细胞被认为是再生牙科中很有前途的细胞群，并且已被证明在体内植入后可产生牙髓-牙本质样组织，除此之外，牙髓干细胞还具有神经元和内皮细胞分化潜能。
天然组织来源的细胞外基质由于其高生物活性及组织特异性被认为是牙髓组织再生的理想支架，与其他生物材料相比存在固有的优势。生物组织的去细胞化降低了免疫原性，保留了原始的脉管结构，允许细胞重新增殖，从而提高植入后的长期性能稳定。脱细胞的人类和动物组织已被广泛用于组织工程，已经证明在再生医学应用中使用脱细胞支架可促进血管形成并为干细胞分化提供指导环境。在牙科领域，已经报告了脱细胞人牙髓、猪牙髓以及牛牙髓支架体内再生功能性牙髓组织，但是，牙髓组织获取来源十分有限，其体量难以满足牙髓再生对支架材料的需求，另外，大量生物材料的长期储存和运输临床应用具有挑战性。
脱细胞人牙髓细胞外基质由于体积小和来源有限严重限制了在实际治疗应用中的可行性，尽管异种来源的猪、牛脱细胞牙髓基质部分解决了这个问题，但其属于大型动物，且组织获取及操作相对复杂。此外，再生功能性牙髓组织形成是牙髓治疗的最终目标，目前通过组织工程再生牙髓的方法尚未应用于临床。确保充足的血液供应，以维持再生过程中组织工程构建体内细胞的活力和功能，仍然是一个挑战。因此，探索新型的相对易获取的支架支持干细胞的附着与生长、制定构建体体内植入后促血管化的策略和实现牙髓牙本质复合体功能性再生对于未来的临床转化有重要意义。
发明内容
本发明提出一种脱细胞基质构建组织工程血管化牙髓的方法及应用，解决了相关技术中的牙髓组织获取来源少、组织获取及操作复杂问题。
本发明的技术方案如下：
一种脱细胞基质构建组织工程血管化牙髓的方法，包括以下步骤：
S1、大鼠颌下腺脱细胞处理得到脱细胞颌下腺基质；
S2、将所述脱细胞颌下腺基质冻干得到冻干支架；
S3、将所述冻干支架体外接种牙髓干细胞得到牙髓干细胞-支架复合体；
S4、将所述牙髓干细胞-支架复合体与牙根切片构建组织工程复合体。
作为进一步的技术方案，所述S1中大鼠颌下腺脱细胞处理包括以下步骤：将颌下腺经去离子水、表面活性剂多次洗涤后酶消，并再次洗涤，灭菌后备用。
作为进一步的技术方案，所述表面活性剂包括十二烷基硫酸钠、聚乙二醇辛基苯基醚中的一种或两种。
作为进一步的技术方案，所述S2中脱细胞颌下腺基质冻干包括以下步骤：将脱细胞颌下腺基质洗涤后经培养基浸泡，进行预冻、冷冻干燥，得到冻干支架。
作为进一步的技术方案，所述预冻时温度为-80℃，时间为2h，所述冷冻干燥时真空压力为0.07mbar，冷阱温度为-51℃，时间为24h。
通过-80℃预冷冻后进行冷冻干燥，更有利于保存支架的组织学组成和形态结构、减少存储过程中支架生长因子的丢失，方便运输。
作为进一步的技术方案，S3前还包括将牙髓干细胞进行原代培养和传代培养；
所述原代培养时所用培养基为补充有20％FBS、100units/ml青霉素、100μg/ml链霉素、0.25μg/ml两性霉素B和2mM L-谷氨酰胺的α-MEM培养基；
所述传代培养时所用培养基为补充有10％FBS、100units/ml青霉素、100μg/ml链霉素、0.25μg/ml两性霉素B和2mM L-谷氨酰胺的α-MEM培养基。
作为进一步的技术方案，所述原代培养包括以下步骤：将牙髓组织用缓冲液洗涤后切割、酶消、制备单细胞悬液，离心，得到细胞沉淀，于所述原代培养基中进行培养；所述酶消时所用酶溶液为补充有3mg/ml I型胶原酶、4mg/ml分散酶的PBS溶液。
作为进一步的技术方案，所述传代培养包括以下步骤：原代细胞生长融合至覆盖80％瓶底壁表面时，吸出培养基并洗涤，加入胰蛋白酶37℃消化2min后，加入传代培养基终止消化，离心，得到细胞沉淀，于传代培养基中进行传代培养。
作为进一步的技术方案，所述S3体外接种牙髓干细胞包括以下步骤：将传代培养后的P3细胞生长融合至80％时，经胰蛋白酶消化，重悬于培养基中，滴加至所述冻干支架上进行细胞培养，得到牙髓干细胞-支架复合体。
作为进一步的技术方案，所述S4构建组织工程复合体包括以下步骤：将牙根切片脱矿处理，得到脱矿处理后的牙根切片，将所述牙髓干细胞-支架复合体培养后，放置于脱矿处理后的牙根切片的扩大根管内。
作为进一步的技术方案，所述脱矿处理，具体为：依次使用17％EDTA、10％EDTA、5％EDTA进行脱矿处理，每次脱矿处理后需超声下去离子水洗；所述培养，具体为：在37℃、含5％CO2的条件下培养3d。
本发明的工作原理及有益效果为：
1、本发明中使用容易大量获取、储存简单及运输方便的异种组织源性冻干脱细胞支架介导牙髓再生，以利于实现临床转化，大鼠脱细胞颌下腺支架可支持牙髓干细胞体外增殖、分化，体内移植可形成血管化牙髓样结构，有极化的成牙本质样细胞排列于牙本质壁，为牙髓再生支架的选择提供了新的思路。
2、本发明中通过对大鼠颌下腺脱细胞基质进行冷冻干燥，有效保存支架组织学组成和形态结构，并且不会改变体内脱细胞移植物的早期血流动力学性能和再增殖潜力。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1中a、b分别为脱细胞颌下腺基质、脱细胞牙髓细胞外基质的扫描电镜图；
图2中a、b、c分别为显示I型胶原、III型胶原、纤连蛋白(绿色)在脱细胞颌下腺基质的免疫荧光成像图，d、e、f分别为显示I型胶原、III型胶原、纤连蛋白(绿色)在脱细胞牙髓基质中的免疫荧光成像图；
图3中a、b、c分别为3天、5天、7天的牙髓干细胞在脱细胞颌下腺支架上黏附、生长的扫描电镜图。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都涉及本发明保护的范围。
实施例1
大鼠颌下腺脱细胞处理：
(a)将8周龄雄性大鼠，使用10％水合氯醛麻醉后，分离周围组织，取双侧颌下腺，并用肝素化的PBS洗涤15min；
(b)使用10％SDS振荡32h，摇床转速为200rpm，每8h更换一次溶液；
(c)无菌去离子水振荡4h，1％Triton X-100振荡2h；
(d)0.02mg/ml的DNAaseI和20mg/ml的RNAase 37℃处理1h；
(e)0.01M PBS洗涤2h，重复3次；
(f)在10kU/ml青霉素、10mg/ml链霉素和25μg/ml两性霉素B中浸泡12小时进行灭菌，得到脱细胞颌下腺基质。
实施例2
人牙髓脱细胞处理：
(a)收集成年人(18-25岁)拔除的健康第三磨牙，凿子沿牙颈部劈开牙齿，取出完整牙髓，并用PBS洗涤10min；
(b)1％SDS振荡16h，摇床转速为130rpm，每8h更换一次溶液；
(c)去离子水振荡4h，1％Triton X-100振荡2h；
(d)0.02mg/ml的DNAaseI和20mg/ml的RNAase 37℃处理1h；
(e)0.01M PBS洗涤2h，重复3次；
(f)10kU/ml青霉素、10mg/ml链霉素和25μg/ml两性霉素B浸泡12小时进行灭菌，得到脱细胞牙髓基质。
将实施例1得到的脱细胞颌下腺基质、实施例2得到的脱细胞牙髓细胞外基质使用扫描电镜观察其结构，结果记录在图1中；使用荧光显微镜观察天然颌下腺和天然牙髓的细胞外基质成分，结果记录在图2中。
由图1可以看出，脱细胞颌下腺基质和牙髓细胞外基质均为多孔、纤维状结构；由图2可以看出，I型胶原、III型胶原、纤连蛋白在天然颌下腺与天然牙髓细胞外基质中均有表达。
实施例3
S1、将实施例1得到的脱细胞颌下腺基质冻干处理：
(a)将上述灭菌后的脱细胞颌下腺基质，PBS洗涤10min，重复3次后，放置于六孔板中，每个孔加入2ml传代培养基(补充有10％FBS、100units/ml青霉素、100μg/ml链霉素、0.25μg/ml两性霉素B 2mM L-谷氨酰胺的α-MEM)，在37℃、含5％CO2的细胞培养箱中培养过夜；
(b)吸出培养基，每个孔加入2mlPBS洗涤2次，放置于25cm2带透气盖的培养瓶中；
(c)冻干前，将上述培养瓶放置于-80℃冰箱进行预冻2h；
(d)冷冻干燥机真空压力值0.07mbar(对应冷阱温度-51℃)，冷冻干燥机冷凝器预冷30min后，将上述培养瓶转移至冷冻干燥机，冻干24h；
(e)取出培养瓶，封口膜封闭，冻干的脱细胞颌下腺基质用作牙髓再生组织工程的生物支架，短期(2个月内)使用保存在4℃，长期保存在-20℃。
S2、牙髓干细胞原代培养：
(a)收集成年人(18-25岁)拔除的健康第三磨牙，保存在4℃的缓冲液(补充有100U/mL青霉素、0.1mg/mL链霉素和0.25μg/mL的PBS)中，4h以内进行细胞培养；
(b)使用70％乙醇消毒牙齿表面，清除牙根上的碎屑和牙周膜，磨开釉牙骨质界，露出牙髓腔，将牙髓组织从牙冠和牙根硬组织中分离，在缓冲液(补充有100units/ml青霉素、100μg/ml链霉素、0.25μg/ml两性霉素B的PBS)中洗涤后，切割成小块；
(c)将小块的牙髓组织转移到酶溶液(补充有3mg/ml I型胶原酶、4mg/ml分散酶的PBS)中，37℃放置1h，其中每30min振荡一次，进行酶消；
(d)上述酶消溶液通过70μm细胞滤器获得单细胞悬液，室温下1100rpm离心6min，吸出上清液，将细胞沉淀重悬在2ml原代培养基(补充有20％FBS、100units/ml青霉素、100μg/ml链霉素、0.25μg/ml两性霉素B和2mM L-谷氨酰胺的α-MEM)中，得到牙髓单细胞悬液，将牙髓单细胞悬液接种到25cm2的培养瓶中，在37℃、含5％CO2的细胞培养箱中培养，第三天加2ml原代培养基，以后每3天更换一次培养基。
S3、牙髓干细胞传代培养：
(a)原代细胞生长融合至覆盖80％瓶底壁表面时，吸出培养基，每个培养瓶加入1ml PBS洗涤，重复2次；
(b)加入1ml 0.25％胰蛋白酶溶液，进行消化，轻轻晃动培养瓶，使消化液流动覆盖所有细胞表面，37℃消化2min，轻拍培养瓶，加入2ml传代培养基终止消化，吸取瓶内培养液，反复轻柔吹打瓶底壁细胞，得到消化后的细胞悬液；
(c)将上述消化后的细胞悬液转移至15ml离心管，室温下1100rpm离心6min，吸出上清液，将细胞沉淀重悬于传代培养基(补充有10％FBS、100units/ml青霉素、100μg/ml链霉素、0.25μg/ml两性霉素B 2mM L-谷氨酰胺的α-MEM)中，按照1:3的比例进行传代培养，每3天更换一次培养基。
S4、冻干支架体外接种牙髓干细胞：
(a)将传代培养后的P3细胞生长融合至80％时，使用0.25％胰蛋白酶消化，全自动细胞计数仪进行细胞计数，以1×107/mL的细胞浓度将细胞重悬于原代培养基中；
(b)将冻干支架脱离瓶壁，取出支架，放置于六孔板中，每个孔放置一个支架；
(c)每个支架滴加100μl 1×107/ml细胞悬液，在37℃、含5％CO2的细胞培养箱中培养2h；
(d)六孔板每个孔添加2ml原代培养基，在37℃、含5％CO2的细胞培养箱中培养，得到牙髓干细胞-支架复合体。
通过扫描电镜观察牙髓干细胞接种于冻干脱细胞颌下腺支架上，分别观察3、5、7天细胞在支架上的黏附与生长情况，结果记录在图3中。
由图3可以看出，冻干脱细胞颌下腺支架可支持牙髓干细胞体外增殖、分化。
实施例4
构建组织工程复合体：
(a)收集因正畸治疗需要拔除的健康前磨牙(14-18岁)，PBS冲洗后-20℃保存；
(b)刮除牙周膜和细胞牙骨质；
(c)截取牙根中上段约3-4mm长的牙根切片，去除牙髓和前期牙本质，扩大根管至牙根直径的1/3-1/2；
(d)牙根切片浸泡在去离子水中，超声清洁20min；
(e)17％EDTA脱矿处理15min，去离子水超声清洁10min，10％EDTA脱矿处理15min，去离子水超声清洁10min，5％EDTA脱矿处理15min，去离子水超声清洁10min，使牙本质部分脱矿，上述脱矿处理过程在摇床上进行；
(f)70％乙醇浸泡12h，去离子水洗涤10min；
(g)10kU/mL青霉素、10mg/mL链霉素和25μg/mL两性霉素B浸泡12h进行灭菌，PBS洗涤10min；
(h)部分脱矿牙根切片在4℃的传代培养基中保存；
(i)将实施例3得到的牙髓干细胞-支架复合体在37℃、含5％CO2的细胞培养箱中培养3d，同时提前1d将(h)得到的部分脱矿牙根切片浸泡于原代培养基中，10cm2培养皿中组合上述生物材料，将牙髓干细胞-支架复合体放置于牙根切片的扩大根管内。
综上所述，本发明实施例提供的基于脱细胞基质构建组织工程血管化牙髓的方法，首先将大鼠颌下腺脱细胞处理得到脱细胞颌下腺基质；其次将脱细胞颌下腺基质冻干得到冻干支架；进一步地将冻干支架体外接种牙髓干细胞得到牙髓干细胞-支架复合体；最后将牙髓干细胞-支架复合体与牙根切片构建组织工程复合体，实现了牙髓组织的完整再生。
以上仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种脱细胞基质构建组织工程血管化牙髓的方法，其特征在于，包括以下步骤：
S1、大鼠颌下腺脱细胞处理得到脱细胞颌下腺基质；
S2、将所述脱细胞颌下腺基质冻干得到冻干支架；
S3、将所述冻干支架体外接种牙髓干细胞得到牙髓干细胞-支架复合体；
S4、将所述牙髓干细胞-支架复合体与牙根切片构建组织工程复合体。
2.根据权利要求1所述的一种脱细胞基质构建组织工程血管化牙髓的方法，其特征在于，所述S1中大鼠颌下腺脱细胞处理包括以下步骤：将颌下腺经去离子水、表面活性剂多次洗涤后酶消，并再次洗涤，灭菌后备用。
3.根据权利要求1所述的一种脱细胞基质构建组织工程血管化牙髓的方法，其特征在于，所述S2中脱细胞颌下腺基质冻干包括以下步骤：将脱细胞颌下腺基质洗涤后经培养基浸泡，进行预冻、冷冻干燥，得到冻干支架。
4.根据权利要求3所述的一种脱细胞基质构建组织工程血管化牙髓的方法，其特征在于，所述预冻时温度为-80℃，时间为2h，所述冷冻干燥时真空压力为0.07mbar，冷阱温度为-51℃，时间为24h。
5.根据权利要求1所述的一种脱细胞基质构建组织工程血管化牙髓的方法，其特征在于，S3前还包括将牙髓干细胞进行原代培养和传代培养；
所述原代培养时所用培养基为补充有20％FBS、100units/ml青霉素、100μg/ml链霉素、0.25μg/ml两性霉素B和2mM L-谷氨酰胺的α-MEM培养基；
所述传代培养时所用培养基为补充有10％FBS、100units/ml青霉素、100μg/ml链霉素、0.25μg/ml两性霉素B和2mM L-谷氨酰胺的α-MEM培养基。
6.根据权利要求5所述的一种脱细胞基质构建组织工程血管化牙髓的方法，其特征在于，所述原代培养包括以下步骤：将牙髓组织用缓冲液洗涤后切割、酶消、制备单细胞悬液，离心，得到细胞沉淀，于所述原代培养基中进行培养；所述酶消时所用酶溶液为补充有3mg/ml I型胶原酶、4mg/ml分散酶的PBS溶液。
7.根据权利要求5所述的一种脱细胞基质构建组织工程血管化牙髓的方法，其特征在于，所述传代培养包括以下步骤：原代细胞生长融合至覆盖80％瓶底壁表面时，吸出培养基并洗涤，加入胰蛋白酶37℃消化2min后，加入传代培养基终止消化，离心，得到细胞沉淀，于传代培养基中进行传代培养。
8.根据权利要求1所述的一种脱细胞基质构建组织工程血管化牙髓的方法，其特征在于，所述S3体外接种牙髓干细胞包括以下步骤：将传代培养后的P3细胞生长融合至80％时，经胰蛋白酶消化，重悬于培养基中，滴加至所述冻干支架上进行细胞培养，得到牙髓干细胞-支架复合体。
9.根据权利要求8所述的一种脱细胞基质构建组织工程血管化牙髓的方法，其特征在于，所述S4构建组织工程复合体包括以下步骤：将牙根切片脱矿处理，得到脱矿处理后的牙根切片，将所述牙髓干细胞-支架复合体培养后，放置于脱矿处理后的牙根切片的扩大根管内。
10.根据权利要求9所述的一种脱细胞基质构建组织工程血管化牙髓的方法，其特征在于，所述脱矿处理具体为：依次使用17％EDTA、10％EDTA、5％EDTA进行脱矿处理，每次脱矿处理后需超声下去离子水洗；所述培养具体为：在37℃、含5％CO2的条件下培养3d。
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