CN113621570A - 基于数字光学处理打印技术的组织工程牙髓及制备方法 - Google Patents
基于数字光学处理打印技术的组织工程牙髓及制备方法 Download PDFInfo
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Abstract
本发明公开了一种基于数字光学处理打印技术的组织工程牙髓，由载牙髓间充质干细胞的水凝胶微球构成。采用基于数字光学处理的3D打印技术，可快速生产高精度的载牙髓间充质干细胞微球，保证形态、速度的基础上保证细胞的活性的功能。微米级直径微球植入物首先有利于进行注射治疗，根管的解剖形态较为细长，无法使用充填器械将预成型的组织结构完全充填至根尖部，而可注射的微球可以带到根尖孔附近；其次，不同微球之间会产生较多间隙，有利于血液再充盈牙髓腔，为再生提供必要养分。合适的微球尺寸为内部细胞摄取营养物质提供了方便，有利于细胞与血液交换营养物质与代谢废物，为细胞生长提供合适的微环境，从而最大限度的促进牙髓再生。
Description
技术领域
本发明属于口腔医疗器械技术领域，具体为一种基于数字光学处理打印技术的组织工程牙髓及制备方法。
背景技术
牙髓发育自外胚间叶，内含有神经、血管、牙髓间充质干细胞、成牙本质细胞等成分，主要起到感觉、营养、形成牙本质、防御及修复的作用[1]。由于牙髓外周无让性的坚硬牙本质的存在，复合根尖孔较小、无有效侧枝循环等因素的协同作用，牙髓一旦发生炎症、受其他刺激和损伤后均较难恢复[2,3]。目前，根管治疗术是临床上针对不可逆性牙髓病变或根尖周病变最为常规的治疗方式，然而失去了牙髓的牙体硬组织有脆性增加、容易染色等问题，活髓切断术、牙髓血运重建、根尖诱导成形术等手段都无法完美解决该问题，寻找一种新的方式刻不容缓。
发明内容
本发明为克服上述现有技术所述的至少一种缺陷(不足)：
本发明的一个目的是提供一种基于数字光学处理打印技术的组织工程牙髓，是载牙髓间充质干细胞的水凝胶微球。
进一步的，所述水凝胶为GelMA水凝胶，所述水凝胶为GelMA水凝胶，所述微球直径为20-999μm。所述微球直径为400μm。
本发明的另一个目的是提供一种基于数字光学处理打印技术的组织工程牙髓及制备方法的制备方法，包括如下步骤：牙髓间充质干细胞的获取与培养；GelMA水凝胶的制备；牙髓间充质干细胞和GelMA水凝胶混合均匀，打印。
进一步的，牙髓间充质干细胞的浓度为1*106-1*108个/mL；优选为1-10*107个/mL。
进一步的，GelMA水凝胶接枝率为1-5M且浓度为5-20％。
进一步的，牙髓间充质干细胞消化离心后，用微量培养基重悬，所得细胞悬液加与GelMA水凝胶混合均匀，打印。
进一步的，将生物墨水转移至已预热至37℃的3D打印机样品槽中，通过数字化光处理3D打印技术逐层打印。
更进一步的，细胞悬液为含有牙髓间充质干细胞的细胞悬液，即生物墨水为含有牙髓间充质干细胞的细胞悬液。
进一步的，打印技术的相关参数设置为10-100μm，曝光时间500-2000ms，曝光强度50％；优选为层厚20μm，曝光时间2000ms，曝光强度50％，表面羽化。
发明的又一个目的是提供以上所述的基于数字光学处理打印技术的组织工程牙髓的应用。
与背景技术相比，本发明的有益效果是：
采用基于数字光学处理的3D打印技术，可快速生产高精度的载牙髓间充质干细胞微球，保证形态、速度的基础上保证细胞的活性的功能。由微球结构构成的植入物首先有利于进行注射治疗，根管的解剖形态较为细长，无法使用充填器械将预成型的组织结构完全充填至根尖部，而可注射的微球可以在注射器与注射头的帮助下将其顺利的带到根尖孔附近；其次，不同微球之间会产生较多空隙，有利于血液再充盈牙髓腔，为再生提供必要养分。合适的微球尺寸为内部细胞摄取营养物质提供了方便，有利于细胞与血液交换营养物质与代谢废物，为细胞生长提供合适的微环境，从而最大限度的促进牙髓再生。
附图说明
图1是本发明组织工程牙髓构建流程示意图。
图2为依照本发明实施例1中制备的载牙髓间充质干细胞的GelMA水凝胶微球的圆率分析图。
图3为依照本发明实施例1中制备的载牙髓间充质干细胞的GelMA水凝胶微球的电子显微镜镜照片。
图4为依照本发明实施例1中制备的载牙髓间充质干细胞的GelMA水凝胶微球的普通光学显微镜照片。
图5为依照本发明实施例2中裸鼠异位牙髓再生模型的构建方法。
图6为依照本发明实施例2中对裸鼠异位牙髓进行牙本质涎磷蛋白DSPP免疫荧光染色的激光共聚焦显微镜照片。
图7为依照本发明实施例3中猪原位牙髓再生模型的构建方法。
图8为依照本发明实施例3中对猪原位再生牙髓的HE染色照片
图9为依照本发明实施例3中对猪原位再生牙髓的HE染色照片的局部放大
其中，1为牙髓；2为牙髓间充质干细胞；3为GelMA水凝胶；4为牙髓间充质干细胞和GelMA水凝胶的混合液；5为基于数字光学处理的3D打印机；6为载牙髓间充质干细胞的GelMA水凝胶微球；7为注射入骨髓腔的载细胞微球；8为牙髓再生。
具体实施方式
附图仅用于示例性说明，不能理解为对本专利的限制；对于本领域技术人员来说，某些公知结构及其说明可能省略是可以理解的。本发明所使用药品及培养基除注明外均为市售品。本发明的目的在于提供一种可用于治疗牙髓病变的组织工程牙髓的制备方法，该组织工程牙髓不仅可以维持牙髓细胞的干性能力，而且可以提高牙髓细胞的成牙本质和成神经的能力，在牙髓腔中形成接近正常牙髓结构的组织，且与人正常牙髓特异性标志物表达的情况一致。
为了实现上述发明目的，本发明提供以下技术方案：
一种可用于治疗牙髓病变的组织工程牙髓，该组织工程牙髓为基于数字光学处理打印技术的载牙髓间充质干细胞的GelMA水凝胶微球。
作为具体实施例的改进，该GelMA水凝胶接枝率为1M且浓度为10％。
作为具体实施例的改进，该牙髓间充质干细胞的浓度为1*106-1*108个/mL，优选为1*107个/mL。
作为具体实施例的改进，该微球直径设计为400μm。
作为具体实施例的改进，该打印技术的相关参数设置为层厚20μm，曝光时间2000ms，曝光强度50％，表面羽化。
一种基于数字光学处理打印技术的组织工程牙髓的制备方法，所述制备方法包括以下步骤：
牙髓间充质干细胞的获取与培养；
GelMA水凝胶的制备；
将上述步骤得到的牙髓间充质干细胞消化离心后，用培养基重悬，所得细胞悬液加与GelMA(1M)水凝胶混合均匀，再将生物墨水转移至3D打印机样品槽中(已预热至37℃)，通过数字化光处理3D打印技术逐层打印。
根据上述的制备方法，其中，细胞悬液为含有牙髓间充质干细胞的细胞悬液。
以上所述的基于数字光学处理打印技术的组织工程牙髓的应用。
由负载牙髓间充质干细胞的GelMA水凝胶微球构成的组织工程牙髓可以通过注射的方式顺利到达根尖孔附近，操作简单且创伤小，另外不同微球间有许多空隙，有利于牙髓间充质干细胞的营养摄入，从而促进牙髓再生。
在一个具体的实施方式中，本发明首先优化GelMA水凝胶浓度，确定为10％，确定合适的浓度后，优化了负载细胞的密度，由于密度过高会影响细胞的存活率，最终确定细胞密度为1*107个/mL，随后优化了数字光处理打印技术的相关参数，包括层厚20μm，曝光时间2000ms，曝光强度50％，表面羽化最后在动物体内的异位牙髓再生模型中取得有良好的牙髓再生效果。基于数字光处理打印技术制造微球速度快，高产，一次10分钟可制造7750个(可满足22个根管的用量)。数字光处理打印技术的精度高达20μm，微球具有极高的直径均一性。
目前，通过组织工程原理构建的牙髓主要分为四类，第一类的主要成分为血液来源的血小板，该材料主要来自于患者自身血液，通过离心后获得血小板或血小板相关浓缩物，因此不具有排异性，资源丰富易得，可以制备成立体的纤维蛋白基质用于搭载细胞[4-6]。第二类组织工程牙髓构建过程中主要采用细胞归巢的方法来募集干细胞及体内其他细胞迁移到牙髓腔中，从而再生牙髓，多位学者已证明特殊的因子可以营造适合内源性细胞归巢的微环境，内源性细胞可以迁移至牙髓腔并发生增殖、分化，从而形成牙髓组织的再生[7-9]。第三类组织工程牙髓构建方法为采用其它载细胞生物材料进行充填，这些生物材料包括天然材料、人工合成材料以及这两者的复合物[10,11]。这些支架一般细胞毒性小、有合适的降解性能、可以为细胞生长提供稳定适宜的微环境、或诱导组织再生。然而，以上方法仍有较多问题存在，如支架塌陷导致无法再生牙髓组织，材料无法降解长期占位影响组织再生，重建的组织为牙骨质或牙周样组织而非牙髓样组织，致密填充导致内部孔隙不足、营养及代谢产物传递能力低下，或体外实验结果尚可但尚未进行体内实验验证等等，牙髓的组织工程重建仍面临着较多挑战[12,13]
本专利提出的微球为包裹患者的智齿干细胞，为细胞和GelMA水凝胶微球一体化打印获得，即可实现带细胞打印。
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下面结合具体实施例，进一步阐述本公开提供的基于数字光学处理打印技术的组织工程牙髓的制备方法。
实施例1：基于数字光学处理打印技术的组织工程牙髓的制备
(1)牙髓细胞的培养与扩增：将患者牙齿迅速储存于含青霉素(100Units/mL)和链霉素(100Units/mL)的α-MEM培养基(Gibco，美国)中，移入超净台内。碘伏消毒涡轮机水、气管道，将牙齿横剖后使用探针完整取出牙髓，使用含青霉素(100Units/mL)和链霉素(100Units/mL)的PBS清洗三遍，转移至EP管内，用组织剪将牙髓组织剪碎，至约1mm*1mm*1mm大小，使用终浓度为3mg/mL的Collagenase与4mg/mL的Dispase消化牙髓组织，在37℃摇床慢速摇动下作用30分钟。室温下1200rpm离心3分钟后，弃去上清，将消化后的组织转移至6孔板中(6孔板中预铺0.2％明胶，37℃过夜)，加入1.5mL含15％FBS、100Units/mL青霉素和100Units/mL链霉素的α-MEM，在5％CO2的37℃孵箱中培养。牙髓间充质干细胞爬出并形成集落后每2-3天换液，当细胞生长至70％-80％时进行传代。
(2)GelMA水凝胶的制备：将10g明胶(猪皮来源)加入到100mL PBS溶液中(pH＝7.4)，保持50℃1000rpm搅拌1小时，直至明胶完全溶解(完全溶解后该溶液呈透明黄色)。将1mL MA用注射器在避光条件下逐滴加入到上述溶液中，磁力搅拌2h，直至反应结束。将反应完全的上述液体灌入12-14kDa的透析袋中(提前煮沸软化)，体积约为透析袋的一半，去除泡沫，用透析袋夹严密封闭。将透析袋转移至40℃的Millipore水中，磁砖子310rpm搅拌透析5天，每12小时更换Millipore水，去除未反应完全的MA与有害中间产物。透析结束后，将透析袋内溶液转移至过滤瓶内真空抽滤(0.22μm滤膜)，将过滤产物-20℃冷冻24小时后转移至-80℃储存。将固态凝胶转移至冻干机内完全冻干至白色海绵样固体，即GelMA。
(3)基于数字光学处理技术的组织工程牙髓打印：0.5g GelMA(1M)水凝胶溶解于Millipore水中至4.6mL，0.22μm滤头过滤，加入LAP至终浓度为0.5％，80μL酚红，加入牙髓细胞悬液至5*107个/mL，将液体补足至5mL，混合均匀后，将生物墨水转移至3D打印机样品槽中(已预热至37℃)。载入用3D MAX软件构建的直径为400μm的微球，切割成20层，层厚20μm(牙髓细胞)消化后直径约14-18μm)，曝光时间2000ms，曝光强度50％，表面羽化，逐层打印，得到组织工程牙髓。
图1示出了本公开实施例中基于数字光学处理技术的组织工程牙髓的构建流程。
如图2所示，所得载牙髓间充质干细胞的GelMA水凝胶微球圆率良好。
如图3所示，所得载牙髓间充质干细胞的GelMA水凝胶微球直径约为400μm，外形圆润饱满，包裹牙髓间充质干细胞，细胞在基础培养基中可增殖、铺展。
如图4所示，SEM显示GelMA水凝胶微球表面存在大量的微孔，有利于细胞粘附。
实施例2：基于数字光学处理打印技术的组织工程牙髓在裸鼠异位牙髓再生模型中的应用
选取实施例1制备的载牙髓间充质干细胞的GelMA水凝胶微球，采用20G(内径为620μm，平头)注射针头将3D打印的带细胞微聚体注射至牙本质片段根管内，根管口覆盖明胶海绵(Microsphere组)，将制备好的移植复合体水平放置入十二孔板中，加入普通完全培养基(Gibco，美国)(含双抗)在37℃，5％CO2环境下孵育一天(静置)，将打印微载体(Microsphere)组的移植复合体埋入全麻的裸鼠皮下。裸鼠饲养8周后，取出移植复合体，用刀片去除牙本质片段外包膜，用镊子及拔髓针取出髓腔内软组织，通过对牙髓移植复合体的免疫荧光染色验证组织工程牙髓的成牙本质能力。
如图5所示，裸鼠异位牙髓再生模型构建步骤，即先预备人牙本质片段，再将微球注射其中构成移植复合体，然后在培养基中孵育一天，最后将移植复合体植入裸鼠皮下。
如图6所示，术后8周成牙本质相关特异性标志物DSPP染色结果显示，本载DPSCs微球注射治疗的方法可获得牙髓再生。
实施例3：基于数字光学处理打印技术的组织工程牙髓在猪原位牙髓再生模型中的应用
使用碘伏棉球对全麻心电监护下的猪口腔内进行消毒，将下前牙开髓，拔除牙髓，根备，用拔髓针扎根尖孔处至出血，棉条压迫止血片刻后，采用20G(直径为620μm，平头)注射针头将实施例1制备的载牙髓间充质干细胞的GelMA水凝胶微球注射至猪牙根管内，光固化树脂封闭根管口并调(牙合)；术后4周，全麻下拔除猪下前牙，将标本固定于4％多聚甲醛中24小时，在脱水仪中使用70％到100％乙醇进行梯度脱水。最终制作成猪牙纵切片，并对切片进行HE染色。
如图7所示，猪原位牙髓再生模型构建，以猪下颌前牙作为实验牙，进行上述GelMA水凝胶微球注射，在4周后进行下颌前牙的拔出。
如图8、9所示，术后4周硬组织切片HE染色显示(图9为图8的局部放大)，可见猪牙靠近根尖处形成了致密组织团块，其中可见较多新生组织，有血管形成，根管壁表面有排列整齐的成牙本质细胞形成，说明本载DPSCs微球注射治疗的方法可获得猪牙髓原位再生。
Claims (10)
1.一种基于数字光学处理打印技术的组织工程牙髓，其特征在于：是载牙髓间充质干细胞的水凝胶微球。
2.根据权利要求1所述的一种基于数字光学处理打印技术的组织工程牙髓，其特征在于：所述水凝胶为GelMA水凝胶，所述微球直径为20-999μm。
3.根据权利要求1所述的一种基于数字光学处理打印技术的组织工程牙髓，其特征在于：所述微球直径为400μm。
4.一种基于数字光学处理打印技术的组织工程牙髓的制备方法，其特征在于包括如下步骤：
牙髓间充质干细胞的获取与培养；GelMA水凝胶的制备；细胞与水凝胶混合均匀，打印。
5.根据权利要求4所述的一种基于数字光学处理打印技术的组织工程牙髓的制备方法，其特征在于，牙髓间充质干细胞的浓度为1*106-1*108个/mL。
6.根据权利要求4所述的一种基于数字光学处理打印技术的组织工程牙髓的制备方法，其特征在于，GelMA水凝胶接枝率为1-5M且浓度为5-20％。
7.根据权利要求4所述的一种基于数字光学处理打印技术的组织工程牙髓的制备方法，其特征在于，牙髓间充质干细胞消化离心后，用培养基重悬，所得细胞悬液加与GelMA水凝胶混合均匀，打印。
8.根据权利要求7所述的一种基于数字光学处理打印技术的组织工程牙髓的制备方法，其特征在于，所述细胞悬液为含有牙髓间充质干细胞的细胞悬液。
9.根据权利要求4所述的一种基于数字光学处理打印技术的组织工程牙髓的制备方法，其特征在于，将生物墨水转移至已预热至37℃的3D打印机样品槽中，通过数字化光处理3D打印技术逐层打印；打印的相关参数设置为层厚10-100μm，曝光时间500-2000ms，曝光强度50％。
10.根据权利要求1-9任一项所述的基于数字光学处理打印技术的组织工程牙髓的应用。
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