CN115382023B - 一种可用于低温沉积3d打印的脱细胞基质及其制备方法和应用 - Google Patents

一种可用于低温沉积3d打印的脱细胞基质及其制备方法和应用 Download PDF

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CN115382023B
CN115382023B CN202210955240.1A CN202210955240A CN115382023B CN 115382023 B CN115382023 B CN 115382023B CN 202210955240 A CN202210955240 A CN 202210955240A CN 115382023 B CN115382023 B CN 115382023B
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陈明学
周一新
郭全义
杨德金
邵宏翊
刘舒云
眭翔
张颂阳
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Beijing Jishuitan Hospital
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Abstract

本发明公开了一种可用于低温沉积3D打印的脱细胞基质及其制备方法和应用，属于组织工程再生医学领域。本发明提供的脱细胞基质的制备方法，包括如下步骤：(1)将动物组织进行脱细胞处理，得到脱细胞基质匀浆；(2)在所述脱细胞基质匀浆中加入乙酸溶液，搅拌，然后让部分乙酸挥发，得到所述脱细胞基质。本发明制备的脱细胞基质具有剪切变稀性和3D可打印性，可以实现脱细胞基质单一材料的3D打印。

Description

一种可用于低温沉积3D打印的脱细胞基质及其制备方法和 应用
技术领域
本发明涉及组织工程再生医学领域，具体涉及的是一种可用于低温沉积3D打印的脱细胞基质及其制备方法和应用。
背景技术
通过组织工程再生医学的方法再生修复损伤的组织或器官已成为发展趋势。3D打印技术由于可以对支架内部结构和形貌实现精确的控制，因此在再生医学领域备受青睐。生物材料墨水目前已成为制约3D打印再生医学领域发展的瓶颈。
脱细胞基质是通过对异体组织进行脱细胞处理，去除移植相关抗原，从而获得的一种具有一定生物活性的生物衍生材料。脱细胞基质由于很好地保留了天然细胞外基质的有效成分，可模拟细胞—细胞、细胞—基质之间相互作用的天然微环境，有效地调控细胞的行为和功能，因此被视为理想的生物材料墨水。但是，生物3D打印技术对生物墨水(生物材料)的性质、粘度、成型方式等均有较高的要求，并非所有的生物材料均具备可打印性。传统方法制备的软骨基质为匀浆状，在不复合其他材料的情况下，单纯的软骨基质依然难以实现3D打印。
发明内容
本发明提供了一种可用于低温沉积3D打印的脱细胞基质及其制备方法和应用，本发明的方法通过在基质匀浆悬浮液中加入乙酸溶液，从而实现了脱细胞基质的3D可打印性。
本发明首先提供了一种脱细胞基质的制备方法，包括如下步骤：
(1)将动物组织进行脱细胞处理，得到脱细胞基质匀浆；
(2)在所述脱细胞基质匀浆中加入乙酸溶液，搅拌，然后让部分乙酸挥发，得到所述脱细胞基质。
上述的制备方法中，所述动物组织为软骨组织、半月板组织、骨组织、肌腱、肌肉、韧带和皮肤中的任一种。
所述动物组织的脱细胞处理方法为物理法、化学法或酶法。
物理法主要指的是差速离心法、超临界流体法、反复冻融法等；化学法主要包括使用各种化学试剂，括酸、碱、非离子型除垢剂、离子型除垢剂、两性离子除垢剂等；酶法为采用胰蛋白酶、DNA酶、RNA酶等进行处理。
上述的制备方法，步骤(2)中，所述乙酸溶液的质量百分浓度为20％～100％；具体可为80％～100％。
所述乙酸溶液为不断滴加到所述脱细胞基质匀浆中，直至所述脱细胞基质匀浆由乳白色变为透明状为止。
上述的制备方法，步骤(2)中，所述搅拌的温度为0～8℃，时间为12～72h；
所述搅拌在密闭容器中进行；
所述乙酸挥发的方法为在抽风机下磁力搅拌挥发乙酸；
所述脱细胞基质的质量百分浓度为5～10％。
本发明还提供了上述制备方法制备得到的脱细胞基质。
所述脱细胞基质在低温沉积3D打印制备组织工程支架中的应用也属于本发明的保护范围。
进一步的，本发明还提供了一种脱细胞基质支架的制备方法，包括如下步骤：所述脱细胞基质作为打印墨水采用3D打印机进行打印；打印后将支架进行冷冻升华干燥，然后将支架置于交联剂中进行交联，再次冷冻升华干燥，得到所述脱细胞基质支架；
所述打印的温度为低温。
上述的制备方法中，所述低温的温度范围为-10～-80℃；具体可为-20℃；
所述冷冻升华干燥的条件如下：真空度<100mTorr，温度-20～-60℃，时间12～72h；
所述交联剂为包括乙基-二甲基胺-丙基碳化二亚胺和n-羟基琥珀酰亚胺的溶液；
具体的，所述交联剂的溶剂为乙醇、水、丙酮和氯仿中的至少一种；具体可为乙醇溶液；
所述交联剂中，所述乙基-二甲基胺-丙基碳化二亚胺的浓度可为10～100mmol/L；具体可为50mmol/L；n-羟基琥珀酰亚胺的浓度可为10～100mmol/L；具体可为20mmol/L；
所述交联的温度为0～25℃，具体可为4℃；时间为12～72h，具体可为24h。
上述的制备方法在交联后还有去除多余交联剂的步骤；具体可为采用PBS缓冲液浸泡。
最后，本发明提供了上述的制备方法制备得到的脱细胞基质支架。
本发明具有如下有益效果：
(1)本发明制备的脱细胞基质具有剪切变稀性和3D可打印性；本发明的方法选择的关键物质为乙酸，其既可以作为溶剂溶解软骨基质，又具有较高的凝固点(16.6℃)；可以使得脱细胞基质在深低温条件下瞬间凝固成型，实现3D打印；
(2)传统的3D打印软骨基质都是需要复合其他材料(如海藻酸等)才具有3D可打印性，而本发明的方法可以实现脱细胞基质单一材料的3D打印；
(3)本发明采用的低温沉积3D打印技术，打印过程都是在深低温过程中实现，可以避免传统打印高温给生物材料造成的活性破坏，充分暴露脱细胞基质的生物活性。
附图说明
图1为获取的软骨组织。
图2为实施例制备的脱细胞软骨基质匀浆。
图3为实施例1中制备的脱细胞软骨基质。
图4为实施例1制备的脱细胞软骨基质黏度-剪切速率曲线。
图5为冷冻平台上的3D打印支架。
图6为实施例2制备的软骨基质支架的大体观和扫描电镜图。
图7为细胞接种到软骨基质支架上的细胞死活染色图；图中，绿色代表活细胞，红色代表死细胞。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所用Dnase和Rnase分别是DNA酶和RNA酶，购自美国Sigma公司；产品货号分别为Dnase 10104159001；Rnase 10109134001。
下述实施例所用磷酸缓冲盐溶液(PBS缓冲液)的配制方法如下：称取8.0g NaCl、0.2g KCl、1.44g Na2HPO4、0.24g KH2PO4溶于800mL蒸馏水中，用HCl调节溶液pH至7.4，最后加蒸馏水定容至1L，即可得0.01M PBS缓冲液。
实施例1、脱细胞软骨基质的制备
(1)软骨基质的脱细胞处理
在实验动物手术台上，无菌条件下切开新鲜的猪膝关节，无菌生理盐水冲洗干净后，取股骨髁、髌骨和胫骨平台的软骨组织(见图1)。将收集到的软骨片置于无菌广口瓶中，加入无菌去离子水(去离子水和软骨组织体积比为5:1)，经过10次循环冻融(冷冻温度为-80℃，解冻温度为室温)处理，每次循环更换一次液体。然后将软骨组织转移到组织匀浆机中，低温条件下间歇性粉碎(粉碎30～60s后放到-20℃的冰箱内降温1～2min)，获得软骨组织匀浆。加入胰蛋白酶-EDTA(0.25％)(美国gibco，货号25200072，)，胰蛋白酶-EDTA(0.25％)和软骨组织匀浆的体积比为1:5；在37℃恒温摇床上处理24小时，磷酸缓冲盐溶液(phosphate buffer saline，PBS)漂洗，10000rpm离心30min后倒掉上清，然后在沉淀中加入浓度为50U/mL DNase和1U/mL RNase的核酸酶溶液，充分搅拌去除核酸物质，最后利用无菌去离子水反复漂洗三天，去除残留的试剂，10000rpm离心60min后所得沉淀即为脱细胞软骨基质匀浆，此时的匀浆为不透明的乳白色(见图2)。
(2)脱细胞软骨基质匀浆的酸处理
由于步骤(1)获得的脱细胞软骨基质匀浆是悬浮液，不具有3D可打印性，因此需要进一步处理。向脱细胞软骨基质匀浆中不断加入质量百分浓度为80％的乙酸，直到基质完全溶解(通过颜色判断，没有溶解前是乳白色的，溶解后是半透明状的)，在4℃条件下，用封口膜封住瓶口，磁力搅拌24小时，让脱细胞软骨基质匀浆充分溶解，此时溶解后的脱细胞软骨基质匀浆为透明溶液，但是此时的浓度和粘稠度较低，依然不具备3D打印性。去除封口膜，在抽风机下继续磁力搅拌，加速醋酸的挥发，待得到粘稠状，质量分数约为5～10％时，即具有3D可打印性；本实施例得到的脱细胞软骨基质的质量分数为6％，照片见图3。
脱细胞软骨基质的质量分数的测定方法为：先测量一定量的匀浆湿重，然后进行60℃烘干过夜，去除溶剂，测量干重，即为溶质质量，干重比湿重即质量浓度。
脱细胞软骨基质的黏度-剪切速率曲线见图4，由图4可见，本发明制备的脱细胞软骨基质具有剪切变稀性(测试温度为室温)。
实施例2、脱细胞软骨基质作为生物材料墨水用于低温沉积3D打印
(1)将实施例1制备的脱细胞软骨基质置于3D打印机(SUNP ALPHA-BP31，上普博源(北京)生物科技有限公司)料筒中，通过活塞挤压的方式进行挤出打印，挤出的脱细胞软骨基质会在冷冻平台上迅速固化成型，得到支架，其照片见图5；
设置打印参数如下：孔径500μm，喷头直径400μm，厚度为2mm，纤维角度为90°，推出速度为0.1mm/s，打印速度为5mm/s，低温冷冻平台温度为-20℃，打印仓温度为-20℃，打印喷头温度为4℃。
(2)打印完成后，将支架放入到冷冻干燥机中升华干燥，升华干燥的条件为真空条件下(<100mTorr)-60℃升华48h。
(3)将升华干燥后的支架放入交联剂中进行交联，交联的温度为4℃，交联的时间为24h；
所述交联剂为含有乙基-二甲基胺-丙基碳化二亚胺(EDAC)与n-羟基琥珀酰亚胺(NHS)的95％(v/v)乙醇溶液，其中，EDAC的浓度为50mmol/L；NHS的浓度为20mmol/L；
(4)交联后用PBS缓冲液浸泡2h，去除多余的交联剂；三蒸水漂洗后，再次冷冻升华干燥处理(真空条件下(<100mTorr)-60℃升华48h)，得到低温沉积3D打印的软骨基质支架。
图6为得到的软骨基质支架的照片和扫描电镜图。由图6可知，本发明制备的支架呈现分级多孔结构。
将脂肪间充质干细胞(来源于SD大鼠脂肪组织)接种到上述制备的软骨基质支架(10×10×1mm3)上，每个支架接种100万个细胞，等细胞充分粘附后再添加培养液(美国Gibco公司，DMEM/F12)，培养7天后取材，PBS溶液漂洗2遍，添加细胞死/活荧光染液(细胞死活染色试剂盒，美国Invitrogen公司，L3224)染色20min，PBS溶液漂洗2遍，共聚焦显微镜观察。激发绿色荧光和红色荧光的相关参数设置如下：激发波长：535nm和355nm，发射波长：585nm和460nm。结果见图7，由图7可知，细胞接种到软骨基质支架上具有很好的细胞活性，说明具有良好的细胞相容性。

Claims (7)

1.一种脱细胞基质支架的制备方法，包括如下步骤：以脱细胞基质作为打印墨水采用3D打印机进行打印；打印后将支架进行冷冻升华干燥，然后将支架置于交联剂中进行交联，再次冷冻升华干燥，得到所述脱细胞基质支架；
所述打印的温度为低温；所述低温的温度范围为-10~-80℃；
所述脱细胞基质的制备方法，包括如下步骤：
（1）将动物组织进行脱细胞处理，得到脱细胞基质匀浆；
（2）在所述脱细胞基质匀浆中加入乙酸溶液，搅拌，然后让部分乙酸挥发，得到所述脱细胞基质；
步骤（2）中，所述乙酸溶液的质量百分浓度为20%~100%；
所述乙酸溶液为不断滴加到所述脱细胞基质匀浆中，直至所述脱细胞基质匀浆由乳白色变为透明状为止；
所述搅拌的温度为0~8℃，时间为12~72 h；
所述脱细胞基质的质量百分浓度为5~10%。
2.根据权利要求1所述的制备方法，其特征在于：所述动物组织为软骨组织、半月板组织、骨组织、肌腱、肌肉、韧带和皮肤中的任一种。
3.根据权利要求1或2所述的制备方法，其特征在于：所述动物组织的脱细胞处理方法为物理法、化学法或酶法。
4.根据权利要求1或2所述的制备方法，其特征在于：步骤（2）中，所述乙酸挥发的方法为在抽风机下磁力搅拌挥发乙酸。
5. 根据权利要求1所述的制备方法，其特征在于：所述冷冻升华干燥的条件如下：真空度<100 mTorr，温度-20~-60℃，时间12~72 h；
所述交联剂为包括乙基-二甲基胺-丙基碳化二亚胺和n-羟基琥珀酰亚胺的溶液；
所述交联的温度为0~25℃，时间为12~72 h。
6.根据权利要求5所述的制备方法，其特征在于：所述交联剂的溶剂为乙醇、水、丙酮和氯仿中的至少一种；
所述交联剂中，所述乙基-二甲基胺-丙基碳化二亚胺的浓度为10~100 mmol/L；n-羟基琥珀酰亚胺的浓度为10~100 mmol/L。
7.权利要求1-6中任一项所述的制备方法制备得到的脱细胞基质支架。
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