CN114045257A - 一种血管化成骨细胞膜片支架复合体及其制备 - Google Patents
一种血管化成骨细胞膜片支架复合体及其制备 Download PDFInfo
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Abstract
本发明提供了一种血管化成骨细胞膜片支架复合体及其制备，采用成骨性细胞和血管生成细胞共培养的方法，通过细胞膜片的技术，消除了传统细胞消化技术而导致的细胞外基质的损失，保留了原本细胞微环境，通过成骨和成血管的协同效应，有利于成骨细胞在骨组织工程支架内的存活和转归；进一步地，将3D打印骨组织工程支架与该细胞膜片的有机结合解决了单纯细胞膜片强度低，不能维持成骨空间的问题。
Description
技术领域
本发明涉及组织工程技术领域，具体地，涉及一种血管化成骨细胞膜片支架复合体及其制备。
背景技术
肿瘤、外伤、感染和先天畸形等常导致严重的颌面部骨缺损，从而影响患者的功能与容貌美观。自体骨移植是检验骨缺损修复的“金标准”，但存在创伤大、供区骨量不足等缺点。目前以种子细胞、3D打印构建的组织工程骨可以无创、安全、个性化的方式修复复杂骨缺损，具有良好的应用前景。
目前可供骨组织工程应用的种子细胞包括成体干细胞、胚胎干细胞和诱导多功能干细胞。理论上胚胎干细胞具有向三种胚层分化和强大的增殖能力，但因为存在伦理争议、癌变、免疫排斥反应等的缺点尚未能被广泛推广研究；而诱导多功能干细胞则是通过基因工程技术将Oct4,Sox2,Nanog和Lin28导入体细胞内获得，但目前还存在成瘤风险；与前两者相比，成体间充质干细胞因具备提取方式简便、增殖能力强、稳定的干性、低免疫原性而广泛应用于骨组织工程，在不同的临床前动物模型和临床骨缺损治疗中取得了一定的疗效，如CN102989041A公开了一种三维多孔骨组织工程材料与人的骨髓间充质干细胞复合而成的骨组织工程支架及其制备方法。牙周膜间充质干细胞位于牙根的牙周组织内，目前认为其在正畸治疗中牙周组织改建和牙周组织再生中扮演重要的作用。牙周膜间充质干细胞主要通过刮下拔除的阻生第三磨牙和正畸减数牙牙根表面的牙周组织分离培养而得。目前，众多的体外实验和动物实验数据表明，牙周膜间充质干细胞可以能够分化为成骨样细胞和成牙骨质细胞，进而形成牙槽骨、牙骨质和牙周膜，实现牙周组织的再生。由于牙周膜间充质干细胞具有很强的矿化基质形成能力，因此也是骨组织工程种子细胞良好替代来源。
但是传统组织工程骨应用的局限性在于其血管化不足，营养和氧气的扩散只能在血管周围100～200μm的范围，且传统的细胞膜片强度较低。
发明内容
本发明旨在克服传统组织工程中，成骨和血管化的不足，以及细胞膜片强度较低的问题，提供一种血管化成骨细胞膜片支架复合体构建的方法。
本发明的首要目的是提供一种血管化成骨细胞膜片支架复合体的制备方法。
本发明的另一目的是提供所述方法制备得到的血管化成骨细胞膜片支架复合体。
本发明的上述目的是通过以下技术方案给予实现的：
本发明提供了一种血管化成骨细胞膜片支架复合体的制备方法，其特征在于，是将血管化成骨细胞膜片包裹在3D打印β-磷酸三钙支架上得到；
所述血管化成骨细胞膜片由人牙周膜间充质干细胞和人脐静脉内皮细胞共培养而得。
将人牙周膜间充质干细胞和人脐静脉内皮细胞两种细胞按不同比例进行混合共培养并进行成膜诱导，通过特定的培养条件得到血管化的成骨细胞膜片；最后与3D打印β-磷酸三钙(β-tricalcium phosphate,β-TCP)支架进行复合构建血管化细胞膜片组织工程骨修复牙槽骨缺损。
通过细胞膜片技术，消除了传统细胞消化技术而导致的细胞外基质的损失，保留了原本细胞微环境，通过成骨和成血管的协同效应，有利于成骨细胞在骨组织工程支架内的存活和转归；进一步将3D打印骨组织工程支架与细胞膜片的有机结合解决了单纯细胞膜片强度低，不能维持成骨空间的问题。
优选地，所述人牙周膜间充质干细胞和人脐静脉内皮细胞的数量比为1～3：1～3。
优选地，所述共培养为将人牙周膜间充质干细胞和人脐静脉内皮细胞在成骨诱导-内皮细胞混合培养基中培养4～21天。
优选地，所述成骨诱导-内皮细胞混合培养基为成骨诱导分化培养基与内皮细胞培养基按照1～3：1～3的体积比混合而成。
最优选地，所述成骨诱导-内皮细胞混合培养基为成骨诱导分化培养基与内皮细胞培养基按照1：1的体积比混合而成。
优选地，所述成骨诱导分化培养基含有如下组分：增殖培养基、地塞米松、β-甘油磷酸、L-抗坏血酸。
优选地，所述成骨诱导分化培养基中各组分的浓度为：50～100nM地塞米松、5～10mMβ-甘油磷酸、10～50μg/mL L-抗坏血酸，余量为增殖培养基。
优选地，所述增殖培养基含有如下组分：10％胎牛血清、1％青霉素-链霉素混合溶液，余量为Dulbecco改良的Eagle培养基-高葡萄糖。
优选地，所述内皮细胞培养基为基础培养基、胎牛血清、内皮细胞生长因子、青霉素-链霉素溶液按照体积比为100：5：1：1配置而成。
优选地，所述3D打印β-磷酸三钙支架的制备方法为：
S1.将量β-磷酸三钙粉末溶于水中制成悬浮液，随后加入2.5wt％聚甲基丙烯酰胺、7mg/ml羟丙基甲基纤维素和0.8％vol聚乙烯亚胺，进行分散、乳化和凝聚，得到打印浆料；
S2.使用生物3D打印机打印尺寸为5mm×10mm×15mm、孔径350～400μm的块状支架；
S3.打印结束后，将支架放在室温下干燥24h，然后在400℃下干燥1h去除有机物，最后在1100℃烧结1h即得到所述3D打印β-磷酸三钙支架。
本发明还请求保护上述方法制备得到的血管化成骨细胞膜片支架复合体。
与现有技术相比，本发明具有以下有益效果：
本发明通过细胞膜片技术，消除了传统细胞消化技术而导致的细胞外基质的损失，保留了原本细胞微环境，通过成骨和成血管的协同效应，有利于成骨细胞在骨组织工程支架内的存活和转归；进一步将3D打印骨组织工程支架与细胞膜片的有机结合，有效增强了细胞膜片的强度，维持成骨空间。
附图说明
图1为实施例2中新生骨组织体积和骨小梁数目。
具体实施方式
以下结合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1一种血管化成骨细胞膜片支架复合体的制备
1、培养基
成骨诱导分化培养基(OM)：
50nM地塞米松、5mMβ-甘油磷酸、10μg/mL L-抗坏血酸，余量为增殖培养基(包括10％胎牛血清、1％青霉素-链霉素混合溶液，余量为Dulbecco改良的Eagle培养基-高葡萄糖)。
内皮细胞培养基(ECM)：
由ScienceCell公司出品，含有如下组分：500mL基础培养基，25mL胎牛血清，5mL内皮细胞生长因子，5mL青霉素-链霉素溶液。
2、制备方法
(1)孔板共培养：将人牙周膜间充质干细胞和人脐静脉内皮细胞分别按3：1、2：2、1：3的数量比混合，分别在OM与ECM的体积比为3：1、2：2、1：3的混合培养基中培养21天得到血管化成骨细胞膜片1～3；人牙周膜间充质干细胞在OM中培养21天作为成骨对照1细胞膜片，人脐静脉内皮细胞在ECM中培养21天作为内皮细胞培养对照2细胞膜片；
(2)3D打印β-TCP支架：将β-TCP粉末溶于蒸馏水中制成悬浮液，随后加入2.5wt％聚甲基丙烯酰胺、7mg/ml羟丙基甲基纤维素和0.8％vol聚乙烯亚胺，进行分散、乳化和凝聚，形成打印浆料，使用生物3D打印机(Regenovo,China)打印尺寸为5mm×10mm×15mm、孔径350～400μm的块状支架；打印结束后，将支架放在室温下干燥24h，然后400℃干燥1h去除有机物，最后在1100℃烧结1h得到最终的支架；
(3)组织工程骨的构建：将血管化成骨细胞膜片1～3和对照1～2细胞膜片分别包裹在3D打印β-TCP支架上，获得细胞膜片TCP支架复合体1～5。
实施例2性能测试
对实施例1中按照人牙周膜间充质干细胞和人脐静脉内皮细胞为3：1制备的细胞膜片、对照1细胞膜片(4：0)进行性能测试。
1、实验方法
将获取的血管化成骨细胞膜片与对照组细胞膜片包裹在5mm×10mm×15mm尺寸的β-TCP支架上，各比例细胞膜片TCP支架复合体各6个样本，而后放置在与支架大小一致的比格犬缺损下颌骨牙槽骨，钛钉固定，盖Bio-Gide胶原膜，分层缝合。术后3月刮取微量下颌骨牙槽骨样本，以4％多聚甲醛固定，送样本进行MicroCT扫描。其中，blank为比格犬缺损下颌骨牙槽骨，scaffold为将单纯的β-TCP支架放置在与支架大小一致的比格犬缺损下颌骨牙槽骨中。
2、实验结果
MicroCT结果如图1所示，结果表明新生骨组织体积和骨小梁数目在3：1比例的细胞膜片TCP支架复合体中最多，与其余各组均有明显的统计学差异。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。
Claims (10)
1.一种血管化成骨细胞膜片支架复合体的制备方法，其特征在于，是将血管化成骨细胞膜片包裹在3D打印β-磷酸三钙支架上得到；
所述血管化成骨细胞膜片由人牙周膜间充质干细胞和人脐静脉内皮细胞共培养而得。
2.根据权利要求1所述方法，其特征在于，所述人牙周膜间充质干细胞和人脐静脉内皮细胞的数量比为1～3：1～3。
3.根据权利要求1所述方法，其特征在于，所述共培养为将人牙周膜间充质干细胞和人脐静脉内皮细胞在成骨诱导-内皮细胞混合培养基中培养4～21天。
4.根据权利要求3所述方法，其特征在于，所述成骨诱导-内皮细胞混合培养基为成骨诱导分化培养基与内皮细胞培养基按照1～3：1～3的体积比混合而成。
5.根据权利要求4所述方法，其特征在于，所述成骨诱导分化培养基含有如下组分：增殖培养基、地塞米松、β-甘油磷酸、L-抗坏血酸。
6.根据权利要求5所述方法，其特征在于，所述成骨诱导分化培养基中各组分的浓度为：50～100nM地塞米松、5～10mMβ-甘油磷酸、10～50μg/mL L-抗坏血酸，余量为增殖培养基。
7.根据权利要求5所述方法，其特征在于，所述增殖培养基含有如下组分：10％胎牛血清、1％青霉素-链霉素混合溶液，余量为Dulbecco改良的Eagle培养基-高葡萄糖。
8.根据权利要求4所述方法，其特征在于，所述内皮细胞培养基为基础培养基、胎牛血清、内皮细胞生长因子、青霉素-链霉素溶液按照体积比为100：5：1：1配置而成。
9.根据权利要求1所述方法，其特征在于，所述3D打印β-磷酸三钙支架的制备方法为：
S1.将量β-磷酸三钙粉末溶于水中制成悬浮液，随后加入2.5wt％聚甲基丙烯酰胺、7mg/ml羟丙基甲基纤维素和0.8％vol聚乙烯亚胺，进行分散、乳化和凝聚，得到打印浆料；
S2.使用生物3D打印机打印尺寸为5mm×10mm×15mm、孔径350～400μm的块状支架；
S3.打印结束后，将支架放在室温下干燥24h，然后在400℃下干燥1h去除有机物，最后在1100℃烧结1h即得到所述3D打印β-磷酸三钙支架。
10.权利要求1～9任一所述方法制备得到的血管化成骨细胞膜片支架复合体。
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| CN114870092A (zh) * | 2022-05-20 | 2022-08-09 | 广州骊贝生物科技有限公司 | 骨替代复合体及其制备方法及用途 |
| CN117363565A (zh) * | 2023-09-28 | 2024-01-09 | 南方医科大学口腔医院 | 一种用于骨再生的血管化干细胞球的构建方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114870092A (zh) * | 2022-05-20 | 2022-08-09 | 广州骊贝生物科技有限公司 | 骨替代复合体及其制备方法及用途 |
| CN114870092B (zh) * | 2022-05-20 | 2023-08-22 | 广州骊贝生物科技有限公司 | 骨替代复合体及其制备方法及用途 |
| CN117363565A (zh) * | 2023-09-28 | 2024-01-09 | 南方医科大学口腔医院 | 一种用于骨再生的血管化干细胞球的构建方法 |
| CN117363565B (zh) * | 2023-09-28 | 2024-05-17 | 南方医科大学口腔医院 | 一种用于骨再生的血管化干细胞球的构建方法 |
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