脐带间充质干细胞对CoCrMo磨损微粒诱导的小鼠颅骨溶解的影响
| 作者: |
1,2王帅,
2邓展涛,
2马元琛,
1,2郑秋坚
1 华南理工大学医学院，再生医学联合研究中心，广州 510006 2 广东省人民医院(广东省医学科学院)骨科中心，广州 510080 |
| 通讯: |
邓展涛
Email: dengzhantao@gdph.org.cn 郑秋坚 Email: zhengqiujian@gdph.org.cn |
| DOI: | 10.3978/j.issn.2095-6959.2022.01.003 |
| 基金: | 国家自然科学基金青年项目(81802222)；广东省自然科学基金(2018A030310694，2020A1515010268，2021A15150110008)；广东省中医药局科研项目(20191004)；广东省人民医院优秀青年人才计划(KJ012019091)；中央高校基本科研业务费专项资金(2020ZYGXZR010)；广东省医学科学技术研究基金(A2019461)；广州市科技计划项目(201904010424)。 |
摘要
目的：探究人脐带间充质干细胞(human umbilical cord mesenchymal stem cells，hUCMSCs)通过抑制炎症反应在CoCrMo磨损微粒(CoCrMo wear particles，CoPs)诱导的小鼠颅骨溶解动物模型中的保护作用。方法：使用扫描电镜(scanning electron microscope，SEM)、透射电镜(transmission electron microscope，TEM)、能谱分析(energy dispersive spectroscopy，EDS)、X射线衍射仪(X-ray diffraction，XRD)探测CoPs的形貌、粒径分布、化学成分百分比以及物相成分分析。使用微型计算机断层扫描(micro-computed tomography，Micro-CT)和三维重建分析来对小鼠颅骨骨质丢失程度进行影像学和定量的分析。通过甲苯胺蓝染色、苏木精-伊红染色(hematoxylin-eosin，HE)和抗酒石酸酸性磷酸酶染色(tartrate-resistant acid phosphatase，TRAP)鉴定小鼠颅骨组织骨溶解的组织学差异。使用ELISA检测试剂盒对CoPs诱导的无菌性松动的小鼠模型的骨膜组织中的炎症因子肿瘤坏死因子α(tumor necrosis factor-α，TNF-α)的表达差异进行鉴定。结果：CoPs主要由C、Cr7C3、Mo2C、Co和CoMoO4组成，并在SEM和TEM中表现为表面较为光滑的球状。为探究hUCMSCs对CoPs诱导小鼠颅骨骨溶解的影响，首先利用Micro-CT扫描小鼠颅骨样本，结果显示：相比于单次hUCMSCs注射组，多次hUCMSCs注射组能明显改善骨质丢失的情况。与单次hUCMSCs注射组相比，多次hUCMSCs注射组HE染色和甲苯胺蓝染色显示颅骨的骨丢失较轻且范围较小，TRAP染色显示破骨细胞活性较低。小鼠颅骨骨膜中的TNF-α表达呈现出hUCMSCs的剂量依赖性降低。结论：hUCMSCs在CoPs诱导的小鼠颅骨溶解动物模型中发挥保护作用，而调节小鼠颅骨骨膜组织的炎症反应水平可能是hUCMSCs影响磨损微粒诱导的无菌性松动的一种调节机制。
关键词：
人脐带间充质干细胞；骨溶解；CoCrMo磨损微粒；无菌性松动；动物模型
Effects of human umbilical cord mesenchymal stem cells in CoCrMo wear particles-induced murine calvarial osteolysis
CorrespondingAuthor: DENG Zhantao Email: dengzhantao@gdph.org.cn
DOI: 10.3978/j.issn.2095-6959.2022.01.003
Foundation: This work was supported by the National Natural Science Foundation of China Youth Science Foundation (81802222), the Natural Science Foundation of Guangdong Province (2018A030310694, 2020A1515010268, 2021A15150110008), the Foundation of Traditional Chinese Medicine of Guangdong Province (20191004), the Outstanding Young Talents Foundation of Guangdong Provincial People’s Hospital (KJ012019091), the Fundamental Research Funds for the Central Universities (2020ZYGXZR010), Medical Science and Technology Research Foundation of Guangdong Province (A2019461), and Program of Science and Technology of Guangzhou (201904010424), China.
Abstract
Objective: To investigate the protective effect of human umbilical cord mesenchymal stem cells (hUCMSCs) in animal model with CoCrMo wear particles (CoPs)-induced murine calvarial osteolysis by inhibited inflammatory reaction. Methods: Scanning electron microscope (SEM), transmission electron microscope (TEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) were used to examine the morphology, particle size distribution, chemical composition ratio and phase analysis of CoPs. Micro-computed tomography (Micro-CT) and 3-dimensional reconstruction analysis were used for quantification of the degree of murine calvarial bone loss. Toluidine blue staining, hematoxylin-eosin (HE) staining and tartrate-resistant acid phosphatase (TRAP) staining were applied to detect histologic changes of murine calvarial osteolysis. The ELISA kit was used to assess the difference in expression of inflammatory cytokine tumor necrosis factor-α (TNF-α) in murine calvarial periosteum tissue induced by CoPs in aseptic osteolysis. Results: CoPs were constituted by C, Cr7C3, Mo2C, Co and CoMoO4, and appeared as a spherulite with a relatively smooth surface detected by SEM and TEM. The implantation of CoPs-induced murine calvarial osteolysis in the mouse model used to investigate the protective effect of hUCMSCs showed that repeated hUCMSCs injection could significantly ameliorate bone loss than single hUCMSCs injection did when scanned with Micro-CT and analysed by 3-dimensional reconstruction. Repeated injection of hUCMSCs significantly showed lower bone loss than single hUCMSCs injection did as detected by HE staining and toluidine blue staining. Repeated hUCMSCs injection also led to less activated osteoclasts by TRAP staining when compared with single hUCMSCs injection. The expression of TNF-α in murine calvarial periosteum tissue represented a hUCMSCs-dose-dependent reduction. Conclusion: hUCMSCs has a protective effect in CoPs-induced murine calvarial osteolysis animal model, while regulating inflammatory responses in murine calvarial periosteum tissue may be the regulatory mechanism for the effect of hUCMSCs injection therapy in wear particles-induced aseptic loosening.
Keywords：
human umbilical cord mesenchymal stem cells; osteolysis; CoCrMo wear particles; aseptic loosening; animal model