人脐带Wharton胶中间充质干细胞体外无支架组织工程软骨的构建

In vitro construction of non-scaffold tissue engineered cartilage using mesenchymal stem cells derived from Wharton's jelly of the human umbilical cord

背景:构建组织工程软骨的方法多为自体种子细胞复合天然或合成物支架,目前多存在种子细胞来源有限、支架安全性和生物相容性、以及细胞在支架中分布不均的问题。目的:探讨人脐带Wharton胶中间充质干细胞向软骨诱导分化并体外构建无支架组织工程软骨的可行性。方法:分离培养人脐带Wharton胶中的间充质干细胞,进行流式细胞学鉴定。对软骨诱导前后的细胞进行组织学和免疫组织化学染色,对其表达的葡萄糖胺聚糖和Ⅱ型胶原进行定量研究,并应用RT-PCR检测软骨诱导前后Ⅱ型胶原和Sox-9mRNA的表达。采用密集诱导培养→离心管培养→生物反应器培养,进行体外构建无支架软骨组织。结果与结论:人脐带Wharton胶富含干细胞,流式细胞仪检测结果显示这些细胞不表达造血干细胞标志,表达CD44,CD105、CD271等间充质干细胞表面标志;HLA-ABC阳性表达,HLA-DPDQDR阴性表达。未进行软骨诱导的细胞弱表达软骨细胞标志,诱导后葡萄糖胺聚糖和Ⅱ型胶原显著增高。RT-PCR结果显示人脐带Wharton胶间充质干细胞诱导前后均表达Sox-9、Ⅱ型胶原mRNA。说明人脐带Wharton胶间充质干细胞具有前软骨细胞的特性。采用密集诱导培养结合生物反应器培养,不用支架,体外可以构建成大块组织工程软骨。表明人脐带Wharton胶间充质干细胞是一种良好的构建组织工程软骨的种子细胞。

BACKGROUND：Autogenous seed cells combined with natural or compositive scaffold are commonly used in construction of tissue engineered cartilage,which face the problems of insufficient seed cells,poor security and biocompatibility,as well as uneven distribution of cells in scaffold.OBJECTIVE：To investigate chondrogenic induction of mesenchymal stem cells（MSCs） derived from Wharton＇s jelly of the human umbilical cord and to fabricate non-scaffold tissue engineered cartilage in vitro.METHODS：MSCs were isolated and cultured from Wharton＇s jelly of the umbilical cord,and the phenotypes were detected by flow cytometry.The Wharton＇s jelly MSCs were cultured with chondrogenic media and detected with histochemistry and immunohistochemistry assay.Expressions of glycosaminoglycan（GAG）,type Ⅱ collagen and Sox-9 were analyzed by reverse transcription-polymerase chain raction（RT-PCR）.Non-scaffold tissue engineered cartilage was fabricated in vitro by high density cell number with chondrogenic induction medium,and then succeeded pellet was culture in bioreactor.RESULTS AND CONCLUSION：Wharton＇s jelly of human umbilical cord was abundant with stem cells.Flow cytometric analysis revealed that Wharton＇s jelly stem cells expressed CD44,CD105 and CD271 rather than hemopoietic stem cell markers.The cells were positive expressed HLA-ABC,and negative expressed HLA-DPDQDR.The histochemistry and immunohistochemistry showed that Wharton＇s jelly MSCs weakly expressed chondrocyte marker,which strongly expressed GAG and type Ⅱ collagen after chondgenic induction.RT-PCR results demonstrated that Wharton＇s jelly MSCs and chondrogenic induction cells were both expressed Sox-9 and type Ⅱ collagen.Non-scaffold tissue engineered cartilage block was formed with high density cell number culture and bioreactor chondrogenic induction in vitro.The results revealed that Wharton＇s jelly MSCs maybe a new seed cells for fabricating tissue engineered cartilage in vitro.