用人牙源性上皮和间充质细胞构建组织工程化牙齿样结构的实验研究

Tissue engineering of tooth-like structures by human dental epithelial and mesenchymal cells

目的 :以人牙源性上皮和间充质细胞作为种子细胞在裸鼠体内构建牙齿样结构。方法 :将体外培养的人牙源性上皮细胞和经生长因子诱导后的牙源性间充质细胞分层接种于经塑形的陶瓷化骨或Colla graft三维支架上 ,将形成的细胞 /支架复合物移植到免疫缺陷小鼠的皮下 ,建立牙源性上皮细胞复合间充质细胞的体内立体培养模型。 14～ 18周后取材 ,采用HE染色、改良丽春红三色法、免疫组化和原位杂交方法检测移植物中的组织新生情况。结果 :细胞在三维支架上形成了包含釉质样组织和牙本质牙髓复合体的牙齿样结构。新生的釉质样组织表达人成釉蛋白 ,其周围残留的牙源性上皮细胞表达人釉原蛋白mRNA。同时 ,人DSP蛋白在牙本质样组织中呈阳性表达。所有对照组标本中均未观察到该现象。结论 :以优选出并经塑形的三维支架为载体 ,在裸鼠体内建立牙源性上皮细胞复合间充质细胞的立体培养模型 ,成功构建出组织工程化牙齿样结构。

AIM:To tissue engineer the tooth-like structu re with human dental epithelial and mesenchymal cells as seed cells in vivo. METHODS:The cultured human dental epithelial cells and induceddental mesenchymal cells were taken as seed cells,and implanted onto processedCBB and Collagraft.Then cell/scaffold constructs were transplanted s.c.into nudemice in order to establish in vivo culture model of dental mesenchymal cell s with dental epithelial cells.14～18 weeks later,implants were excised for hist o logical,immunohistochemical and in situ hybridization analysis.RESU LTS:Tooth-like structures including enamel-like tissue and dentin-pulpcomplex were generated within implants of experimental groups but not of controlgroups. Human ameloblastin was expressed in newly-formed enamel-like tissues,an d the residual dental epithelial cells around enamel-like tissue expressed humanamelogenin mRNA.Human DSP was expressed in dentin-like tissues.CONCLUS ION:The culture model in vivo of human dental mesenchymal cells wit h dental epithelial cells were established with processed CBB and Collagraft ascarriers in nude mice.As a result,tooth-like structures were bioengineered succe ssfully in transplants.[