Formation of Biotooth in Three Dimensional Culture of Dental Pulp Stem Cells Transfected with BMP

在这项研究中，我们试图利用BMP转基因的牙髓干细胞在体外培养出具有牙本质-牙髓复合体的生物牙。在排除脱氧核糖核酸染料Hoechst 33342的基础上，从成人牙髓组织分离出侧群(SP)细胞。CD117、CD90、CD31、CD135染色后，可获得更丰富的干细胞组分。它们表现出干细胞活性，如自我更新能力和分化为神经元、软骨细胞、脂肪细胞和成牙本质细胞的多潜能。与对照组相比，转染骨形成蛋白的牙髓干细胞成牙本质细胞分化标志物的表达增加。将转BMP基因的细胞接种于凹形支架上，并以牙髓干细胞为载体进行分层培养。培养7天后，附着在支架上的成牙本质细胞样细胞将漫长的细胞突起延伸到支架内。Dspp基因在成牙本质细胞层有表达，提示牙本质-牙髓复合体的形成。在狗体内的评估是使用这颗生物牙进行的。生物牙移植于断髓诱导再生牙本质形成。结论：BMP转基因牙髓干细胞结合合适的支架材料可用于牙本质/牙髓复合体的再生治疗，用于根管治疗。

In this investigation, we have tried to develop a biotooth with dentin-pulp complex in vitro using pulp stem cells transfected with Bmp. Side population (SP) cells were isolated from adult pulp tissue based on the exclusion of the DNA dye Hoechst 33342. The more enriched stem cell fractions were obtained after staining with CD117, CD90, CD31 and CD135. They exhibited stem cell activity, such as self-renewal capability and multipotency to differentiate into neurons, chondrocytes and adipocytes as well as odontoblasts. The expression of odontoblast differentiation markers was increased in the pulp stem cells transfected with Bmp compared with control pCAGGS. The Bmp transfected cells were cultured on the scaffold with concaved shape and further layered by pulp stem cells. The odontoblast-like cells attaching to the scaffold extended long cellular processes into the scaffold seven days afterr cultivation. Dspp mRNA was expressed in the odontoblastic layers, suggesting the formation of dentin-pulp complex. The in vivo evaluation in the dog was undertaken using this biotooth. Transplantation of the biotooth on amputated pulp induced regenerative dentin formation. In conclusion, the pulp stem cells transfected with Bmp combined with the proper scaffold can be used for regenerative therapy of dentin/pulp complex for endodontic treatment.