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

在本研究中，我们尝试使用转染BMP的牙髓干细胞在体外开发具有牙本质-牙髓复合体的生物牙。基于排除DNA染料Hoechst 33342，从成人牙髓组织中分离侧群（SP）细胞。用CD 117、CD 90、CD 31和CD 135染色后获得更富集的干细胞组分。它们表现出干细胞活性，如自我更新能力和分化为神经元、软骨细胞和脂肪细胞以及成牙本质细胞的多能性。转染BMP的牙髓干细胞中成牙本质细胞分化标志物的表达较对照组增加。将转染BMP的细胞培养在具有凹陷形状的支架上，并进一步与牙髓干细胞分层。培养7天后，成牙本质细胞样细胞贴附于支架上，并向支架内延伸出较长的细胞突起。Dspp mRNA在成牙本质细胞层中表达，提示牙本质-牙髓复合体的形成。使用该生物牙进行犬体内评价。离体牙髓上移植生物牙诱导再生牙本质形成。结论：转染BMP基因的牙髓干细胞与合适的支架材料结合可用于牙髓牙本质复合体的再生治疗。

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.