课题背景：组织工程构建牙是当前牙再生研究的主要方向，作为牙主体结构的牙髓牙本质复合体是研究重点之一，是组织工程成功构建牙的第一步。关于组织工程化牙髓-牙本质复合体种子细胞的研究，主要以牙间充质细胞和牙髓干细胞为主，对牙乳头细胞的研究较少，对大型哺乳动物猪的牙乳头细胞研究更少。牙乳头细胞是牙髓细胞、牙本质细胞的前体细胞，以牙乳头细胞为种子细胞成功构建组织工程化牙髓牙本质复合体具有重要意义。

应用要点：①以体外培养的猪牙乳头细胞为种子细胞，β－磷酸三钙为支架材料成功构建牙髓牙本质复合体样结构，提示牙乳头来源的间充质细胞可用于构建牙齿硬组织。②阳性对照的实验结果提示，裸鼠皮下不能为牙齿再生提供良好的微环境，动物模型及植入部位有待探索。

同行评价：应用组织工程学技术再生牙齿或牙周组织的研究是目前牙齿再生的研究热点，目前进行牙齿组织工程研究的实验应用牙源性的细胞或组织，该课题应用猪牙乳头细胞再生牙髓牙本质复合体，牙乳头细胞可以作为种子细胞构建牙髓牙本质复合体样结构，是牙髓牙本质复合体再生研究中很好的细胞来源。

摘要
目的: 牙乳头细胞是牙髓细胞、牙本质细胞的前体细胞，应用组织工程方法探讨以猪牙乳头细胞为种子细胞构建牙髓牙本质复合体的可行性。
方法：实验于2004-03/2005-11在上海市口腔医学重点实验室完成。以第1代猪牙乳头细胞为种子细胞与β－磷酸三钙为支架材料复合后接种在裸鼠皮下。移植在裸鼠皮下的β－磷酸三钙支架与牙乳头细胞复合物为实验组，同期移植牙胚作为阳性对照组，同期植入的空白β－磷酸三钙支架材料作阴性对照组。每组３个样本，在同一裸鼠皮下植入。８周后取材进行组织学（苏木精－伊红染色及Goldner'三色法染色）、牙本质涎蛋白免疫组织化学及透射电镜检测。
结果：①移植物组织学切片显示在支架材料孔隙内形成了牙髓牙本质复合体样结构，由牙本质基质样物质、前期牙本质样物质及牙髓样组织组成。在牙本质样物质内部可见少量牙本质小管，牙髓样组织外层细胞较为密集且单层排列，表现出成牙本质细胞样细胞的特征。②免疫组织化学染色显示，在邻近成牙本质细胞样细胞周围的前期牙本质内牙本质涎蛋白呈阳性表达。③透射电镜结果显示，实验组局部可见牙本质小管样结构。
结论：以猪牙乳头细胞为种子细胞、β－磷酸三钙为支架材料，成功构建出牙髓牙本质复合体样结构。
关键词：磷酸三钙；牙乳头细胞；牙本质；牙髓；组织构建；生物材料

张富强，傅远飞，吴伟，翁雨来，李静，张秀丽.猪牙乳头细胞与β－磷酸三钙支架构建牙髓牙本质复合体样结构[J].中国组织工程研究与临床康复，2008，12(6):1009-1012 [www.zglckf.com/zglckf/ejournal/upfiles/08-6/6k-1009(ps).pdf]

Dentin-pulp complex-like structure constructed by porcine dental papilla cells and beta-tricalcium phosphate scaffold

Abstract

AIM：The dental papilla cells are the precursor of dental pulp cells and dentin cells. This study investigated the constructive possibility of constructing dentin-pulp complex by tissue engineering technology, taking porcine dental papilla cells as the seed.
METHODS: This study was performed at Shanghai Key Laboratory of Stomatology from March 2004 to November 2005. Porcine dental papilla cells at the 1st generation were seeded into β-tricalcium phosphate (β-TCP) scaffold, and then the cell-scaffold complex was transplanted subcutaneously into the nude mice. The β-TCP and dental papilla cells compound was served as the experimental group, while the cells transplanted with tooth bud or β-TCP were taken as positive or negative control group respectively. Every group contained 3 samples in the same nude mouse. Eight weeks later, the transplants were detected by the methods of histology (hematoxylin-eosin, Goldner's trichrome), immunohistochemical examinations of dentin sialoprotein and transmission electron microscopy.
RESULTS: ①The histological examination showed that there were dentin-pulp complex-like structure, consisting of dentinal matrix-like tissues, predentin-like tissues and pulp-like tissues, in the hole of β-TCP scaffold. Some dentin tubule-like structures were detectable in the dentin-like tissues. Cells localized along the pulp-like tissues were dense and polarized, which were similar to the odontoblasts.②Dentin sialoprotein expression was demonstrated in pedentin-like tissues nearby the odontoblast-like cells by immunohistochemical examination.③Some dentin tube-like structures were observed in the experimental group through transmission electron microscope scans.
CONCLUSION: Dentin-pulp complex-like structure can be successfully constructed by seeding porcine dental papilla cells into β-TCP scaffold.

Zhang FQ, Fu YF, Wu W, Weng YL, Li J, Zhang XL.Dentin-pulp complex-like structure constructed by porcine dental papilla cells and beta-tricalcium phosphate scaffold.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(6):1009-1012(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-6/6k-1009(ps).pdf]