D'Errico J A, MacNeil R L, Takata T, Berry J, Strayhorn C, Somerman M J
Department of Periodontics/Prevention/Geriatrics and Pharmacology, University of Michigan, Ann Arbor, USA.
Bone. 1997 Feb;20(2):117-26. doi: 10.1016/s8756-3282(96)00348-1.
Periodontal disease is marked by inflammation and subsequent loss and/or damage to tooth-supporting tissues including bone, cementum, and periodontal ligament. A key tissue in the initial process of periodontal development as well as regeneration following periodontal disease is cementum. Research efforts aimed toward understanding mechanisms involved in periodontal development and regeneration, and in particular the formation of root cementum, have been hampered by an inability to isolate and culture cells involved in cementum production (i.e., cementoblasts). Much has been learned regarding the processes and mechanisms involved in bone formation and function from experiments using bone cell cultures. Therefore, the purpose of this study was to develop a strategy whereby cementoblasts could be isolated, cultured, and characterized. As a first step, using in situ hybridization, we determined the timed and spatial expression of mineral-associated proteins during first molar root development in CD-1 mice. These proteins included dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), and type I collagen. During root development in mice BSP, OPN, and OCN mRNAs were expressed selectively by cells lining the tooth root surface--cementoblasts--with high levels of expression at day 41. Importantly, at this time point BSP, OPN, and OCN mRNAs were not expressed throughout the periodontal ligament. These findings provided us with markers selective to root-lining cells, or cementoblasts, in situ, and established the time (day 41) for isolating cells for in vitro studies. To isolate cells from tissues adherent to the root surface, enzymatic digestion was used, similar to what are now considered classical techniques for isolation of osteoblasts. To determine whether cells in vitro contained root-lining cells and cementoblasts, cultured cells were analyzed for expression of mineral-associated proteins. Cells within this heterogeneous primary population expressed type I collagen, BSP, OPN, and OCN as determined by in situ hybridization. In contrast, cells within this population did not express dentin sialoprotein, an odontoblast-specific protein. These procedures have provided a means to obtain root-lining cells in vitro that can now be cloned and used for studies directed at determining the properties of root-lining cells, or cementoblasts, in vitro.
牙周病的特征是炎症以及随后牙齿支持组织（包括骨、牙骨质和牙周韧带）的丧失和/或损伤。牙骨质是牙周发育初始过程以及牙周病后再生过程中的关键组织。由于无法分离和培养参与牙骨质生成的细胞（即成牙骨质细胞），旨在了解牙周发育和再生机制，特别是牙根牙骨质形成机制的研究工作受到了阻碍。从使用骨细胞培养的实验中，我们已经了解了许多关于骨形成和功能的过程及机制。因此，本研究的目的是制定一种策略，以便能够分离、培养和鉴定成牙骨质细胞。第一步，我们使用原位杂交技术，确定了CD-1小鼠第一磨牙牙根发育过程中与矿物质相关蛋白的时间和空间表达。这些蛋白质包括牙本质涎蛋白（DSP）、骨桥蛋白（OPN）、骨涎蛋白（BSP）、骨钙素（OCN）和I型胶原蛋白。在小鼠牙根发育过程中，BSP、OPN和OCN mRNA由牙根表面的细胞——成牙骨质细胞选择性表达，在第41天时表达水平较高。重要的是，在这个时间点，BSP、OPN和OCN mRNA在整个牙周韧带中均未表达。这些发现为我们提供了原位选择性标记牙根内衬细胞或成牙骨质细胞的标志物，并确定了用于体外研究的细胞分离时间（第41天）。为了从附着于牙根表面的组织中分离细胞，我们采用了酶消化法，这类似于现在被认为是分离成骨细胞的经典技术。为了确定体外培养的细胞是否包含牙根内衬细胞和成牙骨质细胞，我们对培养的细胞进行了与矿物质相关蛋白表达的分析。通过原位杂交确定，这个异质性原代细胞群体中的细胞表达I型胶原蛋白、BSP、OPN和OCN。相比之下，这个群体中的细胞不表达牙本质涎蛋白，一种成牙本质细胞特异性蛋白。这些方法提供了一种在体外获得牙根内衬细胞的手段，现在可以对其进行克隆，并用于旨在确定体外牙根内衬细胞或成牙骨质细胞特性的研究。
