Odontoblast cell culture – Characterisation of cells from the dentin-pulp interface

成牙本质细胞作为牙本质-牙髓界面的上皮层，表现出独特的生理特性。这些细胞构建牙本质作为一种硬组织，它们是细胞免疫系统的一部分，作为机械敏感细胞，它们参与伤害性感受。成牙本质细胞是修复牙科材料的靶细胞，也是生物相容性研究必不可少的细胞。不幸的是，目前的科学共识是，原始成牙本质细胞是未分裂的细胞，因此是不可培养的。在初步研究中，我们从年轻患者的几个第三磨牙牙本质-牙髓界面分离出原代细胞，扩增并鉴定其形态。目前这项提议的目的是澄清这些细胞是初级成牙本质细胞的身份。他们的主要困难是缺乏针对这种细胞类型的唯一和特异的标记。因此，与牙髓细胞、牙周韧带细胞、牙乳头细胞、成骨细胞、皮肤成纤维细胞、HeLa和肺上皮细胞等不同来源的细胞相比，分离的原代培养细胞具有不同的特性。我们在初步工作中描述了明显不同的细胞形态。在这里，我们继续分析蛋白质和基因的表达，如DSPP，DMP1，NCAM，Nestin，pAnnexin 2 Runx2，骨钙素或colA1。免疫表型将按照目前的指南进行，以鉴定间充质干细胞，并将进行矿化研究。这些参数的表达将允许评估从牙本质-牙髓界面分离的细胞的身份。如果有可能将原代成牙本质细胞保留在培养中，我们就可以研究成牙本质细胞功能背后的细胞机制。这些发现将是在制定治疗龋损的策略、这些疗法的药物支持的临床相关选择以及局部组织再生方面向前迈出的重要一步。

As an epithelial layer at the dentin-pulp interface odontoblasts express unique physiological properties. These cells build dentin as a hard tissue, they are part of the cellular immune system, and as mechanosensitive cells they are involved in nociception. Odontoblasts are target cells of restorative dental materials and essential for biocompatibility studies as well. Unfortunately, it is current scientific consensus that primary odontoblasts are non-dividing cells and thus inculturable. In preliminary studies we isolated primary cells from the dentin-pulp interface of several third molars of young patients, expanded and characterized the morphology. It is aim of the current proposal to clarify the identity of these cells as primary odontoblasts. They major difficulty is the lack of a unique and specific marker for this cell type. Thus the isolated primary cultures will be characterized by a pattern of various properties in comparison with cells of different origin including pulp cells, cells from the periodontal ligament, dental papilla cells, osteoblasts, skin fibroblasts, HeLa, and lung epithelial cells. We described the clearly different morphology of the cells in a preliminary work. Here we continue with the analysis of the expression of proteins and genes such as DSPP, DMP1, NCAM, nestin, pannexin 2 runx2, osteocalcin or colA1. Immunophenotyping following current guidelines for the identification of mesenchymal stem cells will be performed by cytometry, and mineralization will be investigated as well. The expression of these parameters will allow for the evaluation of the identity of the cells isolated from the dentin-pulp interface. If it was possible to keep primary odontoblasts in culture we could investigate cellular mechanisms behind odontoblast functions. These findings would be a major step forward in the development of strategies for the therapy of carious lesions, clinically relevant options of pharmacological support of these therapies, and local tissues regeneration.