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

作为牙本质-牙髓界面的上皮细胞，成牙本质细胞表现出独特的生理特性。这些细胞将牙本质构建为坚硬的组织，它们是细胞免疫系统的一部分，作为机械敏感细胞，它们参与了伤害感觉。成牙细胞是修复牙材料的靶细胞，也是生物相容性研究的重要组成部分。不幸的是，目前的科学共识是，原代成牙细胞是非分裂细胞，因此不可培养。在初步的研究中，我们从几个年轻患者的第三磨牙的牙本质-牙髓界面分离原代细胞，扩大和表征形态。本文的目的是阐明这些细胞作为原代成牙细胞的身份。他们的主要困难是缺乏这种细胞类型的独特和特定的标记。因此，与不同来源的细胞（包括牙髓细胞、牙周韧带细胞、牙乳头细胞、成骨细胞、皮肤成纤维细胞、HeLa细胞和肺上皮细胞）相比，分离的原代培养物将具有不同特性的模式。我们在初步工作中描述了细胞的明显不同形态。这里我们继续分析蛋白质和基因的表达，如DSPP， DMP1， NCAM, nestin, pannexin2runx2，骨钙素或colA1。采用细胞术对间充质干细胞进行免疫分型，并对矿化进行研究。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。如果有可能在培养中保持原代成牙细胞，我们就可以研究成牙细胞功能背后的细胞机制。这些发现将是发展龋齿病变治疗策略的重要一步，这些疗法的药理支持的临床相关选择，以及局部组织再生。

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.