Background

Dental odontoblasts produce dentin mineralized matrix, trigger immune responses and act as sensory cells. The understanding of the mechanisms of these functions has been particularly restricted due to the lack of odontoblasts being cultivable in vitro. Because of the lack of specific markers to identify cells of the odontoblastic lineage, properties of the cells isolated from the dentin-pulp interface were compared to dental pulp cells, periodontal ligament cells, osteoblasts, skin fibroblasts, epithelial cells (A549) and HeLa in the present study.

Methods

After surgical procedures, the pulp tissue was removed from the tooth crown, and cells were scrapped off the dentin-pulp interface. Explants from teeth of three patients were routinely cultivated, and cells were harvested after several weeks. Cell morphology and ultrastructure was studied by light microscopy (LM), scanning (SEM) or transmission electron microscopy (TEM). Expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), TRPV4, and S100 calcium binding protein A4 (S100A4) were analyzed at the protein level by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using specific antibodies. The differential expression of S100A4 in the various cell lines was further investigated at the gene level by semiquantitative real-time PCR. Mineralization in the various cell types was observed after alizarin red staining after a 28 days incubation period. The immunophenotype of the cells was examined by flow cytometry using monoclonal anti-human antibodies CD90-FITC, CD73-PE, CD105-PE, CD29-PE, CD140a-FITC, CD144-PE, CD45-FITC or CD34-FITC. Differences between median values were statistically analyzed (Mann–Whitney U-test).

Results

Cells from the dentin-pulp interface retain the polarity of odontoblast morphology in culture with an elongated, rounded cell body, and an extended cellular process. Ultrastructural analysis of the cells indicates high secretory activity including the extracellular deposition of fibrillar collagen. An extended rough endoplasmic reticulum is lined by a large number of ribosomes, and a vast number of secretory granules merges with the cell membrane. Protein expression of DSPP, DMP1, and TRPV4 as a transient receptor potential cation was detected in all cell lines. S100A4 was found differentially expressed in cultures of cells from tooth tissues. High expression of S100A4 was observed at the protein and gene level in two fractions of cells isolated from the dentin-pulp interface, but was absent or only weakly expressed in pulp cells. S100A4 expression in cells from the dentin-pulp interface and pulp cells is consistent with the intensity of the formation of mineralized nodules detected by alizarin red staining. Immunophenotyping revealed that a high percentage of CD73 (ecto-5-nucleotidase), an enzyme active on the surface of immune-competent cells, was expressed in cells of the dentin-pulp interface. While 72%–78% of positive cells were detected in dentin-pulp interface fractions, only 28–64% of the cells in pulp cell cultures were stained.

Conclusions

The present findings obtained with a variety of cells of different origin provide experimental evidence that cells isolated from the dentin-pulp interface express unique properties different from dental pulp cells in particular. The differential expression of S100A4 is a relevant marker candidate for differentiating between dental pulp cells and cells of the odontoblast lineage.

中文翻译：

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从牙本质-牙髓界面分离的细胞的显着特性

背景

牙齿成牙本质细胞产生牙本质矿化基质，触发免疫反应并充当感觉细胞。由于缺乏可在体外培养的成牙本质细胞，对这些功能机制的理解特别受到限制。由于缺乏识别成牙本质细胞谱系细胞的特异性标记物，将从牙本质-牙髓界面分离的细胞的特性与牙髓细胞、牙周膜细胞、成骨细胞、皮肤成纤维细胞、上皮细胞 (A549) 和 HeLa 进行比较。目前的研究。

方法

手术后，从牙冠上去除牙髓组织，并从牙本质-牙髓界面上刮下细胞。常规培养三名患者牙齿的外植体，数周后收获细胞。通过光学显微镜 (LM)、扫描 (SEM) 或透射电子显微镜 (TEM) 研究细胞形态和超微结构。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS-PAGE) 和使用特异性抗体的蛋白质印迹在蛋白质水平上分析牙本质唾液酸磷蛋白 (DSPP)、牙本质基质蛋白 1 (DMP1)、TRPV4 和 S100 钙结合蛋白 A4 (S100A4) 的表达. 通过半定量实时 PCR 在基因水平上进一步研究了 S100A4 在各种细胞系中的差异表达。在 28 天的潜伏期后茜素红染色后观察到各种细胞类型的矿化。使用单克隆抗人抗体 CD90-FITC、CD73-PE、CD105-PE、CD29-PE、CD140a-FITC、CD144-PE、CD45-FITC 或 CD34-FITC，通过流式细胞术检查细胞的免疫表型。对中值之间的差异进行统计分析（Mann-Whitney U 检验）。

结果

来自牙本质-牙髓界面的细胞在培养中保留了成牙本质细胞形态的极性，具有细长的圆形细胞体和扩展的细胞过程。细胞的超微结构分析表明具有高分泌活性，包括纤维状胶原的细胞外沉积。延伸的粗面内质网内衬大量核糖体，大量分泌颗粒与细胞膜融合。在所有细胞系中检测到作为瞬时受体电位阳离子的 DSPP、DMP1 和 TRPV4 的蛋白质表达。发现 S100A4 在来自牙齿组织的细胞培养物中差异表达。在从牙本质-牙髓界面分离的两个细胞部分中，在蛋白质和基因水平上观察到 S100A4 的高表达，但在牙髓细胞中不存在或仅微弱表达。S100A4在牙本质-牙髓界面细胞和牙髓细胞中的表达与茜素红染色检测到的矿化结节形成强度一致。免疫表型分析显示，高百分比的 CD73（胞外 5-核苷酸酶），一种在免疫活性细胞表面具有活性的酶，在牙本质 - 牙髓界面的细胞中表达。虽然在牙本质-牙髓界面组分中检测到 72%–78% 的阳性细胞，但牙髓细胞培养物中只有 28–64% 的细胞被染色。在牙本质-牙髓界面的细胞中表达。虽然在牙本质-牙髓界面组分中检测到 72%–78% 的阳性细胞，但牙髓细胞培养物中只有 28–64% 的细胞被染色。在牙本质-牙髓界面的细胞中表达。虽然在牙本质-牙髓界面组分中检测到 72%–78% 的阳性细胞，但牙髓细胞培养物中只有 28–64% 的细胞被染色。

结论

用各种不同来源的细胞获得的本研究结果提供了实验证据，表明从牙本质-牙髓界面分离的细胞表现出与牙髓细胞不同的独特特性。S100A4 的差异表达是区分牙髓细胞和成牙本质细胞谱系细胞的相关标记候选物。