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.

Distinguished properties of cells isolated from the dentin-pulp interface

Gallorini, Marialucia;Cataldi, Amelia;
2021-01-01

Abstract

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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11564/737163
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