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Dentin tubule orientation determines odontoblastic differentiation in vitro: A morphological study.

ABSTRACT: Odontoblasts are post-mitotic cells responsible for maintenance of the dentin, and are therefore important for dental health. In some cases, irreversible pulpitis leads to necrosis and consequently death of odontoblasts. Regenerative endodontics (RE) uses the concept of tissue engineering to restore the root canals to a healthy state, allowing for continued development of the root and surrounding tissue. Human dental pulp stem cells (hDPSCs) have been successfully used in RE to restore odontoblast function. Surface microgeometry is one of the most important factors involved in the induction of differentiation of hDPSCs into odontoblast-like cells. Although different authors have demonstrated the importance of a dentin-like surface with accessible dentin tubules to induce differentiation of hDPSCs, the ultrastructural characteristics of the cells and the secreted extracellular matrix have not been studied in depth. Here, we used an acellular dentin scaffold containing dentin tubules in different spatial geometries, which regulated their accessibility to cells. hDPSCs were cultured on the scaffolds for up to 6 weeks. Systematic characterization of differentiated cells was performed using both optical (hematoxylin and eosin, Masson trichrome, and immunohistochemical determination of dentin sialoprotein [DSSP]) and transmission electron microscopy. The results presented here indicated that cells grown on the dentin surface containing accessible dentin tubules developed a characteristic odontoblastic phenotype, with cellular processes similar to native odontoblasts. The cell organization and characteristics of secreted extracellular matrix were also similar to those of native dentin tissue. Cells grown on non-accessible dentin tubule surfaces secreted a more abundant and dense extracellular matrix, and developed a different phenotype consisting of secretory flat cells organized in layers. Cells grown far from the scaffold, i.e., directly on the culture well surface, developed a secretory phenotype probably influenced by biochemical factors released by the dentin scaffold or differentiated cells. The results presented here support the use of hDPSCs to regenerate dentin and show the utility of scaffold microgeometry for determining the differentiation and secretory phenotype of cultured cells.

SUBMITTER: Martin-de-Llano JJ

PROVIDER: S-EPMC6508697 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Dentin tubule orientation determines odontoblastic differentiation in vitro: A morphological study.

Martín-de-Llano José Javier JJ Mata Manuel M Peydró Santiago S Peydró Amando A Carda Carmen C

PloS one 20190509 5

Odontoblasts are post-mitotic cells responsible for maintenance of the dentin, and are therefore important for dental health. In some cases, irreversible pulpitis leads to necrosis and consequently death of odontoblasts. Regenerative endodontics (RE) uses the concept of tissue engineering to restore the root canals to a healthy state, allowing for continued development of the root and surrounding tissue. Human dental pulp stem cells (hDPSCs) have been successfully used in RE to restore odontobla ...[more]

PMID: 31071116

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Project description:The presence of stem cells within the dental-pulp tissue as well as their differentiation into a new generation of functional odontoblast-like cells constitutes an important step of the dentin-pulp regeneration. Recent investigations demonstrated that the complement system activation participates in 2 critical steps of dentin-pulp regeneration: pulp progenitor's recruitment and pulp nerve sprouting. Surprisingly, its implication in odontoblastic differentiation has not been addressed yet. Since the complement receptor C5a receptor-like 2 (C5L2) is expressed by different stem cells, the aim of this study is to investigate if the dental pulp stem cells express C5L2 and if this receptor participates in odontoblastic differentiation. Immunohistochemistry performed on human third molar pulp sections showed a perivascular co-localization of the mesenchymal stem cell markers STRO1 and C5L2. In vitro immunofluorescent staining confirmed that hDPSCs express C5L2. Furthermore, we determined by real-time polymerase chain reaction that the expression of C5L2 is highly modulated in human dental pulp stem cells (hDPSCs) undergoing odontoblastic differentiation. Moreover, we showed that this odontogenesis-regulated expression of C5L2 is specifically potentiated by the proinflammatory cytokine TNFα. Using a C5L2-siRNA silencing strategy, we provide direct evidence that C5L2 constitutes a negative regulator of the dentinogenic marker DMP1 (dentin matrix protein 1) expression by hDPSCs. Our findings suggest a direct correlation between the odontoblastic differentiation and the level of C5L2 expression in hDPSCs and identify C5L2 as a negative regulator of DMP1 expression by hDPSCs during the odontoblastic differentiation and inflammation processes. This work is the first to demonstrate the involvement of C5L2 in the biological function of stem cells, provides an important knowledge in understanding odontoblastic differentiation of dental pulp stem cells, and may be useful in future dentin-pulp engineering strategies.

Project description:Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.

Dataset Information

Dentin tubule orientation determines odontoblastic differentiation in vitro: A morphological study.

Publications

Dentin tubule orientation determines odontoblastic differentiation in vitro: A morphological study.

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