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Clinical Oral Investigations Jun 2008This review summarizes the in vivo experiments carried out by our group after implantation of bioactive molecules (matricellular molecules) into the exposed pulp of the...
This review summarizes the in vivo experiments carried out by our group after implantation of bioactive molecules (matricellular molecules) into the exposed pulp of the first maxillary molar of the rat or the mandibular incisor of rats and mice. We describe the cascade of recruitment, proliferation and terminal differentiation of cells involved in the formation of reparative dentin. Cloned immortalized odontoblast progenitors were also implanted in the incisors and in vitro studies aimed at revealing the signaling pathways leading from undifferentiated progenitors to fully differentiated polarized cells. Together, these experimental approaches pave the way for controlled dentin regenerative processes and repair.
Topics: Amelogenin; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Clone Cells; Dental Pulp Exposure; Dentin; Dentin, Secondary; Extracellular Matrix; Integrin-Binding Sialoprotein; Mice; Odontoblasts; Peptide Fragments; Rats; Regeneration; Sialoglycoproteins; Signal Transduction; Stem Cells; Transforming Growth Factor beta; Wound Healing
PubMed: 18157557
DOI: 10.1007/s00784-007-0172-6 -
Stem Cell Research & Therapy Apr 2019Odontoblast is a unique progenitor that plays a role in dentin formation. So far, the dentinogenic differentiation of dental pulp stem cells and the role of surface...
BACKGROUND
Odontoblast is a unique progenitor that plays a role in dentin formation. So far, the dentinogenic differentiation of dental pulp stem cells and the role of surface molecules of odontoblasts in dentinogenesis are not well known yet. In this study, we obtained odontoblast-like cells from human dental pulp cells and screened odontoblast-specific cell surface antigens by decoy immunization.
METHODS
Through decoy immunization with intact odontoblast-like cells derived from human dental pulp cells, we constructed 12 monoclonal antibodies (mAbs) of IgG type, and their binding affinities for cell surface of odontoblast-like cells were analyzed by flow cytometry. Immunoprecipitation, mass spectrometry, and immunohistochemistry were performed to demonstrate odontoblast-specific antigens. Odontoblasts were sorted by these mAbs using magnetic-activated cell sorting system, and their mineralization efficiency was increased after sorting.
RESULTS
We constructed 12 mAbs of IgG type, which had a strong binding affinity for cell surface antigens of odontoblast-like cells. In human adult tooth, these mAbs accumulated in the odontoblastic layer between dentin and pulp and in the perivascular region adjacent to the blood vessels in the pulp core. Cell surface expression of the antigenic molecules was increased during odontogenic cytodifferentiation and decreased gradually as dentinogenic maturation progressed. Proteomic analysis showed that two representative antigenic molecules, OD40 and OD46, had the potential to be components for cell adhesion and extracellular matrix structures.
CONCLUSION
These results suggest that mAbs will be useful for detecting and separating odontoblasts from the primary pulp cells and other lineage cells and will provide information on the structures of extracellular matrix and microenvironment that appears during the dentinogenic differentiation.
Topics: Adult; Adult Stem Cells; Antigens, Differentiation; Cell Differentiation; Dental Pulp; Humans; Odontoblasts
PubMed: 31029165
DOI: 10.1186/s13287-019-1232-y -
Journal of Endodontics Apr 2021Oxytocin (OT) is a neurohypophysial hormone that plays a role in lactation and parturition and exerts diverse biological actions via the OT receptor. Recently, several...
INTRODUCTION
Oxytocin (OT) is a neurohypophysial hormone that plays a role in lactation and parturition and exerts diverse biological actions via the OT receptor. Recently, several studies have reported that OT stimulates bone formation by osteoblasts in osteoporosis. We focused on OT and hypothesized that OT can stimulate the differentiation of odontoblasts as well as osteoblasts. The aim of this study was to verify whether OT is an essential factor in dentinogenesis; we examined the effects of OT on dentinogenesis using a long-term culture system of rat dental pulp cells.
METHODS
Using a culture system of rat dental pulp cells with Otr knocked out by CRISPR-Cas9 genome editing, we examined the effects of OT on odontoblastlike cell differentiation as reflected by dentin formation.
RESULTS
We confirmed that OT stimulated mineralized nodule formation and the expression of both dentin sialoprotein and bone Gla protein messenger RNAs (mRNAs) in the culture system. Interestingly, the cultured cells treated with OT also exhibited an increase of both Wnt10a and Lef-1 mRNA. The Otr knockout cells showed inhibition of nodule formation and mRNA expression, and these phenomena remained despite OT treatment. These results indicate the following: OT regulates odontoblastlike cell differentiation via the OT receptor, it stimulates dentin formation, and the Wnt canonical pathway is closely related to these effects.
CONCLUSIONS
The present results suggest that OT can promote odontoblastlike cell differentiation, resulting in increased dentin formation, and that OT could be an important factor for dentinogenesis.
Topics: Animals; Cell Differentiation; Dental Pulp; Dentin; Dentinogenesis; Female; Lymphoid Enhancer-Binding Factor 1; Odontoblasts; Oxytocin; Phosphoproteins; Rats; Sialoglycoproteins; Wnt Proteins
PubMed: 33422572
DOI: 10.1016/j.joen.2020.12.017 -
The Bulletin of Tokyo Dental College Jun 2022Serum serves as a source of rich nutrients during in vitro cell culture, facilitating cell adhesion, growth, and differentiation. When culturing stem cells for...
Serum serves as a source of rich nutrients during in vitro cell culture, facilitating cell adhesion, growth, and differentiation. When culturing stem cells for transplantation, however, it must be remembered that such culture medium may contain substances potentially harmful to the proposed recipient and may even induce cellular damage. The purpose of this study was to determine whether KnockOut Serum Replacement (KSR), a chemically defined medium supplement, enhanced in vitro differentiation of induced pluripotent stem cells into odontoblasts. Cranial neural crest cells, precursors of odontoblasts, were generated from mouse-induced pluripotent stem cells. They were then cultured in serum-free Dulbecco's modified Eagle's/F12 medium containing fibroblast growth factor 8 with or without KSR. The cells cultured with KSR showed strong proliferation, acquired a spindle-like morphology, and connected with the surrounding cells. KnockOut Serum Replacement also boosted expression of odontoblast markers as measured by qRT-PCR, and increased dentin sialoprotein as assessed by immunostaining. These results confirmed that mouse-induced pluripotent stem cells differentiated into odontoblasts under serum-free conditions, and that KSR enhanced the efficiency of this process.
Topics: Animals; Cell Differentiation; Induced Pluripotent Stem Cells; Mice; Odontoblasts
PubMed: 35613864
DOI: 10.2209/tdcpublication.2021-0042 -
Scientific Reports Jan 2022Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that...
Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/β-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/β-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and β-catenin expression and BCL9-β-catenin co-localization. In addition, BCL9 formed a complex with β-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/β-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/β-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury.
Topics: Animals; Calcification, Physiologic; Cell Differentiation; Cells, Cultured; Dental Pulp; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Odontoblasts; Signal Transduction; Stem Cells; Transcription Factors; Wnt Proteins; beta Catenin
PubMed: 35027586
DOI: 10.1038/s41598-021-04453-8 -
Zhong Nan Da Xue Xue Bao. Yi Xue Ban =... Jun 2020To explore the difference in odontoblast differentiation capacity between stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs), and...
OBJECTIVES
To explore the difference in odontoblast differentiation capacity between stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs), and to examine the expression level of ephrinB1 in odontoblast differentiation of these stem cells.
METHODS
The stems cells were divided into a SHED group and a DPSCs group. After odontoblast differentiation induction, the above 2 groups were also randomly divided into a 3 d group and a 7 d group, respectively.The calcium deposition was detected by alkaline phosphatase (ALP) staining and alizarin red staining.The mRNA and protein expressions of ephrinB1, dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) were detected by real-time PCR and Western blotting.
RESULTS
ALP staining and alizarin red staining showed that there was stronger mineralization capacity in the SHED group than that in the DPSCs group. The relative mRNA and protein expressions of DMP-1, DSPP, and ephrinB1 in the SHED group were higher than those in the DPSCs group except for the protein expression of DMP-1 in the SHED 3 d group (all <0.05).
CONCLUSIONS
SHED has stronger odontoblast differentiation capacity than DPSCs. In addition, ephrinB1 may be involved in the processes of odontoblast differentiation in the SHED and DPSCs.
Topics: Cell Differentiation; Cell Proliferation; Cells, Cultured; Dental Pulp; Humans; Odontoblasts; Osteogenesis; Stem Cells; Tooth, Deciduous
PubMed: 32879125
DOI: 10.11817/j.issn.1672-7347.2020.190102 -
International Journal of Molecular... May 2022FUN14 domain‑containing 1 (FUNDC1) is a receptor that has been previously reported to activate hypoxia‑induced mitophagy. However, the potential role of FUNDC1 in...
FUN14 domain‑containing 1 (FUNDC1) is a receptor that has been previously reported to activate hypoxia‑induced mitophagy. However, the potential role of FUNDC1 in the pathophysiology of dental pulp diseases remains unknown. Therefore, present study first collected tissue specimens from patients with pulpitis and from healthy individuals. The results of reverse transcription‑quantitative PCR and immunohistochemical staining revealed markedly increased FUNDC1 and hypoxia‑inducible factor‑1α expression in pulpitis tissue specimens compared with those from healthy individuals. To provide a theoretical basis for the study of the occurrence, development and reparative mechanisms in the dental pulp after tissue injury, the present study then investigated the role of hypoxia‑induced mitophagy in the regulation of proliferation, migration and odontoblastic differentiation in human dental pulp cells (HDPCs), in addition, to the possible involvement of FUNDC1. The surface markers and multipotent differentiation capabilities of HDPCs were performed by flow cytometry (surface markers), alizarin red (osteogenic capabilities), alcian blue (chondrogenic capabilities) and oil red O (adipogenic capabilities). Following culture under hypoxia conditions (1% O) for varying time periods, the proliferation, migration and odontoblastic differentiation of HDPCs were measured using Cell Counting Kit‑8, wound healing and Transwell migration assays, alkaline phosphatase staining and activity tests and western blotting (runt‑related transcription factor 2, collagen I, osterix and osteopontin), respectively. Immunofluorescence and western blotting were performed to measure the expression levels of hypoxia‑inducible factor‑1α, pro‑fission dynamin‑related protein 1, mitochondria‑related proteins translocase of inner mitochondrial membrane 23 and translocase of outer mitochondrial membrane 20, in addition to those of autophagy markers (p62, LC3II, Beclin‑1 and autophagy‑related 5). Transmission electron microscopy was also used to image the autophagosomes and mitochondrial morphology. In addition, to study the functional role of FUNDC1, its expression was silenced by liposome‑mediated transfection with small interfering RNA into HDPCs. Compared with those in HDPCs cultured under normoxic conditions (21% O), the ability of autophagy in HDPCs cultured under hypoxic conditions for 18 h was markedly increased, whilst the proliferation, migration and odontoblastic differentiation were also enhanced. Increased numbers of autophagosomes could also be observed in the hypoxic group. However, FUNDC1 knockdown in HDPCs reversed the aforementioned effects. Overall, data from the present study suggest that hypoxia can promote the proliferation, migration and odontoblastic differentiation of HDPCs, where the underlying mechanism may be associated with the activation of mitophagy downstream of FUNDC1.
Topics: Cell Proliferation; Cells, Cultured; Dental Pulp; Humans; Hypoxia; Mitophagy; Odontoblasts
PubMed: 35362539
DOI: 10.3892/ijmm.2022.5128 -
Archives of Oral Biology Sep 2021Our goal was to define trigeminal nerve ending quantities and patterns in rat molar dentine, their responses to attrition (tooth wear), and their associated odontoblasts...
OBJECTIVE
Our goal was to define trigeminal nerve ending quantities and patterns in rat molar dentine, their responses to attrition (tooth wear), and their associated odontoblasts and connections with pulpal plexuses.
DESIGN
Trigeminal ganglia were labeled for axonal transport of H-proteins to dentinal nerve endings in male rats (3-13 months old). Autoradiography detected radio-labeled dentinal tubules as indicators of nerve ending locations. Quantitative morphometry was done (ANOVA, t-tests), and littermates were compared for attrition and innervation.
RESULTS
There were six dentinal patterns, only two of which had an associated neural plexus of Raschkow and cell-free zone (Den-1, Den-2). Other nerves entered dentin from bush-like endings near elongated odontoblasts (Den-B), as single fibers (Den-X), as networks in predentine (PdN), or as single fibers in tertiary dentine at cusp tips (Den-S). There were at least 186,600 innervated dentinal tubules within the set of three right maxillary molars of the best-labeled rat, and similar densities were found in other rats. Attrition levels differed among cusps and in littermates (t-test p < 0.02-0.0001), but the matched right/left cusps per rat were similar. Innervations of tertiary and enamel-free dentine (Den-S, Den-X) were preserved in all rats. Den-B and Den-2 coronal patterns were unchanged unless displaced by dentinogenesis. Den-1 losses occurred in older cusps, while Den-2 patterns increased near cervical and intercuspal odontoblasts.
CONCLUSIONS
The extensive molar dentinal innervation had unique distributions per rat per cusp that depended on region (buccal, middle, palatal) and attrition, but only two of six patterns connected to a plexus of Raschkow.
Topics: Animals; Dental Pulp; Dentin; Male; Molar; Odontoblasts; Rats; Trigeminal Nerve
PubMed: 34146928
DOI: 10.1016/j.archoralbio.2021.105197 -
Journal of Dental Research Jan 2012Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKO(od)) mouse model, we have observed severe defects in odontogenesis and dentin formation with the removal of the Bmp2 gene...
Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKO(od)) mouse model, we have observed severe defects in odontogenesis and dentin formation with the removal of the Bmp2 gene in early-polarizing odontoblasts. The odontoblasts in the Bmp2-cKO(od) do not mature properly and fail to form proper dentin with normal dentinal tubules and activate terminal differentiation, as reflected by decreased Osterix, Col1a1, and Dspp expression. There is less dentin, and the dentin is hypomineralized and patchy. We also describe an indirect effect of the Bmp2 gene in odontoblasts on formation of the vascular bed and associated pericytes in the pulp. This vascular niche and numbers of CD146+ pericytes are likely controlled by odontogenic and Bmp2-dependent VegfA production in odontoblasts. The complex roles of Bmp2, postulated to be both direct and indirect, lead to permanent defects in the teeth throughout life, and result in teeth with low quantities of dentin and dentin of poor quality.
Topics: Animals; Basigin; Bone Morphogenetic Protein 2; Cell Differentiation; Collagen Type I; Dental Pulp; Dentin; Dentinogenesis; Extracellular Matrix Proteins; Gene Deletion; Mice; Mice, Transgenic; Neovascularization, Physiologic; Odontoblasts; Pericytes; Phosphoproteins; Sialoglycoproteins; Sp7 Transcription Factor; Stem Cells; Transcription Factors; Vascular Endothelial Growth Factor A; X-Ray Microtomography
PubMed: 21984706
DOI: 10.1177/0022034511424409 -
Biomolecules Jul 2021Intracellular Ca signaling engendered by Ca influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface....
Intracellular Ca signaling engendered by Ca influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca is exported by the Na-Ca exchanger (NCX) and plasma membrane Ca-ATPase (PMCA) to maintain Ca homeostasis. We previously demonstrated a functional coupling between Ca extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca concentration ([Ca]). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca] was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1-4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca] in odontoblasts in both rat and human odontoblasts. The Ca extrusion efficiency during the hypotonic or alkaline solution-induced [Ca] increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4-8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca] homeostasis in odontoblasts by Ca extrusion following [Ca] elevation. In addition, PMCA participates in dentinogenesis by transporting Ca to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.
Topics: Animals; Calcium; Cell Line; Dentin; Humans; Odontoblasts; Plasma Membrane Calcium-Transporting ATPases; Rats; Rats, Wistar; Tooth Calcification
PubMed: 34356633
DOI: 10.3390/biom11071010