RICK regulates the odontogenic differentiation of dental pulp stem cells through activation of TNF-α via the ERK and not through NF-κB signaling pathway

Dental pulp stem cells (DPSCs) are capable of both self-renewal and multilineage differentiation, which play a positive role in dentinogenesis. Studies have shown that tumor necrosis factor-α (TNF-α) is involved in the differentiation of DPSCs under pro-inflammatory stimuli, but the mechanism of action of TNF-α is unknown. Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) is a biomarker of an early inflammatory response that plays a key role in modulating cell differentiation, but the role of RICK in DPSCs is still unclear. In this study, we identified that RICK regulates TNF-α-mediated odontogenic differentiation of DPSCs via the ERK signaling pathway. The expression of the biomarkers of odontogenic differentiation dental matrix protein-1 (DMP-1), dentin sialophosphoprotein (DSPP), biomarkers of odontogenic differentiation, increased in low concentration (1–10 ng/ml) of TNF-α and decreased in high concentration (50–100 ng/ml). Odontogenic differentiation increased over time in the odontogenic differentiation medium. In the presence of 10 ng/L TNF-α, the expression of RICK increased gradually over time, along with odontogenic differentiation. Genetic silencing of RICK expression reduced the expression of odontogenic markers DMP-1 and DSPP. The ERK, but not the NF-κB signaling pathway, was activated during the odontogenic differentiation of DPSCs. ERK signaling modulators decreased when RICK expression was inhibited. PD98059, an ERK inhibitor, blocked the odontogenic differentiation of DPSCs induced by TNF-α. These results provide a further theoretical and experimental basis for the potential use of RICK in targeted therapy for dentin regeneration.     

Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells

Background information. Although adult bone‐marrow‐derived cell populations have been used to make teeth when recombined with embryonic oral epithelium, the differences between dental and non‐dental stem‐cell‐mediated odontogenesis remain an open question. Results. STRO‐1⁺ (stromal precursor cell marker) DPSCs (dental pulp stem cells) and BMSSCs (bone marrow stromal stem cells) were isolated from rat dental pulp and bone marrow respectively by magnetic‐activated cell‐sorting techniques. Their odontogenic capacity was compared under the same inductive microenvironment produced by ABCs (apical bud cells) from 2‐day‐old rat incisors. Co‐cultured DPSCs/ABCs in vitro showed more active odontogenic differentiation ability than mixed BMSSCs/ABCs, as indicated by the accelerated matrix mineralization, up‐regulated alkaline phosphatase activity, cell‐cycle modification, and the expression of tooth‐specific proteins and genes. After cultured for 14 days in the renal capsules of rat hosts, recombined DPSC/ABC pellets formed typical tooth‐shaped tissues with balanced amelogenesis and dentinogenesis, whereas BMSSC/ABC recombinants developed into atypical dentin—pulp complexes without enamel formation. DPSC and BMSSC pellets in vivo produced osteodentin‐like structures and fibrous connective tissues respectively. Conclusions. DPSCs presented more striking odontogenic capability than BMSSCs under the induction of postnatal ABCs. This report provides critical insights into the selection of candidate cells for tooth regeneration between dental and non‐dental stem cell populations.     

Priming integrin α5 promotes human dental pulp stem cells odontogenic differentiation due to extracellular matrix deposition and amplified extracellular matrix-receptor activity

Our previous study showed that knocking down integrin α5 (ITGA5) expression by using a lentiviral vector in human dental pulp stem cells (DPSCs) led to weakening proliferation and migration capacity while enhanced odontogenic differentiation. To seek for possible clinical application, we investigated the effect of the ITGA5 priming synthetic cyclic peptide (SCP; GA-CRRETAWAC-GA) on proliferation, migration, and the odontogenic differentiation of DPSCs. Remarkably, the involved mechanism was explored by isobaric tag for relative and absolute quantitation proteomic technique, and the in vivo effect of ITGA5 was investigated by nude mice subcutaneous transplantation of cell and hydroxyapatite/β-tricalcium phosphate complex. Results showed that SCP weakened the proliferation and migration capacity while enhanced odontogenic differentiation of DPSCs as lentivirus. The phosphorylation of FAK, PI3K/AKT, and MEK1/2/ERK1/2, along with IGF2/IGFBP2 and Wnt/β-catenin signaling pathway play an important role in this process. Proteomic Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed the key role of extracellular matrix (ECM) and ECM-receptor activity pathway were involved. ECM constituents, secreted protein acidic and cysteine-rich (SPARC), lumican, vitronectin, prolargin, decorin, collagen type VI α1 chain (COL6A1), COL6A2, COL14A1, and COL5A1 were upregulated in the ITGA5-silenced group. Inhibited expression of ITGA5 in DPSCs increased osteoid tissue formation and stronger related genes expression in vivo. In conclusion, the ITGA5 priming peptide could promote DPSCs odontogenic differentiation as lentivirus. Proteomics and bioinformatic analysis revealed that this may be due to the deposition of ECM and amplified ECM-receptor activity, which could fuel the application process of utilizing priming ITGA5 on dental clinical practice.     

Mineral trioxide aggregate promotes odontoblastic differentiation via mitogen-activated protein kinase pathway in human dental pulp stem cells

Mitogen-activated protein kinase (MAPK) pathways are involved in stem cell differentiation. However, the odontoblastic differentiation-inducing effects by mineral trioxide aggregate (MTA) via MAPK pathways have not been clarified in human dental pulp stem cells (DPSCs). In this study we investigated the effects of MTA on cell viability and production of differentiation markers, and the involvement of MAPK signaling pathways in cultured human DPSCs. Cells were cultured with MTA, and the viability and differentiation productions of the cells were determined using the MTT assay and real-time PCR analysis, respectively. MAPK activation was measured by western blotting. MTA at concentrations of 20 and 10 mg/ml was toxic for human DPSCs. MTA significantly increased the expression of alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP), type I collagen (COLI), osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs and induced the phosphorylation of p42 and p44 (p42/44), p38 and c-Jun N-terminal kinases 1 and 2 (JNK1/2) MAPK. Furthermore, the inhibitor of p42/44 MAPK attenuated the MTA-induced odontoblastic differentiation. These data indicated that MTA-induced odontoblastic differentiation of human DPSCs was via MAPK pathways, which may play a key role in the repair responses of dentin-pulp-like complexes.     

Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cells

The masticatory apparatus absorbs high occlusal forces, but uncontrolled parafunctional or orthodontic forces damage periodontal ligament (PDL), cause pulpal calcification, pulp necrosis and tooth loss. Morphology and functional differentiation of connective tissue cells can be controlled by mechanical stimuli but effects of uncontrolled forces on intra-pulpal homeostasis and ability of dental pulp stem cells (DPSCs) to withstand direct external forces are unclear. Using dynamic hydrostatic pressure (HSP), we tested the hypothesis that direct HSP disrupts DPSC survival and odontogenic differentiation. DPSCs from four teenage patients were subjected to HSP followed by assessment of cell adhesion, survival and recovery capacity based on odontogenic differentiation, mineralization and responsiveness to bone morphogenetic protein-2 (BMP-2). HSP down-regulated DPSC adhesion and survival but promoted differentiation by increasing mineralization, in vivo hard tissue regeneration and BMP-2 responsiveness despite reduced cell numbers. HSP-treated DPSCs displayed enhanced odontogenic differentiation, an indication of favorable recovery from HSP-induced cellular stress.     

In Vitro Reparative Dentin: a Biochemical and Morphological Study

In this study, starting from human dental pulp cells cultured in vitro, we simulated reparative dentinogenesis using a medium supplemented with different odontogenic inductors. The differentiation of dental pulp cells in odontoblast-like cells was evaluated by means of staining, and ultramorphological, biochemical and biomolecular methods. Alizarin red staining showed mineral deposition while transmission electron microscopy revealed a synthesis of extracellular matrix fibers during the differentiation process. Biochemical assays demonstrated that the differentiated phenotype expressed odontoblast markers, such as Dentin Matrix Protein 1 (DMP1) and Dentin Sialoprotein (DSP), as well as type I collagen. Quantitative data regarding the mRNA expression of DMP1, DSP and type I collagen were obtained by Real Time PCR. Immunofluorescence data demonstrated the various localizations of DSP and DMP1 during odontoblast differentiation. Based on our results, we obtained odontoblast-like cells which simulated the reparative dentin processes in order to better investigate the mechanism of odontoblast differentiation, and dentin extracellular matrix deposition and mineralization.Key words: dental tissue, in vitro differentiation, DMP1, DSP, type I collagen     

Effects of FGF2 and TGFbeta1 on the differentiation of human dental pulp stem cells in vitro

Two crucial growth factors, FGF2 and TGFbeta1, were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta1 and FGF2+TGFbeta1. The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta1 or FGF2+TGFbeta1 can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta1 on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta1 and FGF2+TGFbeta1 mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta1.     

Estrogen deficiency inhibits the odonto/osteogenic differentiation of dental pulp stem cells via activation of the NF-κB pathway

Various factors can affect the functions of dental pulp stem cells (DPSCs). However, little knowledge is available about the effects of estrogen deficiency on the differentiation of DPSCs. In this study, an estrogen-deficient rat model was constructed and multi-colony-derived DPSCs were obtained from the incisors of ovariectomized (OVX) or sham-operated rats. Odonto/osteogenic differentiation and the possible involvement of the nuclear factor kappa B (NF-κB) pathway in the OVX-DPSCs/Sham-DPSCs of these rats were then investigated. OVX-DPSCs presented decreased odonto/osteogenic capacity and an activated NF-κB pathway, as compared with Sham-DPSCs. When the cellular NF-κB pathway was specifically inhibited by BMS345541, the odonto/osteogenic potential in OVX-DPSCs was significantly upregulated. Thus, estrogen deficiency down-regulated the odonto/osteogenic differentiation of DPSCs by activating NF-κB signaling and inhibition of the NF-κB pathway effectively rescued the decreased differentiation potential of DPSCs.     

MEPE-Derived ASARM Peptide Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in Tooth Models of X-Linked Hypophosphatemia

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide − a substrate for PHEX and a strong inhibitor of mineralization − derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.     

Role of epithelial-stem cell interactions during dental cell differentiation

Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin. We also found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies.     

Differentiation and regenerative capacities of human odontoma-derived mesenchymal cells

Regenerating human tooth ex vivo and biological repair of dental caries are hampered by non-viable odontogenic stem cells that can regenerate different tooth components. Odontoma is a developmental dental anomaly that may contain putative post-natal stem cells with the ability to differentiate and regenerate in vivo new dental structures that may include enamel, dentin, cementum and pulp tissues. We evaluated odontoma tissues from 14 patients and further isolated and characterized human odontoma-derived mesenchymal cells (HODCs) with neural stem cell and hard tissue regenerative properties from a group of complex odontoma tissues from 1 of 14 patients. Complex odontoma was more common (9 of 14) than compound type and females (9 of 14) were more affected than males in our set of patients. HODCs were highly proliferative like dental pulp stem cells (DPSCs) but demonstrated stronger neural immunophenotype than both DPSCs and mandible bone marrow stromal cells (BMSCs) by expressing higher levels of nestin, Sox 2 and βIII-tubulin. When transplanted with hydroxyapatite/tricalcium phosphate into immunocompromised mice, HODCs differentiated and regenerated calcified hard tissues in vivo that were morphologically and quantitatively comparable to those generated by DPSCs and BMSCs. When transplanted with polycaprolactone (biodegradable carrier), HODCs differentiated to form new predentin on the surface of a dentin platform. Newly formed predentin contained numerous distinct dentinal tubules and an apparent dentin–pulp arrangement. HODCs represent unique odontogenic progenitors that readily commit to formation of dental hard tissues.     

Conditioned medium derived from 3D tooth germs: A novel cocktail for stem cell priming and early in vivo pulp regeneration

ObjectivesConditioned medium (CM) from 2D cell culture can mitigate the weakened regenerative capacity of the implanted stem cells. However, the capacity of 3D CM to prime dental pulp stem cells (DPSCs) for pulp regeneration and its protein profile are still elusive. We aim to investigate the protein profile of CM derived from 3D tooth germs, and to unveil its potential for DPSCs‐based pulp regeneration.Materials and MethodsWe prepared CM of 3D ex vivo cultured tooth germ organs (3D TGO‐CM) and CM of 2D cultured tooth germ cells (2D TGC‐CM) and applied them to prime DPSCs. Influences on cell behaviours and protein profiles of CMs were compared. In vivo pulp regeneration of CMs‐primed DPSCs was explored using a tooth root fragment model on nude mice.ResultsTGO‐CM enhanced DPSCs proliferation, migration, in vitro mineralization, odontogenic differentiation, and angiogenesis performances. The TGO‐CM group generated superior pulp structures, more odontogenic cells attachment, and enhanced vasculature at 4 weeks post‐surgery, compared with the TGC‐CM group. Secretome analysis revealed that TGO‐CM contained more odontogenic and angiogenic growth factors and fewer pro‐inflammatory cytokines. Mechanisms leading to the differential CM profiles may be attributed to the cytokine–cytokine receptor interaction and PI3K‐Akt signalling pathway.ConclusionsThe unique secretome profile of 3D TGO‐CM made it a successful priming cocktail to enhance DPSCs‐based early pulp regeneration.