Search

Rodent animal models for vital pulp therapy are commonly used in dental research because their tooth anatomy and cellular processes are similar to the anatomy and processes in humans. However, most studies have been conducted using uninfected sound teeth, which makes it difficult to adequately assess the inflammatory shift after vital pulp therapy. In the present study, we aimed to establish a caries-induced pulpitis model based on the conventional rat caries model and then evaluate inflammatory changes during the wound-healing process after pulp capping in a model of reversible pulpitis induced by carious infection. To establish the caries-induced pulpitis model, the pulpal inflammatory status was investigated at different stages of caries progression by immunostaining targeted to specific inflammatory biomarkers. Immunohistochemical staining revealed that both Toll-like receptor 2 and proliferating cell nuclear antigen were expressed in moderate and severe caries-stimulated pulp, indicating that an immune reaction occurred at both stages of caries progression. M2 macrophages were predominant in moderate caries-stimulated pulp, whereas M1 macrophages were predominant in the severe caries-stimulated pulp. Pulp capping in teeth with moderate caries (i.e., teeth with reversible pulpitis) led to complete tertiary dentin formation within 28 d after treatment. Impaired wound healing was observed in teeth with severe caries (i.e., teeth with irreversible pulpitis). During the wound-healing process in reversible pulpitis after pulp capping, M2 macrophages were predominant at all time points; their proliferative capacity was upregulated in the early stage of wound healing compared with healthy pulp. In conclusion, we successfully established a caries-induced pulpitis model for studies of vital pulp therapy. M2 macrophages have an important role in the early stages of the wound-healing process in reversible pulpitis. [ABSTRACT FROM AUTHOR]

Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography–tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies. [ABSTRACT FROM AUTHOR]

Dentin is a major mineralized component of teeth. Odontoblasts are responsible for synthesis and secretion of dentin matrix. Previously, it has been demonstrated in a cell culture system that the E3 ubiquitin ligase, murine double minute 2 (Mdm2), promotes odontoblast-like differentiation of mouse dental papilla cells (mDPCs) by ubiquitinating p53 and the odontoblast-specific substrate Dlx3. However, whether Mdm2 plays an essential role in vivo in odontoblast differentiation and dentin formation remains unknown. In this study, we investigated the in vivo functions of Mdm2 using Dmp1-Cre;Mdm2 flox/flox mice combined with multiple histological and molecular biological methods. The results showed that Mdm2 deletion in the odontoblast layer led to defects in odontoblast differentiation and dentin formation. Unexpectedly, specific inhibition of the Mdm2-p53 axis in wild-type mice by injection of a small-molecule inhibitor Nutlin-3a indicated that the role of Mdm2 in dentinogenesis was p53 independent, which was inconsistent with the previous in vitro study. In situ proximity ligation assay (PLA) showed that Mdm2 interacted with and ubiquitinated Dlx3 in the odontoblast nucleus of mouse molars. Dlx3 promoted the translocation of Mdm2 to the nucleus, and in turn, the nuclear Mdm2 mediated ubiquitination of Dlx3 and promoted the odontoblast-like differentiation of mDPCs. Dlx3 interacted with Mdm2 through its C-terminal domain. Deletion of the C-terminal domain of Dlx3 reversed the enhanced odontoblast-like differentiation and the activation of Dspp promoter mediated by overexpression of wild-type or nuclear Mdm2. Our findings suggest that nuclear Mdm2 mediates ubiquitination of the transcription factor Dlx3, which is essential for Dlx3 transcriptional activity on Dspp as well as subsequent odontoblast differentiation and dentin formation. [ABSTRACT FROM AUTHOR]

Small-molecule drugs targeting glycogen synthase kinase 3 (GSK3) as inhibitors of the protein kinase activity are able to stimulate reparative dentine formation. To develop this approach into a viable clinical treatment for exposed pulp lesions, we synthesized a novel, small-molecule noncompetitive adenosine triphosphate (ATP) drug that can be incorporated into a biodegradable hydrogel for placement by syringe into the tooth. This new drug, named NP928, belongs to the thiadiazolidinone (TDZD) family and has equivalent activity to similar drugs of this family such as tideglusib. However, NP928 is more water soluble than other TDZD drugs, making it more suitable for direct delivery into pulp lesions. We have previously reported that biodegradable marine collagen sponges can successfully deliver TDZD drugs to pulp lesions, but this involves in-theater preparation of the material, which is not ideal in a clinical context. To improve surgical handling and delivery, here we incorporated NP928 into a specifically tailored hydrogel that can be placed by syringe into a damaged tooth. This hydrogel is based on biodegradable hyaluronic acid and can be gelled in situ upon dental blue light exposure, similarly to other common dental materials. NP928 released from hyaluronic acid–based hydrogels upregulated Wnt/β-catenin activity in pulp stem cells and fostered reparative dentine formation compared to marine collagen sponges delivering equivalent concentrations of NP928. This drug-hydrogel combination has the potential to be rapidly developed into a therapeutic procedure that is amenable to general dental practice. [ABSTRACT FROM AUTHOR]

WW domain-containing E3 Ub-protein ligase 2 (WWP2) belongs to the homologous to E6AP C-terminus (HECT) E3 ligase family. It has been explored to regulate osteogenic differentiation, chondrogenesis, and palatogenesis. Odontoblasts are terminally differentiated mesenchymal cells, which contribute to dentin formation in tooth development. However, it remained unknown whether WWP2 participated in odontoblast differentiation. In this study, WWP2 was found to be expressed in mouse dental papilla cells (mDPCs), odontoblasts, and odontoblastic-induced mDPCs by immunohistochemistry and Western blotting. Besides, WWP2 expression was decreased in the cytoplasm but increased in the nuclei of differentiation-induced mDPCs. When Wwp2 was knocked down, the elevated expression of odontoblast marker genes (Dmp1 and Dspp) in mDPCs induced by differentiation medium was suppressed. Meanwhile, a decrease of alkaline phosphatase (ALP) activity was observed by ALP staining, and reduced formation of mineralized matrix nodules was demonstrated by Alizarin Red S staining. Overexpression of WWP2 presented opposite results to knockdown experiments, suggesting that WWP2 promoted odontoblastic differentiation of mDPCs. Further investigation found that WWP2 was coexpressed and interacted with KLF5 in the nuclei, leading to ubiquitination of KLF5. The PPPSY (PY2) motif of KLF5 was essential for its physical binding with WWP2. Also, cysteine 838 (Cys838) of WWP2 was the active site for ubiquitination of KLF5, which did not lead to proteolysis of KLF5. Then, KLF5 was confirmed to be monoubiquitinated and transactivated by WWP2, which promoted the expression of KLF5 downstream genes Dmp1 and Dspp. Deletion of the PY2 motif of KLF5 or mutation of Cys838 of WWP2 reduced the upregulation of Dmp1 and Dspp. Besides, lysine (K) residues K31, K52, K83, and K265 of KLF5 were verified to be crucial to WWP2-mediated KLF5 transactivation. Taken together, WWP2 promoted odontoblastic differentiation by monoubiquitinating KLF5. [ABSTRACT FROM AUTHOR]

Odontoblast differentiation is a complex and multistep process regulated by signaling pathways, including the Wnt/β-catenin signaling pathway. Both positive and negative effects of Wnt/β-catenin signaling on dentinogenesis have been reported, but the underlying mechanisms of these conflicting results are still unclear. To gain a better insight into the role of Wnt/β-catenin in dentinogenesis, we used dental pulp cells from a panel of transgenic mice, in which fluorescent protein expression identifies cells at different stages of odontoblast and osteoblast differentiation. Our results showed that exposure of pulp cells to WNT3a at various times and durations did not induce premature differentiation of odontoblasts. These treatments supported the survival of undifferentiated cells in dental pulp and promoted the formation of 2.3GFP+ preodontoblasts and their rapid transition into differentiated odontoblasts expressing DMP1-Cherry and DSPP-Cerulean transgenes. WNT3a also promoted osteogenesis in dental pulp cultures. These findings provide critical information for the development of improved treatments for vital pulp therapy and dentin regeneration. [ABSTRACT FROM AUTHOR]

Autophagy is an intracellular self-degradation process that is essential for tissue development, cell differentiation, and survival. Nevertheless, the role of autophagy in tooth development has not been definitively identified. The goal of this study was to investigate how autophagy is involved in midkine (MK)-mediated odontoblast-like differentiation, mineralization, and tertiary dentin formation in a mouse tooth pulp exposure model. In vitro studies show that MK and LC3 have similar expression patterns during odontoblast-like cell differentiation. Odontoblast-like cell differentiation is promoted through MK-mediated autophagy, which leads to increased mineralized nodule formation. Subcutaneous transplantation of hydroxyapatite/tricalcium phosphate with rMK-treated human dental pulp cells led to dentin pulp-like tissue formation through MK-mediated autophagy. Furthermore, MK-mediated autophagy induces differentiation of dental pulp cells into odontoblast-like cells that form DSP-positive tertiary dentin in vivo. Our findings may provide 1) novel insight into the role of MK in regulating odontoblast-like differentiation and dentin formation in particular via autophagy and 2) potential application of MK in vital pulp therapy. [ABSTRACT FROM AUTHOR]

Dentin is an important structural component of the tooth. Odontoblast differentiation is an essential biological process that guarantees normal dentin formation, which is precisely regulated by various proteins. Murine double minute 2 (Mdm2) is an E3 ubiquitin ligase, and it plays a pivotal role in the differentiation of different cell types, such as osteoblasts and myoblasts. However, whether Mdm2 plays a role in odontoblast differentiation remains unknown. Here, we investigated the spatiotemporal expression of Mdm2 by immunostaining and found that Mdm2 was highly expressed in the odontoblasts and slightly in the dental papilla cells of mouse incisors and molars. Gene knockdown and overexpression experiments verified that Mdm2 promoted the odontoblast-like differentiation of mouse dental papilla cells (mDPCs). Intranuclear colocalization and physical interaction between Mdm2 and distal-less 3 (Dlx3), a transcription factor important for odontoblast differentiation, was found during the odontoblast-like differentiation of mDPCs by double immunofluorescence and immunoprecipitation. Mdm2 was proved to monoubiquitinate Dlx3, which enhanced the expression of Dlx3 target gene Dspp. In addition, p53, the canonical substrate of Mdm2, was validated to be also ubiquitinated but degraded by Mdm2 during the odontoblast-like differentiation of mDPCs. Gene knockdown experiments confirmed that p53 inhibited the odontoblast-like differentiation of mDPCs. p53 and Mdm2 double knockdown partially rescued the reduced odontoblast-like differentiation by knockdown of Mdm2 alone. Taken together, our study revealed that Mdm2 promoted the odontoblast-like differentiation of mDPCs by ubiquitinating both Dlx3 and p53. On one hand, the monoubiquitination of Dlx3 by Mdm2 led to upregulation of Dspp, which is a marker of the odontoblast differentiation. On the other hand, ubiquitination of p53 by Mdm2 resulted in its degradation, which eliminated the inhibitory effect of p53 on the odontoblast-like differentiation of mDPCs. [ABSTRACT FROM AUTHOR]

Bone sialoprotein (BSP) is a member of the SIBLING family with essential roles in skeletogenesis. In the developing teeth, although the expression and function of BSP in the formation of acellular cementum and periodontal attachment are well documented, there are uncertainties regarding the expression and function of BSP by odontoblasts and dentin. Reporter mice are valuable animal models for biological research, providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. In the present study, we examined the expression of a BSP-GFPtpz reporter mouse line during odontoblast differentiation, reparative dentinogenesis, and bone. In the developing teeth, BSP-GFPtpz was expressed at high levels in cementoblasts but not in odontoblasts or dentin. In bones, the transgene was highly expressed in osteoblasts at an early stage of differentiation. Interestingly, despite its lack of expression in odontoblasts and dental pulp during tooth development, the BSP-GFPtpz transgene was detected during in vitro mineralization of primary pulp cultures and during reparative dentinogenesis following pulp exposures. Importantly, under these experimental contexts, the expression of BSP-GFPtpz was still exclusive to DSPP-Cerulean, an odontoblast-specific reporter gene. This suggests that the combinatorial use of BSP-GFPtpz and DSPP-Cerulean can be a valuable experimental tool to distinguish osteogenic from dentinogenic cells, thereby providing an avenue to investigate mechanisms that distinctly regulate the lineage progression of progenitors into odontoblasts versus osteoblasts. [ABSTRACT FROM AUTHOR]

Dentin hypersensitivity commonly occurs due to opened dentinal tubules for many reasons. In our previous study, copine 7 (CPNE7) could induce dentin formation for an indirect pulp-capping model in vivo. This study aims to investigate the formation of tertiary dentin when CPNE7 is applied to intentionally exposed dentin with nothing over it in vivo, whether it affects microleakage of the teeth, and the penetration ability of CPNE7 molecules through dentinal tubules in vitro. Cervical dentin areas of 6 maxillary incisors of 5 beagles were exposed to a class V-like lesion, and 1 side of 3 maxillary incisors was adapted with recombinant CPNE7 protein for 5 min as the experimental group. The other side was the control group, and there was no treatment of ethylenediaminetetraacetic acid (EDTA) and CPNE7 after preparation. The defects were exposed without any restorations, and all beagles were sacrificed after 4 wk. The fluid penetration of exposed dentin areas was investigated by a microleakage-testing device and confocal laser scanning microscope. Tertiary dentin formation was confirmed with histological scanning electronic microscopic analysis. Tertiary dentin formation reduces dentinal fluid flow due to occluded tubules or discontinuity with primary or secondary dentin. The in vivo hypersensitivity model with the anterior teeth of beagle dogs showed newly formed tertiary dentin at the dentin-pulp boundary in recombinant CPNE7-treated teeth when compared with the untreated control group in histologic analysis. Scanning electronic microscopic analysis revealed occluded sites with mineral deposition of intratubular dentin. In the permeability test, the mean microleakage value of the CPNE7-treated group was significantly lower than that of the control group (P < 0.05). The tubular penetration of rhodamine B-combined CPNE7 was confirmed under confocal laser scanning microscope. CPNE7 induces formation of tertiary dentin through shallowly exposed dentinal tubules, which decreases dentin permeability. [ABSTRACT FROM AUTHOR]

The objective of our experiments was to identify new therapeutic strategies to stimulate dentin formation in an adult tooth. To address this objective, we evaluated dentin production in 2 acute trauma models: one involving a pulp exposure and the other involving a superficial dentin injury. Molecular, cellular, and histologic analyses revealed that in response to a severe injury, where the pulp is exposed to the oral cavity, cell death is rampant and the repair response initiates from surviving pulp cells and, to a lesser extent, surviving odontoblasts. When an injury is superficial, as in the case of a dentin injury model, then disturbances are largely confined to pulp tissue immediately underneath the damaged dentin tubules. We found that the pulp remained vital and innervated; primary odontoblasts upregulated HIF1α; and the rate of mineralization was significantly increased. A tamoxifen-inducible Axin2CreERT2/+; R26R mTmG/+ reporter strain was then used to demonstrate that a population of long-lived Wnt-responsive odontoblasts, which secreted dentin throughout the life of the animal, were responsible for depositing new dentin in response to a superficial injury. Amplifying Wnt signaling in the pulp stimulates dentin secretion, and in the dentin injury model, we show that a liposomal formulation of human WNT3A protein passes through dentinal tubules and is capable of upregulating Wnt signaling in the pulp. These data provide strong proof of concept for a therapeutic pulp-capping material to stimulate Wnt signaling in odontoblasts and thus improve the pulp repair response. [ABSTRACT FROM AUTHOR]

The enhancer of zeste homolog 2 (EZH2) is a catalytic subunit of PRC2 (polycomb repressor complex 2). It mediates gene silencing via methyltransferase activity and is involved in the determination of cell lineage. However, the function of EZH2 and the underlying mechanisms by which it affects the differentiation of human dental pulp cell (hDPC) have remained underexplored. In this research, we found that EZH2 expression decreased during the mineralization of hDPCs, with attenuated H3K27me3 (trimethylation on lysine 27 in histone H3). Overexpression of EZH2 impaired the odontogenic differentiation of hDPCs, while EZH2 without methyltransferase activity mutation (mutation of suppressed variegation of 3 to 9, enhancer of zeste and trithorax domain, EZH2ΔSET) did not display this phenotype. In addition, siRNA knockdown studies showed that EZH2 negatively modulated hDPC differentiation in vitro and inhibited mineralized nodule formation in transplanted β-tricalcium phosphate / hDPC composites. To further investigate the underlying mechanisms, we explored the Wnt/β-catenin signaling pathway in view of the fact that previous research had documented the essential role that it plays during hDPC mineralization, as well as its links to EZH2 in other cells. We demonstrated for the first time that EZH2 depletion activated the Wnt/β-catenin signaling pathway and enhanced the accumulation of β-catenin in hDPCs. Chromatin immunoprecipitation analysis suggested that these effects are attributable to the level of the EZH2-regulated H3K27me3 on the β-catenin promoter. We conclude that EZH2 plays a negative role during the odontogenic differentiation of hDPCs. Suppression of EZH2 could promote hDPC mineralization by epigenetically regulating the expression of β-catenin and activating the Wnt canonical signaling pathway. [ABSTRACT FROM AUTHOR]

Primary cilia, with highly regulated cellular sensory functions, play key roles in tissue development and function maintenance. Intraflagellar transport 140 (IFT140) is a subunit of IFT complex A, which is specialized for retrograde transportation in cilia. Mutations of Ift140 are usually associated with syndromic ciliopathy and may cause isolated diseases such as retinal dystrophy, short ribs, and polycystic kidney. However, the role of IFT140 in tooth development has not been well investigated. In this study, a close relationship between IFT140 and dentin formation is disclosed. During tooth development, IFT140 was highly expressed in odontoblasts. To further understand the role of IFT140 in dentinogenesis, Ift140flox/flox/Osx-Cre mouse was generated. The dentin thickness of Ift140flox/flox/Osx-Cre mouse is thinner and the dentin formation is slower than that in control. In vitro, deletion of IFT140 in odontoblasts led to poor odontogenic differentiation, abnormal primary cilia, and decreased Sonic hedgehog signaling molecules. More important, due to loss of primary cilia in odontoblasts by IFT140 deletion, reparative dentin formation was impaired in a tooth-drilling model. These results suggest that cilia gene IFT140 is essential in promoting dentin formation and reparation. [ABSTRACT FROM AUTHOR]

The induced pluripotent stem cells (iPSCs) have an intrinsic capability for indefinite self-renewal and large-scale expansion and can differentiate into all types of cells. Here, we tested the potential of iPSCs from dental pulp stem cells (DPSCs) to differentiate into functional odontoblasts. DPSCs were reprogrammed into iPSCs via electroporation of reprogramming factors OCT-4, SOX2, KLF4, LIN28, and L-MYC. The iPSCs presented overexpression of the reprogramming genes and high protein expressions of alkaline phosphatase, OCT4, and TRA-1-60 in vitro and generated tissues from 3 germ layers in vivo. Dentin discs with poly-L-lactic acid scaffolds containing iPSCs were implanted subcutaneously into immunodeficient mice. After 28 d from implantation, the iPSCs generated a pulp-like tissue with the presence of tubular dentin in vivo. The differentiation potential after long-term expansion was assessed in vitro. iPSCs and DPSCs of passages 4 and 14 were treated with either odontogenic medium or extract of bioactive cement for 28 d. Regardless of the passage tested, iPSCs expressed putative markers of odontoblastic differentiation and kept the same mineralization potential, while DPSC P14 failed to do the same. Analysis of these data collectively demonstrates that human iPSCs can be a source to derive human odontoblasts for dental pulp research and test bioactivity of materials. [ABSTRACT FROM AUTHOR]

Eph receptors belong to a subfamily of receptor tyrosine kinases that are activated by membrane-spanning ligands called ephrins. Previously, we demonstrated that the ephrinB1-EphB2 interaction regulates odontogenic/osteogenic differentiation from dental pulp cells (DPCs) in vitro. The goal of this study was to identify the molecular mechanisms regulated by the EphB2/ephrinB1 system that govern tertiary dentin formation in vitro and in vivo. During tooth development, ephrinB1, and EphB2 were expressed in preodontoblast and odontoblasts at postnatal day 4. EphrinB1 was continuously expressed in odontoblasts and odontoblastic processes until the completion of tooth eruption. In addition, ephrinB1 was expressed in odontoblastic processes 2 wk following tooth injury without pulp exposure, whereas EphB2 was expressed in the center of pulp niches but not odontoblasts. In a model of tooth injury with pulp exposure, ephrinB1 was strongly expressed in odontoblasts 4 wk postinjury. In vitro studies with human and mouse DPCs treated with calcium hydroxide (CH) or mineral trioxide aggregate (MTA) showed an increased expression of insulin-like growth factor 1 (IGF-1). Experiments using several inhibitors of IGF-1 receptor signaling revealed that inhibiting the Ras/Raf-1/MAPK pathway inhibited EphB2 expression, and inhibiting the PI3K/Akt/mTOR pathway specifically inhibited ephrinB1 gene expression. Tooth injury in mice with odontoblast-specific IGF-1 receptor ablation exhibited a reduced tertiary dentin volume, mineral density, and ephrinB1 expression 4 wk following injury. We conclude that the IGF-1/ephrinB1 axis plays significant roles in the early stages of tooth injury. Further research is needed to fully understand the potential of targeting ephrinB1 as a regenerative pulp therapy. [ABSTRACT FROM AUTHOR]

In humans, the SOST gene encodes sclerostin, an inhibitor of bone growth and remodeling, which also negatively regulates the bone repair process. Sclerostin has also been implicated in tooth formation, but its potential role in pulp healing remains unknown. The aim of this study was to explore the role of sclerostin in reparative dentinogenesis using Sost knockout mice ( Sost-/-). The pulps of the first maxillary molars were mechanically exposed in 3-mo-old Sost-/- and wild-type (WT) mice ( n = 14 mice per group), capped with mineral trioxide aggregate cement, and the cavities were filled with a bonded composite resin. Reparative dentinogenesis was dynamically followed up by micro-computed tomography and characterized by histological analyses. Presurgical analysis revealed a significantly lower pulp volume in Sost-/- mice compared with WT. At 30 and 49 d postsurgery, a large-forming reparative mineralized bridge, associated with osteopontin-positive mineralization foci, was observed in the Sost-/- pulps, whereas a much smaller bridge was detected in WT. At the longer time points, the bridge, which was associated with dentin sialoprotein-positive cells, had expanded in both groups but remained significantly larger in Sost-/- pulps. Sclerostin expression in the healing WT pulps was detected in the cells neighboring the forming dentin bridge. In vitro, mineralization induced by Sost-/- dental pulp cells (DPCs) was also dramatically enhanced when compared with WT DPCs. These observations were associated with an increased Sost expression in WT cells. Taken together, our data show that sclerostin deficiency hastened reparative dentinogenesis after pulp injury, suggesting that the inhibition of sclerostin may constitute a promising therapeutic strategy for improving the healing of damaged pulps. [ABSTRACT FROM AUTHOR]

The goal of this study was to examine the contribution of perivascular cells to odontoblasts during the development, growth, and repair of dentin using mouse molars as a model. We used an inducible, Cre-loxP in vivo fate-mapping approach to examine the contributions of the descendants of cells expressing the αSMA-CreERT2 transgene to the odontoblast lineage. In vivo lineage-tracing experiments in molars showed the contribution of αSMA-tdTomato+ cells to a small number of newly formed odontoblasts during primary dentinogenesis. Using an experimental pulp exposure model in molars to induce reparative dentinogenesis, we demonstrate the contribution of αSMA-tdTomato+ cells to cells secreting reparative dentin. Our results demonstrate that αSMA-tdTomato+ cells differentiated into Col2.3-GFP+ cells composed of both Dspp+ odontoblasts and Bsp+ osteoblasts. Our findings identify a population of mesenchymal progenitor cells capable of giving rise to a second generation of odontoblasts during reparative dentinogenesis. This population also makes a small contribution to odontoblasts during primary dentinogenesis. [ABSTRACT FROM AUTHOR]

Liberation of the sequestrated bioactive molecules from dentine by the action of applied dental materials has been proposed as an important mechanism in inducing a dentinogenic response in teeth with viable pulps. Although adhesive restorations and dentine-bonding procedures are routinely practiced, clinical protocols to improve pulp protection and dentine regeneration are not currently driven by biological knowledge. This study investigated the effect of dentine (powder and slice) conditioning by etchants/conditioners relevant to adhesive restorative systems on growth factor solubilization and odontoblast-like cell differentiation of human dental pulp progenitor cells (DPSCs). The agents included ethylenediaminetetraacetic acid (EDTA; 10%, pH 7.2), phosphoric acid (37%, pH <1), citric acid (10%, pH 1.5), and polyacrylic acid (25%, pH 3.9). Growth factors were detected in dentine matrix extracts drawn by EDTA, phosphoric acid, and citric acid from powdered dentine. The dentine matrix extracts were shown to be bioactive, capable of stimulating odontogenic/osteogenic differentiation as observed by gene expression and phenotypic changes in DPSCs cultured in monolayer on plastic. Polyacrylic acid failed to solubilize proteins from powdered dentine and was therefore considered ineffective in triggering a growth factor-mediated response in cells. The study went on to investigate the effect of conditioning dentine slices on growth factor liberation and DPSC behavior. Conditioning by EDTA, phosphoric acid, and citric acid exposed growth factors on dentine and triggered an upregulation in genes associated with mineralized differentiation, osteopontin, and alkaline phosphatase in DPSCs cultured on dentine. The cells demonstrated odontoblast-like appearances with elongated bodies and long extracellular processes extending on dentine surface. However, phosphoric acid-treated dentine appeared strikingly less populated with cells, suggesting a detrimental impact on cell attachment and growth when conditioning by this agent. These findings take crucial steps in informing clinical practice on dentine-conditioning protocols as far as treatment of operatively exposed dentine in teeth with vital pulps is concerned. [ABSTRACT FROM AUTHOR]

In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation of DPSC clones. In addition, Sema3A treatment of DPSC clones increased β-catenin nuclear accumulation, upregulated expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping. These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/β-catenin signaling. Sema3A might be an alternative agent for direct pulp capping, which requires further study. [ABSTRACT FROM AUTHOR]

Osteopontin (OPN) is a highly phosphorylated glycoprotein that is a prominent component of the mineralized extracellular matrix of bone. The secretion of OPN by immunocompetent cells plays a role in the differentiation of odontoblast-like cells during pulpal healing following tooth transplantation. This study aimed to clarify the role of OPN during reparative dentinogenesis. A groove-shaped cavity was prepared on the mesial surface of the upper first molars of wild-type (WT) and Opn knockout (KO) mice, and the samples were collected at intervals of 1 to 14 d. The demineralized sections were processed for immunohistochemistry for Ki67, nestin, OPN, dentin sialoprotein (DSP), integrin αvβ3, and type I collagen; in situ hybridization for Opn, col1a1, and dentin sialophosphoprotein (Dspp); and apoptosis assay. For the loss and gain of function experiments, an in vitro culture assay for evaluating dentin-pulp complex regeneration was performed. On day 1 in WT mice, odontoblasts beneath the affected dentin lost nestin immunoreactivity. On day 3, the expression of Opn was recognized at the mesial dental pulp, and OPN was deposited along the predentin-dentin border. Nestin-positive newly differentiated odontoblast-like cells expressed both Dspp and col1a1 and showed positive immunoreactivity for integrin αvβ3, DSP, and type I collagen. Until day 14, reparative dentin formation continued next to the preexisting dentin at the mesial coronal pulp. In contrast, there was no reparative dentin in the Opn KO mice where nestin- and DSP-positive newly differentiated odontoblast-like cells lacked immunoreaction for type I collagen. The in vitro organ culture demonstrated that the administration of recombinant OPN rescued the type I collagen secretion by odontoblast-like cells in the Opn KO mice. The results suggested that the deposition of OPN at the calcification front is essential for the type I collagen secretion by newly differentiated odontoblast-like cells to form reparative dentin during pulpal healing following cavity preparation. [ABSTRACT FROM AUTHOR]

Calcium hydroxide and mineral trioxide aggregate are widely used for indirect and direct pulp capping and root canal filling. Their dissociation into Ca(2+) and OH(-) in dental pulp creates an alkaline environment, which activates reparative/reactionary dentinogenesis. However, the mechanisms by which odontoblasts detect the pH of the extracellular environment remain unclear. We examined the alkali-sensitive intracellular Ca(2+) signaling pathway in rat odontoblasts. In the presence or absence of extracellular Ca(2+), application of alkaline solution increased intracellular Ca(2+) concentration, or [Ca(2+)]i Alkaline solution-induced [Ca(2+)]i increases depended on extracellular pH (8.5 to 10.5) in both the absence and the presence of extracellular Ca(2+) The amplitude was smaller in the absence than in the presence of extracellular Ca(2+) Each increase in [Ca(2+)]i, activated by pH 7.5, 8.5, or 9.5, depended on extracellular Ca(2+) concentration; the equilibrium binding constant for extracellular Ca(2+) concentration decreased as extracellular pH increased (1.04 mM at pH 7.5 to 0.11 mM at pH 9.5). Repeated applications of alkaline solution did not have a desensitizing effect on alkali-induced [Ca(2+)]i increases and inward currents. In the presence of extracellular Ca(2+), alkaline solution-induced [Ca(2+)]i increases were suppressed by application of an antagonist of transient receptor potential ankyrin subfamily member 1 (TRPA1) channels. Ca(2+) exclusion efficiency during alkaline solution-induced [Ca(2+)]i increases was reduced by a Na(+)-Ca(2+) exchanger antagonist. Alizarin red and von Kossa staining revealed increased mineralization levels under repeated high pH stimulation, whereas the TRPA1 antagonist strongly reduced this effect. These findings indicate that alkaline stimuli-such as the alkaline environment inside dental pulp treated with calcium hydroxide or mineral trioxide aggregate-activate Ca(2+) mobilization via Ca(2+) influx mediated by TRPA1 channels and intracellular Ca(2+) release in odontoblasts. High pH-sensing mechanisms in odontoblasts are important for activating dentinogenesis induced by an alkaline environment. [ABSTRACT FROM AUTHOR]

If there is a partial loss of dentin, the exposed dentinal surface should be protected by an indirect pulp capping (IPC) procedure to preserve pulp vitality and prevent symptoms of dentin hypersensitivity. In our previous study, copine7 (CPNE7) induced odontoblast differentiation in vitro and promoted dentin formation in vivo. The aim of this study was to investigate the possibility of IPC therapy using the CPNE7 protein at the exposed dentinal surface and the resulting effects on tertiary dentin formation in a beagle model. CPNE7 promoted mineralization of odontoblasts and had high calcium ion-binding capacity. The in vivo IPC model with canine teeth showed that regeneration of physiologic reactionary dentin with dentinal tubule structures was clearly observed beneath the remaining dentin in the CPNE7 group, whereas irregular features of reparative dentin were generated in the mineral trioxide aggregate (MTA) group. The CPNE7+MTA group also showed typical reactionary dentin without reparative dentin, showing synergistic effects of CPNE7 with MTA. A scanning electron microscopy analysis showed that dentinal tubules beneath the original dentin were occluded by the deposition of peritubular dentin in the CPNE7 and CPNE7+MTA groups, whereas those in the control group were opened. Therefore, CPNE7 may be able to serve as a novel IPC material and improve symptoms of dentin hypersensitivity. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is closely related to oral-complicated diseases by oxidative stress. This study investigates whether cellular myeloblastosis (c-myb) could protect human dental pulp cells against glucose oxidative stress and regulate autophagy activity for pulp vitality. Diabetes mellitus was induced by streptozotocin in Sprague-Dawley rats, and their pulp tissue in teeth was analyzed in terms of pulp cavity and molecules by hematoxylin and eosin and immunohistochemistry staining. Human dental pulp cells were serially subcultured and treated with glucose oxidase in the presence of elevated glucose to generate glucose oxidative stress. The replication-deficient adenovirus c-myb and small interfering RNA c-myb were introduced for c-myb expression. The pulp tissue from the diabetic rats was structurally different from normal tissue in terms of narrow pulp capacity, reduced c-myb, and dentinogenesis molecules. Glucose oxidase treatment decreased c-myb and dentinogenesis molecules (bone morphogenetic protein 2 and 7, dentin matrix protein 1, and dentin sialophosphoprotein) in human dental pulp cells. However, overexpression of c-myb by adenovirus c-myb increased dentinogenesis, autophagy molecules (autophagy protein 5, microtubule-associated protein 1A/1B-light chain 3, and Beclin-1), and cell survival via p-AMPK/AKT signaling even with glucose oxidative stress. In contrast, the lack of c-myb decreased the above molecules and cell survival by downregulating p-AMPK/AKT signaling. The results indicate that diabetes leads to irreversible damage to dental pulp, which is related to downexpression of autophagy via the p-AMPK/AKT pathway by decline of c-myb. The findings of this study provide a new insight that c-myb could ameliorate autophagy activity and that it is applicable for monitoring complicated diseases of dental pulp. The involvement of c-myb in pulp pathology could serve a therapeutic target in oral-complicated diseases. [ABSTRACT FROM AUTHOR]

Members of the fibroblast growth factor (FGF) family play essential and important roles in primary and reparative dentinogenesis. Although there appears to be a general agreement on the effects of FGF signaling on the proliferation of pulp cells, there are conflicting results regarding its effects on odontoblast differentiation. We recently examined the effects of continuous exposure of dental pulp cells to FGF2 and showed that the effects of FGF2 on differentiation of progenitor cells into odontoblasts were stage specific and dependent on the stage of cell maturity. The purpose of this study was to gain further insight into cellular and molecular mechanisms regulating the stimulatory effects of FGF2 on odontoblast differentiation. To do so, we examined the effects of early and limited exposure of pulp cells from a series of green fluorescent protein (GFP) reporter transgenic mice that display stage-specific activation of transgenes during odontoblast differentiation to FGF2. Our results showed that early and limited exposure of pulp cells to FGF2 did not have significant effects on the extent of mineralization but induced significant increases in the expression of Dmp1 and Dspp and the number of DMP1-GFP(+) and DSPP-Cerulean(+) odontoblasts. Our results also showed that the stimulatory effects of FGF2 on odontoblast differentiation were mediated through FGFR/MEK/Erk1/2 signaling, increases in Bmp2, and activation of the BMP/BMPR signaling pathway. These observations show that early and limited exposure of pulp cells to FGF2 alone promotes odontoblast differentiation and provides critical insight for applications of FGF2 in dentin regeneration. [ABSTRACT FROM AUTHOR]

The Trps1 transcription factor is highly expressed in dental mesenchyme and preodontoblasts, while in mature, secretory odontoblasts, it is expressed at low levels. Previously, we have shown that high Trps1 levels in mature odontoblasts impair their function in vitro and in vivo. Col1a1-Trps1 transgenic (Trps1-Tg) mice demonstrate defective dentin secretion and mineralization, which are associated with significantly decreased Dspp expression due to direct repression of the Dspp gene by Trps1. Here, by crossing Trps1-Tg and Col1a1-Dspp transgenic (Dspp-Tg) mice, we generated Col1a1-Trps1;Col1a1-Dspp double transgenic (double-Tg) mice in which Dspp was restored in odontoblasts overexpressing Trps1. Comparative micro–computed tomography analyses revealed partial correction of the dentin volume and no improvement of dentin mineralization in double transgenic mice in comparison with Trps1-Tg and wild-type (WT) mice. In addition, dentin of double-Tg mice has an irregular mineralization pattern characteristic for dentin in hypophosphatemic rickets. Consistent with this phenotype, decreased levels of Phex, Vdr, and Fam20c proteins are detected in both Trps1-Tg and double-Tg odontoblasts in comparison with WT and Dspp-Tg odontoblasts. This suggests that the Dspp-independent dentin mineralization defects in Trps1-Tg mice are a result of downregulation of a group of proteins critical for mineral deposition within the dentin matrix. In summary, by demonstrating that Trps1 functions as a repressor of later stages of dentinogenesis, we provide functional significance of the dynamic Trps1 expression pattern during dentinogenesis. [ABSTRACT FROM AUTHOR]

In organized tissues, the precise geometry and the overall shape are critical for the specialized functions that the cells carry out. Odontoblasts are major matrix-producing cells of the tooth and have also been suggested to participate in sensory transmission. However, refined morphologic data on these important cells are limited, which hampers the analysis and understanding of their cellular functions. We took advantage of fluorescent color-coding genetic tracing to visualize and reconstruct in 3 dimensions single odontoblasts, pulp cells, and their assemblages. Our results show distinct structural features and compartments of odontoblasts at different stages of maturation, with regard to overall cellular shape, formation of the main process, orientation, and matrix deposition. We demonstrate previously unanticipated contacts between the processes of pulp cells and odontoblasts. All reported data are related to mouse incisor tooth. We also show that odontoblasts express TRPM5 and Piezo2 ion channels. Piezo2 is expressed ubiquitously, while TRPM5 is asymmetrically distributed with distinct localization to regions proximal to and within odontoblast processes. [ABSTRACT FROM AUTHOR]

This study investigated the relevance between pulp vitality and autophagy in aged human dental pulp cells (HDPCs) and whether peroxisome proliferator-activated receptor gamma (PPARγ) affects autophagy regulation for homeostasis in the aging progress. In vivo experiments were used in human and Sprague-Dawley rat teeth obtained from young and adult individuals. Aging- and autophagy-related molecules were determined by immunohistochemistry and hematoxylin and eosin staining. HDPCs were serially subcultured until spontaneously arrested for in vitro aging, and the replication deficiency adenovirus was introduced for PPARγ overexpression. Subsequently, the effect of PPARγ on regulation of autophagy molecules, mitochondria activity, and cell viability was assessed using Western blotting, confocal microscopy, and the MTT assay, respectively. In adult pulp tissue, autophagy molecules (autophagy protein 5, microtubule-associated protein 1A/1B light chain, and Beclin-1) were increased, but aging-related (PPARγ and heme oxygenase 1 [HO-1]) and dentinogenesis (dentin sialophosphoprotein and dentin matrix acidic phosphoprotein) molecules were decreased. In aged HDPCs, autophagy and intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 were increased, while PPARγ and HO-1 were decreased. Under stimulation with lipopolysaccharide, autophagy- and aging-related molecules were differentially expressed between young and aged cells. PPARγ induced HO-1 and autophagy molecules but reduced inflammatory molecules in aged cells. In addition, PPARγ activated strong mitochondrial activity and cell viability in aging cells. Inhibition of HO-1 by tin protoporphyrin IX exacerbated autophagy and mitochondrial activity as well as cell viability in young cells. This study indicates that PPARγ maintains pulp homeostasis through the regulation of autophagy molecules during the life span of HDPCs. [ABSTRACT FROM AUTHOR]

Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. However, it remains unclear if Wnt ligands, produced from dental mesenchyme, are necessary for odontoblast differentiation and dentin formation. Here, we show that odontoblast-specific disruption of Wntless (Wls), a chaperon protein that regulates Wnt sorting and secretion, leads to severe defects in dentin formation and root elongation. Dentin thickness decreased remarkably and pulp chambers enlarged in the mandibular molars of OC-Cre;WlsCO/CO mice. Although the initial odontoblast differentiation was normal in the mutant crown, odontoblasts became cuboidal and dentin thickness was reduced. In immunohistochemistry, Wnt10a, β-catenin, type I collagen, and dentin sialoprotein were significantly down-regulated in the odontoblasts of mutant crown. In addition, roots were short and root canals were widened. Cell proliferation was reduced in the developing root apex of mutant molars. Furthermore, Wnt10a and Axin2 expression was remarkably decreased in the odontoblasts of mutant roots. Deletion of the Wls gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation and root elongation. [ABSTRACT FROM PUBLISHER]

Bone and dentin share similar biochemical compositions and physiological properties. Dentin, a major tooth component, is formed by odontoblasts; in contrast, bone is produced by osteoblasts. Osterix (Osx), a zinc finger-containing transcription factor, has been identified as an essential regulator of osteoblast differentiation and bone formation. However, it has been difficult to establish whether Osx functions in odontoblast differentiation and dentin formation. To understand the role of Osx in dentin formation, we analyzed mice in which Osx was subjected to tissue-specific ablation under the control of either the Col1a1 or the OC promoter. Two independent Osx conditional knockout mice exhibited similar molar abnormalities. Although no phenotype was found in the crowns of these teeth, both mutant lines exhibited short molar roots due to impaired root elongation. Furthermore, the interradicular dentin in these mice showed severe hypoplastic features, which were likely caused by disruptions in odontoblast differentiation and dentin formation. These phenotypes were closely related to the temporospatial expression pattern of Osx during tooth development. These findings indicate that Osx is required for root formation by regulating odontoblast differentiation, maturation, and root elongation. Cumulatively, our data strongly indicate that Osx is a site-specific regulator in tooth root formation. [ABSTRACT FROM PUBLISHER]

FAM20C is an evolutionarily reserved molecule highly expressed in mineralized tissues. Previously we demonstrated that Sox2-Cre;Fam20Cfl/fl mice, in which Fam20C was ubiquitously inactivated, had dentin and enamel defects as well as hypophosphatemic rickets. We also showed that K14-Cre;Fam20Cfl/fl mice, in which Fam20C was specifically inactivated in the epithelium, had enamel defects but lacked hypophosphatemia and defects in the bone and dentin. These results indicated that the enamel defects in the Sox2-Cre;Fam20Cfl/fl mice were independent of dentin defects and hypophosphatemia. To determine if the dentin defects in the Sox2-Cre;Fam20Cfl/fl mice were associated with the enamel defects and hypophosphatemia, we crossed Fam20Cfl/fl mice with Wnt1-Cre and Osr2-Cre transgenic mice to inactivate Fam20C in the craniofacial mesenchymal cells that form dentin and alveolar bone. The resulting Wnt1-Cre;Fam20Cfl/fl and Osr2-Cre;Fam20Cfl/fl mice showed remarkable dentin and alveolar bone defects, while their enamel did not show apparent defects. The serum FGF23 levels in these mice were higher than normal but lower than those in the Sox2-Cre;Fam20Cfl/fl mice; they developed a mild type of hypophosphatemia that did not cause major defects in long bones. These results indicate that the dentin defects in the Sox2-Cre;Fam20Cfl/fl mice were independent of the enamel defects. [ABSTRACT FROM PUBLISHER]

Tissue loss due to oral diseases requires the healing and regeneration of tissues of multiple lineages. While stem cells are native to oral tissues, a current major limitation to regeneration is the ability to direct their lineage-specific differentiation. This work utilizes polymeric scaffold systems with spatiotemporally controlled morphogen cues to develop precise morphogen fields to direct mesenchymal stem cell differentiation. First, a simple three-layer scaffold design was developed that presented two spatially segregated, lineage-specific cues (Dentinogenic TGF-β1 and Osteogenic BMP4). However, this system resulted in diffuse morphogen fields, as assessed by the in vitro imaging of cell-signaling pathways triggered by the morphogens. Mathematical modeling was then exploited, in combination with incorporation of specific inhibitors (neutralizing antibodies or a small molecule kinase inhibitor) into each morphogen in an opposing spatial pattern as the respective morphogen, to design a five-layer scaffold that was predicted to yield distinct, spatially segregated zones of morphogen signaling. To validate this system, undifferentiated MSCs were uniformly seeded in these scaffold systems, and distinct mineralized tissue differentiation were noted within these morphogen zones. Finally, to demonstrate temporal control over morphogen signaling, latent TGF-β1 was incorporated into one region of a concentric scaffold design, and laser treatment was used to activate the morphogen on-demand and to induce dentin differentiation solely within that specific spatial zone. This study demonstrates a significant advance in scaffold design to generate precise morphogen fields that can be used to develop in situ models to explore tissue differentiation and may ultimately be useful in engineering multi-lineage tissues in clinical dentistry. [ABSTRACT FROM PUBLISHER]

Reactionary dentin formation is an adaptive secretory response mediated by odontoblasts to moderate dentin injury. The implications of this process for neuroimmune interactions operating to contain pathogens have not been fully appreciated. The purpose of the present study was to describe the relationship between reactionary dentinogenesis, the neurogenic changes of dental pulp innervation, and dendritic cell recruitment to caries progression, using a comparative immunohistochemical approach in human teeth from young adult individuals. Reactionary dentin formation during dentin caries progression is associated with changes in the integrity of junctional complexes within the odontoblast layer. Diminished coexpression of Cx43 and zonula occludens 1 implies a reduced level of intercellular connectivity between odontoblasts. Dentin caries also causes overexpression of growth-associated protein 43, a modulator of neural plasticity that promotes extensive sprouting of nerve endings into the reactionary dentin matrix. At the same time, an elevated number of HLA-DR-positive dendritic cells infiltrate the odontoblast layer and subsequently invade reactionary dentin formed underneath the early caries-affected regions. Simultaneous odontoblast layer remodeling, nerve fiber sprouting, and activation of dendritic cells during caries progression suggest a coordinated neuroimmune response to fight caries pathogen invasion and to promote dentin-pulp healing. We propose that reactionary dentin formation hinders pathogen invasion and supports defensive neuroimmune interactions against infection. The eventual understanding of this complex scenario may contribute to the development of novel approaches to dental caries treatment. [ABSTRACT FROM AUTHOR]

While transforming growth factor-β1 (TGF-β1) can regulate odontoblast differentiation in tooth crown morphogenesis, its effects on cells including stem cells from the apical papilla (SCAPs) involved in root formation are unclear. Nuclear factor I-C (NFIC) has been implicated in the regulation of root development, and interplay with TGF-β1 signaling has been reported in some cell types. We hypothesize that NFIC and TGF-β1 are important to the behavior of SCAPs and that the interplay between these molecules controls the regulation of the odontogenic differentiation of SCAPs. TGF-β1 inhibited the proliferation of SCAPs and their mineralization. Real-time polymerase chain-reaction (RT-PCR) and Western blot results showed that TGF-β1 significantly decreased osteogenic/dentinogenic gene expression. The inhibition of TGF-β/Smad signaling (SIS3) attenuated the suppressive effect of TGF-β1 on SCAPs. Importantly, overexpression of NFIC antagonized the effects of TGF-β1 on SCAPs, while knockdown of NFIC enhanced these effects, demonstrating a key regulatory role for NFIC in modulating TGF-β1 signaling in SCAPs. We conclude that this interplay between NFIC and TGF-β1 regulates SCAPs behavior and can determine the differentiation of these cells. These signaling interactions help inform the development of regenerative strategies aimed at root growth and development in immature teeth for endodontic treatment. [ABSTRACT FROM PUBLISHER]

The clinical translation of stem-cell-based dental pulp regeneration will require the use of injectable scaffolds. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) can generate a functional dental pulp when injected into full-length root canals. SHED survived and began to express putative markers of odontoblastic differentiation after 7 days when mixed with Puramatrix™ (peptide hydrogel), or after 14 days when mixed with recombinant human Collagen (rhCollagen) type I, and injected into the root canals of human premolars in vitro. Roots of human premolars injected with scaffolds (Puramatrix™ or rhCollagen) containing SHED were implanted subcutaneously into immunodeficient mice (CB-17 SCID). We observed pulp-like tissues with odontoblasts capable of generating new tubular dentin throughout the root canals. Notably, the pulp tissue engineered with SHED injected with either Puramatrix™ or rhCollagen type I presented similar cellularity and vascularization when compared with control human dental pulps. Analysis of these data, collectively, demonstrates that SHED injected into full-length human root canals differentiate into functional odontoblasts, and suggests that such a strategy might facilitate the completion of root formation in necrotic immature permanent teeth. [ABSTRACT FROM PUBLISHER]

Previously, we showed that Sox2-Cre;Fam20Cfl/fl mice in which Fam20C was ubiquitously inactivated had severe defects in dentin, enamel, and bone, along with hypophosphatemia. It remains to be determined if the enamel defects in the mice with universal inactivation of Family with sequence similarity 20-C (FAM20C) were associated with the dentin defects and whether hypophosphatemia in the knockout mice contributed to the enamel defects. In this study, we crossed Fam20Cfl/fl mice with keratin 14-Cre (K14-Cre) transgenic mice to specifically inactivate Fam20C in the epithelial cells, including the dental epithelial cells that are responsible for forming tooth enamel. X-ray, backscattered scanning electron microscopic, and histological analyses showed that the K14-Cre;Fam20Cfl/fl mice had severe enamel and ameloblast defects, while their dentin and alveolar bone were not significantly affected. Accordingly, serum biochemistry of the K14-Cre;Fam20Cfl/fl mice showed normal phosphate and FGF23 levels in the circulation. Analysis of these data indicates that, while FAM20C is a molecule essential to amelogenesis, its inactivation in the dental epithelium does not significantly affect dentinogenesis. Hypophosphatemia makes no significant contribution to the enamel defects in the mice with the ubiquitous deletion of Fam20C. [ABSTRACT FROM PUBLISHER]

Phosphatases are involved in bone and tooth mineralization, but their mechanisms of action are not completely understood. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) regulates inhibitory extracellular pyrophosphate through its pyrophosphatase activity to control mineral propagation in the matrix; mice without TNAP lack acellular cementum, and have mineralization defects in dentin, enamel, and bone. PHOSPHO1 is a phosphatase found within membrane-bounded matrix vesicles in mineralized tissues, and double ablation of Alpl and Phospho1 in mice leads to a complete absence of skeletal mineralization. Here, we describe mineralization abnormalities in the teeth of Phospho1-/- mice, and in compound knockout mice lacking Phospho1 and one allele of Alpl (Phospho1-/-;Alpl+/-). In wild-type mice, PHOSPHO1 and TNAP co-localized to odontoblasts at early stages of dentinogenesis, coincident with the early mineralization of mantle dentin. In Phospho1 knockout mice, radiography, micro-computed tomography, histology, and transmission electron microscopy all demonstrated mineralization abnormalities of incisor dentin, with the most remarkable findings being reduced overall mineralization coincident with decreased matrix vesicle mineralization in the Phospho1-/- mice, and the almost complete absence of matrix vesicles in the Phospho1-/-;Alpl+/- mice, whose incisors showed a further reduction in mineralization. Results from this study support prominent non-redundant roles for both PHOSPHO1 and TNAP in dentin mineralization. [ABSTRACT FROM PUBLISHER]

Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKOod) 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-cKOod 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. [ABSTRACT FROM PUBLISHER]

Dickkopf-related protein 1 (DKK1) is a potent inhibitor of Wnt/β-catenin signaling. Dkk1-null mutant embryos display severe defects in head induction. Conversely, targeted expression of Dkk1 in dental epithelial cells leads to the formation of dysfunctional enamel knots and subsequent tooth defects during embryonic development. However, its role in post-natal dentinogenesis is largely unknown. To address this issue, we studied the role of DKK1 in post-natal dentin development using 2.3-kb Col1a1-Dkk1 transgenic mice, with the following key findings: (1) The Dkk1 transgene was highly expressed in pulp and odontoblast cells during post-natal developmental stages; (2) the 1st molar displayed short roots, an enlarged pulp/root canal region, and a decrease in the dentin formation rate; (3) a small malformed second molar and an absent third molar; (4) an increase of immature odontoblasts, few mature odontoblasts, and sharply reduced dentinal tubules; and (5) a dramatic change in Osx and nestin expression. We propose that DKK1 controls post-natal mandibular molar dentin formation either directly or indirectly via the inhibition of Wnt signaling at the following aspects: (i) post-natal dentin formation, (ii) formation and/or maintenance of the dentin tubular system, (iii) mineralization of the dentin, and (iv) regulation of molecules such as Osx and nestin. [ABSTRACT FROM PUBLISHER]

Dentin matrix protein 1 (DMP1), an acidic protein that is essential to the mineralization of bone and dentin, exists as proteolytically processed fragments in the mineralized tissues. In this study, we characterized the tooth and jaw phenotypes in transgenic mice containing no wild-type DMP1, but expressing a mutant DMP1 in which Asp213, a residue at one cleavage site, was replaced by Ala213 (named “Dmp1-KO/D213A-Tg” mice). The teeth and mandible of Dmp1-KO/D213A-Tg mice were compared with those of wild-type, Dmp1-knockout (Dmp1-KO), and Dmp1 -KO mice expressing the normal Dmp1 transgene. The results showed that D213A-DMP1 was not cleaved in dentin, and the expression of D213A-DMP1 failed to rescue the defects in the dentin, cementum, and alveolar bones in the Dmp1-KO mice. These findings indicate that the proteolytic processing of DMP1 is essential to the formation and mineralization of dentin, cementum, and jaw bones. [ABSTRACT FROM PUBLISHER]

Odontoblasts are long-lived post-mitotic cells in the dental pulp, whose function is to form and maintain dentin. The survival mechanisms that preserve the viability of terminally differentiated odontoblasts during the life of a healthy tooth have not been described. In the present study, we characterized the autophagic-lysosomal system of human odontoblasts with transmission electron microscopy and immunocytochemistry, to analyze the mechanisms that maintain the functional viability of these dentinogenic cells. Odontoblasts were found to develop an autophagic-lysosomal system organized mainly by large autophagic vacuoles that are acid-phosphatase-positive to various degrees. These vacuoles expressed the autophagosomal and lysosomal markers LC3 and LAMP2, respectively, in an age-related pattern indicating the organization of a dynamic autophagic machinery. Progressive accumulation of lipofuscin within lysosomes indicates reduced lysosomal activity as a function of odontoblast aging. Our results suggest that autophagic activity in odontoblasts is a fundamental mechanism to ensure turnover and degradation of subcellular components. A reduction in the efficacy of this system might compromise cell viability and dentinogenic secretory capacity. In adult teeth, this condition is described as an ‘old odontoblast’ stage. [ABSTRACT FROM PUBLISHER]

Important factors involved in odontogenesis in mouse dental papillae disappear between the preand post-natal stages of development. Therefore, we hypothesized that certain genes involved in odontogenesis in dental papillae were subject to pre-/post-natal down-regulation. Our goal was to identify, by microarray analysis, which genes were down-regulated. Dental papillae were isolated from embryonic 16-day-, 18-day- (E16, E18), and post-natal 3-day-old (P3) murine first mandibular molar germs and analyzed by microarray. The number of down-regulated genes was 2269 between E16 and E18, and 3130 between E18 and P3. Drastic down-regulation (fold change > 10.0) of Adamts4, Aldha1a2, and Lef1 was observed at both E16 and E18, and quantitative RT-PCR revealed a post-natal reduction in their expression (Adamts4, 1/3; Aldh1a2, 1/13; and Lef1, 1/37). These results suggest that down-regulation of these three genes is an important factor in normal odontogenesis in dental papillae. [ABSTRACT FROM AUTHOR]