The aim of this study was to understand the roles of Sonic Hedgehog (SHH) signaling during tooth root and periodontium formation. In this study, we generated the dental mesenchyme-specific Smoothened (Smo) activated/inactivated mice with the activity of Cre recombinase under the control of osteocalcin promoter.In the Smo activated mutant molar sections at the postnatal 28 days, we found extremely thin root dentin and widened pulp chamber. Picrosirius red staining showed loosely arranged fibers in the periodontal space and decreased cellular cementum with some root resorption. Immunohistochemical staining showed less localization of matrix proteins such as Bsp, Dmp1, Pstn, and Ank in the cementum, periodontal ligament, and/or cementoblast.In the Smo inactivated mutant mouse, there was not any remarkable differences in the localization of these matrix proteins compared with the wild type. These findings suggest that adequate suppressing regulation of SHH signaling is required in the development of tooth root and periodontium.
Animals ; Dental Cementum ; Dental Pulp Cavity ; Dentin ; Hedgehogs ; Mice ; Molar ; Osteocalcin ; Periodontal Ligament ; Periodontium ; Recombinases ; Root Resorption ; Tooth Root ; Tooth

Regulation of osteoblast and osteocyte viability is essential for bone homeostasis. Smad4, a major transducer of bone morphogenetic protein and transforming growth factor-β signaling pathways, regulates apoptosis in various cell types through a mitochondrial pathway. However, it remains poorly understood whether Smad4 is necessary for the regulation of osteoblast and osteocyte viability. In this study, we analyzed Smad4Δ(Os) mice, in which Smad4 was subjected to tissue-specific disruption under the control of the 2.3-kb Col1a1 promoter, to understand the functional significance of Smad4 in regulating osteoblast/osteocyte viability during bone formation and remodeling. Smad4Δ(Os) mice showed a significant increase in osteoblast number and osteocyte density in the trabecular and cortical regions of the femur, whereas osteoclast activity was significantly decreased. The proliferation of osteoblasts/osteocytes did not alter, as shown by measuring 5′-bromo-2′deoxyuridine incorporation. By contrast, the percentage of TUNEL-positive cells decreased, together with a decrease in the Bax/Bcl-2 ratio and in the proteolytic cleavage of caspase 3, in Smad4Δ(Os) mice. Apoptosis in isolated calvaria cells from Smad4Δ(Os) mice decreased after differentiation, which was consistent with the results of the TUNEL assay and western blotting in Smad4Δ(Os) mice. Conversely, osteoblast cells overexpressing Smad4 showed increased apoptosis. In an apoptosis induction model of Smad4Δ(Os) mice, osteoblasts/osteocytes were more resistant to apoptosis than were control cells, and, consequently, bone remodeling was attenuated. These findings indicate that Smad4 has a significant role in regulating osteoblast/osteocyte viability and therefore controls bone homeostasis.
Animals ; Apoptosis ; Blotting, Western ; Bone Morphogenetic Proteins ; Bone Remodeling ; Caspase 3 ; Femur ; Homeostasis* ; In Situ Nick-End Labeling ; Mice ; Osteoblasts ; Osteoclasts ; Osteocytes ; Osteogenesis ; Skull ; Transducers

Dentin is the major part of tooth and formed by odontoblasts. Under the influence of the inner enamel epithelium, odontoblasts differentiate from ectomesenchymal cells of the dental papilla and secrete pre-dentin which then undergo mineralization into dentin. Transforming growth factor-beta (TGF-β)/bone morphogenetic protein (BMP) signaling is essential for dentinogenesis; however, the precise molecular mechanisms remain unclear. To understand the role of TGF-β/BMP signaling in odontoblast differentiation and dentin formation, we generated mice with conditional ablation of Smad4, a key intracellular mediator of TGF-β/BMP signaling, using Osr2 or OC-Cre mice. Here we found the molars of Osr2(Cre)Smad4 mutant mice exhibited impaired odontoblast differentiation, and normal dentin was replaced by ectopic bone-like structure. In Osr2(Cre)Smad4 mutant mice, cell polarity of odontoblast was lost, and the thickness of crown dentin was decreased in later stage compared to wild type. Moreover, the root dentin was also impaired and showed ectopic bone-like structure similar to Osr2(Cre)Smad4 mutant mice. Taken together, our results suggest that Smad4-dependent TGF-β/BMP signaling plays a critical role in odontoblast differentiation and dentin formation during tooth development.
Animals ; Cell Polarity ; Crowns ; Dental Enamel ; Dental Papilla ; Dentin* ; Dentinogenesis ; Epithelium ; Mice ; Miners ; Molar ; Odontoblasts* ; Tooth

PURPOSE: Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. Deletion of the Wntless (Wls) gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation. However, it remains unclear if autonomous Wnt ligands are necessary for differentiation of dental pulp cells into odontoblast-like cells to induce reparative dentinogenesis, one of well-known feature of pulp repair to form tertiary dentin. MATERIALS AND METHODS: To analyze the autonomous role of Wls for differentiation of dental pulp cells into odontoblast-like cells, we used primary dental pulp cells from unerupted molars of Wls-floxed allele mouse after infection with adenovirus for Cre recombinase expression to knockout the floxed Wls gene or control GFP expression. The differentiation of dental pulp cells into odontoblast-like cells was analyzed by quantitative real-time polymerase chain reaction. RESULT: Proliferation rate was significantly decreased in dental pulp cells with Cre expression for Wls knockout. The expression levels of Osterix (Osx), runt-related transcription factor 2 (Runx2), and nuclear factor I-C (Nfic) were all significantly decreased by 0.3-fold, 0.2-fold, and 0.3-fold respectively in dental pulp cells with Wls knockout. In addition, the expression levels of Bsp, Col1a1, Opn, and Alpl were significantly decreased by 0.7-fold, 0.3-fold, 0.8-fold, and 0.6-fold respectively in dental pulp cells with Wls knockout. CONCLUSION: Wnt ligands produced autonomously are necessary for proper proliferation and odontoblastic differentiation of mouse dental pulp cells toward further tertiary dentinogenesis.
Adenoviridae ; Alleles ; Animals ; Dental Pulp* ; Dentin ; Dentinogenesis ; Epithelium ; Ligands ; Mesoderm ; Mice* ; Molar ; Morphogenesis ; NFI Transcription Factors ; Odontoblasts* ; Real-Time Polymerase Chain Reaction ; Recombinases ; Tooth ; Transcription Factors

Molecular interactions between epithelium and mesenchyme are important for root formation. Nuclear factor I-C (Nfic) has been identified as a key regulator of root formation. However, the mechanisms of root formation and their interactions between Hertwig's epithelial root sheath (HERS) and mesenchyme remain unclear. In this study, we investigated the role of Nfic in root patterning and growth during molar root development. The molars of Nfic knockout mice exhibited an enlarged pulp chamber and apical displacement of the pulpal floor, characteristic features of taurodontism, due to delayed furcation formation. In developing molar roots of mutant mice at P14, BrdU positive cells decreased in the apical mesenchyme of the elongation region whereas those cells increased in the dental papilla of the furcation region. Whereas cytokeratin 14 and laminin were localized in HERS cells of mutant molars, Smoothened (Smo) and Gli1 were downregulated in preodontoblasts. In contrast, cytokeratin 14 and Smo were localized in the cells of the furcation region of mutant molars. These results indicate that Nfic regulates cell proliferation in the dental mesenchyme and affects the fate of HERS cells in a site-specific manner. From the results, it is suggested that Nfic is required for root patterning and growth during root morphogenesis.
Animals ; Bromodeoxyuridine ; Cell Proliferation ; Dental Papilla ; Dental Pulp Cavity ; Epithelium ; Keratin-14 ; Laminin ; Mesoderm ; Mice ; Mice, Knockout ; Molar ; Morphogenesis ; NFI Transcription Factors ; Tooth Root* ; Tooth*

Genetically engineered mice have provided much information about gene function in the field of developmental biology. Recently, conditional gene targeting using the Cre/loxP system has been developed to control the cell type and timing of the target gene expression. The increase in number of kidney-specific Cre mice allows for the analysis of phenotypes that cannot be addressed by conventional gene targeting. The mammalian kidney is a vital organ that plays a critical homeostatic role in the regulation of body fluid composition and excretion of waste products. The interactions between epithelial and mesenchymal cells are very critical events in the field of developmental biology, especially renal development. Kidney development is a complex process, requiring inductive interactions between epithelial and mesenchymal cells that eventually lead to the growth and differentiation of multiple highly specialized stromal, vascular, and epithelial cell types. Through the use of genetically engineered mouse models, the molecular bases for many of the events in the developing kidney have been identified. Defective morphogenesis may result in clinical phenotypes that range from complete renal agenesis to diseases such as hypertension that exist in the setting of grossly normal kidneys. In this review, we focus on the growth and transcription factors that define kidney progenitor cell populations, initiate ureteric bud branching, induce nephron formation within the metanephric mesenchyme, and differentiate stromal and vascular progenitors in the metanephric mesenchyme.
Animals ; Body Fluids ; Congenital Abnormalities ; Developmental Biology ; Epithelial Cells ; Gene Expression ; Gene Targeting ; Hypertension ; Kidney ; Kidney Diseases ; Mesoderm ; Mice ; Morphogenesis ; Nephrons ; Phenotype ; Stem Cells ; Transcription Factors ; Ureter ; Waste Products

Ameloblasts ; Animals ; Dental Enamel ; Dentin ; Epithelial Cells ; Inclusion Bodies ; Mice ; Mice, Knockout ; Morphogenesis ; Neural Crest ; Odontoblasts ; Tooth ; Tooth Germ

Wnt/beta-catenin signaling plays a critical role in bone formation and regeneration. Dentin and cementum share many similarities with bone in their biochemical compositions and biomechanical properties. Whether Wnt/beta-catenin signaling is involved in the dento-alveolar complex formation is unknown. To understand the roles of Wnt/beta-catenin signaling in the dento-alveolar complex formation, we generated conditional beta-catenin activation mice through intercross of Catnb+/lox(ex3) mice with Col1a1-cre mice. In mutant mice, tooth formation and eruption was disturbed. Lower incisors and molars did not erupt. Bone formation was increased in the mandible but tooth formation was severely disturbed. Hypomineralized dentin was deposited in the crown but roots of molars were extremely short and distorted. In the odontoblasts of mutant molars, expression of dentin matrix proteins was obviously downregulated following the activation of beta-catenin whereas that of mineralization inhibitor was increased. Cementum and periodontal ligament were hypoplastic but periodontal space was narrow due to increased alveolar bone formation. While cementum matrix proteins were decreased, bone matrix proteins were increased in the cementum and alveolar bone of mutant mice. These results indicate that local activation of beta-catenin in the osteoblasts and odontoblasts leads to aberrant dento-alveolar complex formation. Therefore, appropriate inhibition of Wnt/beta-catenin signaling is important for the dento-alveolar complex formation.
Animals ; beta Catenin ; Bone Matrix ; Crowns ; Dental Cementum ; Dentin ; Incisor ; Mandible ; Mice ; Molar ; Odontoblasts ; Osteoblasts ; Osteogenesis ; Periodontal Ligament ; Proteins ; Regeneration ; Tooth

Under some pathological conditions as bile flow obstruction or liver diseases with the enterohepatic circulation being disrupted, regurgitation of bile acids into the systemic circulation occurs and the plasma level of bile acids increases. Bile acids in circulation may affect the nervous system. We examined this possibility by studying the effects of bile acids on gating of neuronal (N)-type Ca2+ channel that is essential for neurotransmitter release at synapses of the peripheral and central nervous system. N-type Ca2+ channel currents were recorded from bullfrog sympathetic neuron under a cell-attached mode using 100 mM Ba2+ as a charge carrier. Cholic acid (CA, 10(-6) M) that is relatively hydrophilic thus less cytotoxic was included in the pipette solution. CA suppressed the open probability of N-type Ca2+ channel, which appeared to be due to an increase in null (no activity) sweeps. For example, the proportion of null sweep in the presence of CA was ~40% at +40 mV as compared with ~8% in the control recorded without CA. Other single channel properties including slope conductance, single channel current amplitude, open and shut times were not significantly affected by CA being present. The results suggest that CA could modulate N-type Ca2+ channel gating at a concentration as low as 10(-6) M. Bile acids have been shown to activate nonselective cation conductance and depolarize the cell membrane. Under pathological conditions with increased circulating bile acids, CA suppression of N-type Ca2+ channel function may be beneficial against overexcitation of the synapses.
Bile ; Bile Acids and Salts ; Calcium Channels, N-Type ; Cell Membrane ; Central Nervous System ; Cholic Acid ; Enterohepatic Circulation ; Fees and Charges ; Ganglia, Sympathetic ; Liver Diseases ; Nervous System ; Neurons ; Neurotransmitter Agents ; Plasma ; Rana catesbeiana ; Synapses

OBJECTIVE: The purpose of this study was to analyze the stress distribution and the displacement pattern of mandibular anterior teeth under various intrusive force vectors according to the position of orthodontic miniscrews and hooks, using three-dimensional finite element analysis. METHODS: A three-dimensional finite element model was constructed to simulate mandibular teeth, periodontal ligament, and alveolar bone. The displacement of individual tooth on three-dimensional planes and the von Mises stress distribution were compared when various intrusion force vectors were applied. RESULTS: Intrusive forces applied to 4 mandibular anterior teeth largely resulted in remarkable labial tipping of the segment according to the miniscrew position. All 6 mandibular anterior teeth were labially tipped and the stress concentrated on the labiogingival area by intrusive force from miniscrews placed mesial to the canine. The distointrusive force vector led to pure intrusion and the stress was evenly distributed in the whole periodontal ligament when the hook was placed between the central and lateral incisors and the miniscrew was placed distal to the canine. CONCLUSIONS: Within the limits of this study, it can be concluded that predictable pure intrusion of the 6 anterior teeth segment may be accomplished using miniscrews placed distal to the canine and hooks located between the central and lateral incisors.
Displacement (Psychology) ; Incisor ; Periodontal Ligament ; Tooth