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Mesenchymal stem cells (MSCs) are typically defined by their in vitro characteristics, and as a consequence the in vivo identity of MSCs and their niches are poorly understood. To address this issue, we used lineage tracing in a mouse incisor model and identified the neurovascular bundle (NVB) as an MSC niche. We found that NVB sensory nerves secrete Shh protein, which activates Gli1 expression in periarterial cells that contribute to all mesenchymal derivatives. These periarterial cells do not express classical MSC markers used to define MSCs in vitro. In contrast, NG2(+) pericytes represent an MSC subpopulation derived from Gli1+ cells; they express classical MSC markers and contribute little to homeostasis but are actively involved in injury repair. Likewise, incisor Gli1(+) cells, but not NG2(+) cells, exhibit typical MSC characteristics in vitro. Collectively, we demonstrate that MSCs originate from periarterial cells and are regulated by Shh secretion from an NVB., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The rodent incisor is one of a number of organs that grow continuously throughout the life of an animal. Continuous growth of the incisor arose as an evolutionary adaptation to compensate for abrasion at the distal end of the tooth. The sustained turnover of cells that deposit the mineralized dental tissues is made possible by epithelial and mesenchymal stem cells residing at the proximal end of the incisor. A complex network of signaling pathways and transcription factors regulates the formation, maintenance, and differentiation of these stem cells during development and throughout adulthood. Research over the past 15 years has led to significant progress in our understanding of this network, which includes FGF, BMP, Notch, and Hh signaling, as well as cell adhesion molecules and micro-RNAs. This review surveys key historical experiments that laid the foundation of the field and discusses more recent findings that definitively identified the stem cell population, elucidated the regulatory network, and demonstrated possible genetic mechanisms for the evolution of continuously growing teeth., (Copyright © 2013 Wiley Periodicals, Inc.)

Aim of the Study: Establishment of Ki67, p53 and CD34 expression in human tooth buds of different stages of odontogenetic development., Materials and Methods: Tissue samples containing tooth buds were removed from the incisor areas of human fetuses in different stages of development (weeks 9-10, 12-13, 13-16, 21-24), and from the canine and molar areas of 21-24 weeks fetuses. The tissue fragments were fixed using formalin and were processed using common histological techniques with paraffin embedding. Immunostaining for Ki67, p53 and CD34 has been performed using the dextran method and moist heat antigen retrieval (except for CD34). The resulting slides were photographed and quantitatively evaluated., Results: Ki67 immunoexpression decreases with advancement of the developmental stage of the tooth bud: in the inner enamel epithelium, between weeks 9 and 16 (IEE), in the preameloblasts (PB) between weeks 13 and 16, in the ameloblasts (AB) between weeks 21 and 24; outer enamel epithelium (OEE); stratum intermedium (SI); in the dental papilla: between weeks 9 and 10 in the dental papilla (DP), between weeks 13 and 16 in the outer layer of the dental papilla (DP1) and in the central layer of the dental papilla (DP2). Likewise, we noted Ki67 expression in the odontoblast layer (O) and pulp (P), between weeks 21 and 24. Concerning CD34 expression, we observed a decrease from weeks 9-10 until weeks 13-16, followed by an increase until weeks 21-24 of intrauterine life. From weeks 9-10, we observed a constant decrease of expression until weeks 13-16, followed by an increase during weeks 21-24., Conclusions: All Ki67, p53 and CD34 have been identified in the tooth bud. Ki67 expression gradually decreases with the embryonic development of the tooth, while p53 and CD34 expression decreases from weeks 9-10 to weeks 13-16 of intrauterine life, followed by an increase until weeks 21-24.

Tooth development relies on the interaction between the oral ectoderm and underlying mesenchyme, and is regulated by a complex genetic cascade. This transcriptional cascade is regulated by the spatiotemporal activation and deactivation of transcription factors. The specificity proteins 6 (Sp6) and chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (Ctip2) were identified in loss-of-function studies as key transcription factors required for tooth development. Ctip2 binds to the Sp6 promoter in vivo; however, its role in Sp6 expression remains unclear. In this study, we investigated Sp6 transcriptional regulation by Ctip2. Immunohistochemical analysis revealed that Sp6 and Ctip2 colocalize in the rat incisor during tooth development. We examined whether Ctip2 regulates Sp6 promoter activity in dental epithelial cells. Cotransfection experiments using serial Sp6 promoter-luciferase constructs and Ctip2 expression plasmids showed that Ctip2 significantly suppressed the Sp6 second promoter activity, although the Sp6 first promoter activity was unaffected. Ctip2 was able to bind to the proximal region of the Sp6 first promoter, as previously demonstrated, and also to the novel distal region of the first, and second promoter regions. Our findings indicate that Ctip2 regulates Sp6 gene expression through direct binding to the Sp6 second promoter region. J. Med. Invest. 61: 126-136, February, 2014.

Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells isolated from the exfoliated human deciduous incisor that can differentiate into a many cell types. In this study, we evaluated the effect of liquorice or angelica extracts on the hepatic differentiation potential of SHEDs cells. SHEDs cells cultured in medium containing liquorice extracts were analyzed for 1) changes in cellular morphology, 2) changes in hepatic gene expression, AFP (Alpha-fetoprotein) and ALB (Albumin), and 3) albumin secretion and urea synthesis activity. Our data show that the hepatic differentiation potential of SHEDs cells is enhanced by the presence of liquorice or angelica extracts in the culture medium. Our findings present new therapeutic possibilities for liver damage repair.

The differentiation of dental epithelia into enamel-producing ameloblasts or the root epithelial lineage compartmentalizes teeth into crowns and roots. Bmp signaling has been linked to enamel formation, but its role in root epithelial lineage differentiation is unclear. Here we show that cessation of epithelial Bmp signaling by Bmpr1a depletion during the differentiation stage switched differentiation of crown epithelia into the root lineage and led to formation of ectopic cementum-like structures. This phenotype is related to the upregulation of Wnt/β-catenin signaling and epithelial-mesenchymal transition (EMT). Although epithelial β-catenin depletion during the differentiation stage also led to variable enamel defect and precocious/ectopic formation of fragmented root epithelia in some teeth, it did not cause ectopic cementogenesis and inhibited EMT in cultured dental epithelia. Concomitant epithelial β-catenin depletion rescued EMT and ectopic cementogenesis caused by Bmpr1a depletion. These data suggested that Bmp and Wnt/β-catenin pathways interact antagonistically in dental epithelia to regulate the root lineage differentiation and EMT. These findings will aid in the design of new strategies to promote functional differentiation in the regeneration and tissue engineering of teeth and will provide new insights into the dynamic interactions between the Bmp and Wnt/β-catenin pathways during cell fate decisions.

Signals of perlecan, an extracellular matrix molecule, which accumulates within the intercellular spaces of the stellate reticulum of the enamel organ, are mediated by at least two receptors, dystroglycan (DG) and integrin β1, in a case-dependent manner in various events in embryogenesis and pathogenesis. This study aims to understand the expression profiles of these two perlecan receptors at both protein and gene levels in murine enamel organ development. Before birth, α-DG was immunolocalized in stellate reticulum cells, in which perlecan was colocalized, while integrin β1 was mainly distributed in the peripheral enamel organ cells as well as the dental mesenchymal cells. On and after postnatal Day 1, the expression of α-DG was dramatically decreased in the stellate reticulum, while integrin β1 was enhanced around blood vessels within the enamel organ. Furthermore, biosyntheses of α-DG and integrin β1 by dental epithelial and pulp mesenchymal cells were confirmed in vitro by using immunofluorescence and reverse-transcriptase polymerase chain reaction. The results suggest that DG is a perlecan receptor that specifically functions in the stellate reticulum of the embryonic stage, but that dental epithelial and mesenchymal cells are maturated by capturing perlecan signals differentially through integrin β1., (Copyright © 2013 Elsevier B.V. All rights reserved.)

FAM20C, also known as Golgi casein kinase (G-CK), is proposed to be the archetype for a family of secreted kinases that phosphorylate target proteins in the Golgi and in extracellular matrices, but FAM20C serving an extracellular function is controversial. FAM20C phosphorylates secretory calcium-binding phosphoproteins (SCPPs), which are associated with the evolution of biomineralization in vertebrates. Current models of biomineralization assume SCPP proteins are secreted as phosphoproteins and their phosphates are essential for protein conformation and function. It would be a radical departure from current theories if proteins in mineralizing matrices were dephosphorylated as part of the mineralization mechanism and rephosphorylated in the extracellular milieu by FAM20C using ATP. To see if such mechanisms are possible in the formation of dental enamel, we tested the hypothesis that FAM20C is secreted by ameloblasts and accumulates in the enamel extracellular matrix during tooth development. FAM20C localization was determined by immunohistochemistry in day 5 mouse incisors and molars and by Western blot analyses of proteins extracted from pig enamel organ epithelia (EOE) and enamel shavings. FAM20C localized intracellularly within ameloblasts and odontoblasts in a pattern consistent with Golgi localization. Western blots detected FAM20C in the EOE extracts but not in the enamel matrix. We conclude that FAM20C is not a constituent of the enamel extracellular matrix and functions intracellularly within ameloblasts., (© 2013 American Society for Bone and Mineral Research.)

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies., (© 2013.)

Rodent incisors maintain the ability to grow continuously and their labial dentin is covered with enamel. Bcl11b zinc-finger transcription factor is expressed in ameloblast progenitors in mouse incisors and its absence in Bcl11b(KO/KO) mice results in a defect in embryonic tooth development. However, the role of Bcl11b in incisor maintenance in adult tissue was not studied because of death at birth in Bcl11b(KO/KO) mice. Here, we examined compound heterozygous Bcl11b(S826G/KO) mice, one allele of which has an amino acid substitution of serine at position 826 for glycine, that exhibited hypoplastic maxillary incisors with lower concentrations of minerals at the enamel and the dentin, accompanying the maxillary bone hypoplasia. Histological examinations revealed hypoplasia of the labial cervical loop in incisors, shortening of the ameloblast progenitor region, and impairment in differentiation and proliferation of ameloblast-lineage cells. Interestingly, however, juvenile mice at 5days after birth did not show marked change in these phenotypes. These results suggest that attenuated Bcl11b activity impairs ameloblast progenitors and incisor maintenance. The number of BrdU label-retaining cells, putative stem cells, was lower in Bcl11b(S826G/KO) incisors, which suggests the incisor hypoplasia may be in part a result of the decreased number of stem cells. Interestingly, the level of Shh and FGF3 expressions, which are assumed to play key roles in the development and maintenance of ameloblasts and odontoblasts, was not decreased, though the expressed areas were more restricted in ameloblast progenitor and mesenchyme regions of Bcl11b(S826G/KO) incisors, respectively. Those data suggest that the incisor maintenance by Bcl11b is not directly related to the FGF epithelial-mesenchymal signaling loop including Shh but is intrinsic to ameloblast progenitors and possibly stem cells., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The mouse incisor is a remarkable tooth that grows throughout the animal's lifetime. This continuous renewal is fueled by adult epithelial stem cells that give rise to ameloblasts, which generate enamel, and little is known about the function of microRNAs in this process. Here, we describe the role of a novel Pitx2:miR-200c/141:noggin regulatory pathway in dental epithelial cell differentiation. miR-200c repressed noggin, an antagonist of Bmp signaling. Pitx2 expression caused an upregulation of miR-200c and chromatin immunoprecipitation assays revealed endogenous Pitx2 binding to the miR-200c/141 promoter. A positive-feedback loop was discovered between miR-200c and Bmp signaling. miR-200c/141 induced expression of E-cadherin and the dental epithelial cell differentiation marker amelogenin. In addition, miR-203 expression was activated by endogenous Pitx2 and targeted the Bmp antagonist Bmper to further regulate Bmp signaling. miR-200c/141 knockout mice showed defects in enamel formation, with decreased E-cadherin and amelogenin expression and increased noggin expression. Our in vivo and in vitro studies reveal a multistep transcriptional program involving the Pitx2:miR-200c/141:noggin regulatory pathway that is important in epithelial cell differentiation and tooth development.

The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell-cycle inhibitors p16(Ink4a) and p19(Arf). However, deletion of Ink4a/Arf only partially rescues Bmi1-null phenotypes, indicating that other important targets of BMI1 exist. Here, using the continuously growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation.

The aim of this study was to further define the relationship between cell proliferation and the rate of tooth eruption in the rat incisor. Vinblastine is a drug that blocks cellular mitosis and was used to inhibit cell proliferation in the odontogenic region of rat incisors that were submitted to a shortening treatment or to higher masticatory forces. Male Wistar rats were divided into five groups: normofunctional (control group for incisor eruption), hypofunctional (incisor submitted to eruption acceleration), hyperfunctional (incisors under higher masticatory forces), hypofunctional with vinblastine and hyperfunctional with vinblastine. In incisors submitted to shortening procedures, a significant decrease in the eruption rate and cell proliferation was observed two days after vinblastine injection, suggesting that incisor eruption is dependent on cell proliferation., (Copyright © 2013 Wiley Periodicals, Inc.)

We hypothesize that a population of migrating cells can form patterns when changes in local strains owing to relative cell motions induce changes in cell motility. That the mechanism originates in competing rates of motion distinguishes it from mechanisms involving strain energy gradients, e.g. those generated by surface energy effects or eigenstrains among cells, and diffusion-reaction mechanisms involving chemical signalling factors. The theory is tested by its ability to reproduce the morphological characteristics of enamel in the mouse incisor. Dental enamel is formed during amelogenesis by a population of ameloblasts that move about laterally within an expanding curved sheet, subject to continuously evolving spatial and temporal gradients in strain. Discrete-cell simulations of this process compute the changing strain environment of all cells and predict cell trajectories by invoking simple rules for the motion of an individual cell in response to its strain environment. The rules balance a tendency for cells to enhance relative sliding motion against a tendency to maintain uniform cell-cell separation. The simulations account for observed waviness in the enamel microstructure, the speed and shape of the 'commencement front' that separates domains of migrating secretory-stage ameloblasts from those that are not yet migrating, the initiation and sustainment of layered, fracture-resistant decussation patterns (cross-plied microstructure) and the transition from decussating inner enamel to non-decussating outer enamel. All these characteristics can be correctly predicted with the use of a single scalar adjustable parameter.

Demineralized bone or dentine implanted intramuscularly induce endochondral bone formation. This phenomenon, termed "bone induction" is triggered by non-collagenous signal molecules, named "Bone Morphogenetic Proteins" (BMPs), released from bone or dentine. Demineralization of bone/dentine prior their implantation facilitates the release of BMPs from the extracellular matrix allowing to reach a BMP threshold level needed to initiate the process of differentiation of mesenchymal cells towards an osteogenic/chondrogenic lineage. Unprocessed, mineralized tissues usually fail to induce cartilage/bone. Isolated BMPs are commercially available, and in clinical practice are an alternative for demineralized tissues, however, in many cases demineralized bone has advantages over soluble BMPs, as it combines both bone inducing principles and mechanical properties, a feature important for bridging bone fracture and filling bone defects. Demineralized bones are an inexpensive source of bone forming agents for bone-fracture healing or filling bone defects. In this report we demonstrated that storage of lyophilized demineralized murine incisors for 30 months does not deteriorate its osteoinductive potency and colonizing induced bone by bone marrow. Lyophylized incisors, stored for 0-30 months at refrigator were implanted intramuscularly and recovered, together with surrounding tissues at various time intervals ranging 10-450 days. Bone closely associated with implant was observed in about 87% of cases, regardless the storage duration. It is concluded that storage of demineralized and lyophilized incisor matrices for at least 30 months does not change their osteoinductive potency.

Objectives: The rodent incisor cervical loop and molar Hertwig's epithelial root sheath (HERS) are common models used for investigating tooth root generation. The purpose of the present study was to gain a better understanding of the molecular mechanisms mediating root development by determining the distinctive gene and protein expression profiles of each during different stages of development., Methods: In this study, we used quantitative real time reverse transcription-PCR and immunohistochemistry to analyse the expression levels of high mobility group AT-hook 2, ameloblastin, amelogenin, dentine sialoprotein, dentine matrix protein 1, osteocalcin, and bone sialoprotein in rat epithelial and mesenchymal cells isolated at postnatal days 4 and 8., Results: Results showed that the expression of these genes and proteins was up-regulated in cervical loop epithelial cells, but decreased or unchanged in other cells during development. This increase in expression in the incisor cervical loop may be due to the interaction of the inner incisor dental papilla cells, which are the niche cells of cervical loop epithelial cells and demonstrated up-regulated expression of the corresponding proteins, revealing a complex and dynamic interplay of these molecules during neonatal tooth development., Conclusion: These findings provide novel insights into the molecular processes underlying crown development of rodent incisors, and contribute to our overall understanding of the pathogenic processes of tooth root dysontogenesis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Dental epithelial stem cells (DESCs) drive continuous growth in the adult mouse incisors. To date, a robust system for the primary culture of these cells has not been reported, and little is known about the basic molecular architecture of these cells or the minimal extracellular scaffolding that is necessary to maintain the epithelial stem cell population in an undifferentiated state. We report a method of isolating DESCs from the cervical loop of the mouse mandibular incisor. Cells were viable in a two-dimensional culture system and did not demonstrate preferential proliferation when grown on top of various substrates. Characterization of these cells indicated that E-cadherin, integrin alpha-6, and integrin beta-4 mark the DESCs both in vivo and in vitro. We also grew these cells in a three-dimensional microenvironment and obtained spheres with an epithelial morphology and expression patterns. Insights into the mechanisms of stem cell maintenance in vitro will help lay the groundwork for the successful generation of bioengineered teeth from adult DESCs.

Epiprofin/Specificity Protein 6 (Epfn) is a Krüppel-like family (KLF) transcription factor that is critically involved in tooth morphogenesis and dental cell differentiation. However, its mechanism of action is still not fully understood. We have employed both loss-of-function and gain-of-function approaches to address the role of Epfn in the formation of cell junctions in dental cells and in the regulation of junction-associated signal transduction pathways. We have evaluated the expression of junction proteins in bell-stage incisor and molar tooth sections from Epfn(-/-) mice and in dental pulp MDPC-23 cells overexpressing Epfn. In Epfn(-/-) mice, a dramatic reduction occurs in the expression of tight junction and adherens junction proteins and of the adherens-junction-associated β-catenin protein, a major effector of canonical Wnt signaling. Loss of cell junctions and β-catenin in Epfn(-/-) mice is correlated with a clear decrease in bone morphogenetic protein 4 (BMP-4) expression, a decrease in nestin in the tooth mesenchyme, altered cell proliferation, and failure of ameloblast cell differentiation. Overexpression of Epfn in MDPC-23 cells results in an increased cellular accumulation of β-catenin protein, indicative of upregulation of canonical Wnt signaling. Together, these results suggest that Epfn enhances canonical Wnt/β-catenin signaling in the developing dental pulp mesenchyme, a condition that promotes the activity of other downstream signaling pathways, such as BMP, which are fundamental for cellular induction and ameloblast differentiation. These altered signaling events might underlie some of the most prominent dental defects observed in Epfn(-/-) mice, such as the absence of ameloblasts and enamel, and might throw light on developmental malformations of the tooth, including hyperdontia.

The transcription factor Sox2 plays important roles in maintaining the pluripotency of embryonic stem cells and adult progenitors. However, whether Sox2 is involved in odontogenesis has not been reported. In this study, we examined the expression pattern of Sox2 during mouse incisor and molar development using real-time PCR, in situ hybridization and immunohistochemistry. Sox2 mRNA was expressed in the dental epithelium and mesenchyme while Sox2 protein was mainly detected in the epithelium from embryonic day (E) 11.5 to postnatal (PN) day 20. In the case of incisor, Sox2 mRNA and protein were expressed in most of dental epithelial cells from E11.5 to E14.5, and they were both highly expressed in the labial cervical loop area from E16.5 to PN20. During molar development, we observed an asymmetrical distribution of Sox2 protein in the epithelium from E13.5 to E16.5, with stronger signals in the lingual side. From E18.5 to PN2, Sox2 was expressed within the cervical loop area, and the stellate intermediate layer. From PN6 to PN14, Sox2 expression was confined mainly to the apical end of hertwig's epithelium root sheath (HERS) cells. Sox2 was also detected within the perivascular region of the dental pulp at PN14 and PN20. Our results suggested that: (1) Sox2 was involved in mouse odontogenesis, and (2) it might participate in maintaining the pluripotency of the epithelial stem cells of labial cervical loop in mouse incisor development and the epithelium progenitors during molar development, (3) Sox2 might be regulated at post-transcription level during mouse odontogenesis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The epithelial cell rests of Malassez (ERM) are odontogenic epithelial cells located within the periodontal ligament matrix. While their function is unknown, they may support tissue homeostasis and maintain periodontal ligament space or even contribute to periodontal regeneration. We investigated the notion that ERM contain a subpopulation of stem cells that could undergo epithelial-mesenchymal transition and differentiate into mesenchymal stem-like cells with multilineage potential. For this purpose, ERM collected from ovine incisors were subjected to different inductive conditions in vitro, previously developed for the characterization of bone marrow mesenchymal stromal/stem cells (BMSC). We found that ex vivo-expanded ERM expressed both epithelial (cytokeratin-8, E-cadherin, and epithelial membrane protein-1) and BMSC markers (CD44, CD29, and heat shock protein-90β). Integrin α6/CD49f could be used for the enrichment of clonogenic cell clusters [colony-forming units-epithelial cells (CFU-Epi)]. Integrin α6/CD49f-positive-selected epithelial cells demonstrated over 50- and 7-fold greater CFU-Epi than integrin α(6)/CD49f-negative cells and unfractionated cells, respectively. Importantly, ERM demonstrated stem cell-like properties in their differentiation capacity to form bone, fat, cartilage, and neural cells in vitro. When transplanted into immunocompromised mice, ERM generated bone, cementum-like and Sharpey's fiber-like structures. Additionally, gene expression studies showed that osteogenic induction of ERM triggered an epithelial-mesenchymal transition. In conclusion, ERM are unusual cells that display the morphological and phenotypic characteristics of ectoderm-derived epithelial cells; however, they also have the capacity to differentiate into a mesenchymal phenotype and thus represent a unique stem cell population within the periodontal ligament.

Rodent incisors are capable of growing continuously and the renewal of dental epithelium giving rise to enamel-forming ameloblasts and dental mesenchyme giving rise to dentin-forming odontoblasts and pulp cells is achieved by stem cells residing at their proximal ends. Although the dental epithelial stem cell niche (cervical loop) is well characterized, little is known about the dental mesenchymal stem cell niche. Ring1a/b are the core Polycomb repressive complex1 (PRC1) components that have recently also been found in a protein complex with BcoR (Bcl-6 interacting corepressor) and Fbxl10. During mouse incisor development, we found that genes encoding members of the PRC1 complex are strongly expressed in the incisor apical mesenchyme in an area that contains the cells with the highest proliferation rate in the tooth pulp, consistent with a location for transit amplifying cells. Analysis of Ring1a(-/-);Ring1b(cko/cko) mice showed that loss of Ring1a/b postnatally results in defective cervical loops and disturbances of enamel and dentin formation in continuously growing incisors. To further characterize the defect found in Ring1a(-/-);Ring1b(cko/cko) mice, we demonstrated that cell proliferation is dramatically reduced in the apical mesenchyme and cervical loop epithelium of Ring1a(-/-);Ring1b(cko/cko) incisors in comparison to Ring1a(-/-);Ring1b(fl/fl)cre- incisors. Fgf signaling and downstream targets that have been previously shown to be important in the maintenance of the dental epithelial stem cell compartment in the cervical loop are downregulated in Ring1a(-/-);Ring1b(cko/cko) incisors. In addition, expression of other genes of the PRC1 complex is also altered. We also identified an essential postnatal requirement for Ring1 proteins in molar root formation. These results show that the PRC1 complex regulates the transit amplifying cell compartment of the dental mesenchymal stem cell niche and cell differentiation in developing mouse incisors and is required for molar root formation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Stem cells are essential for the regeneration and homeostasis of many organs, such as tooth, hair, skin, and intestine. Although human tooth regeneration is limited, a number of animals have evolved continuously growing teeth that provide models of stem cell-based organ renewal. A well-studied model is the mouse incisor, which contains dental epithelial stem cells in structures known as cervical loops. These stem cells produce progeny that proliferate and migrate along the proximo-distal axis of the incisor and differentiate into enamel-forming ameloblasts. Here, we studied the role of E-cadherin in behavior of the stem cells and their progeny. Levels of E-cadherin are highly dynamic in the incisor, such that E-cadherin is expressed in the stem cells, downregulated in the transit-amplifying cells, re-expressed in the pre-ameloblasts and then downregulated again in the ameloblasts. Conditional inactivation of E-cadherin in the cervical loop led to decreased numbers of label-retaining stem cells, increased proliferation, and decreased cell migration in the mouse incisor. Using both genetic and pharmacological approaches, we showed that Fibroblast Growth Factors regulate E-cadherin expression, cell proliferation and migration in the incisor. Together, our data indicate that E-cadherin is an important regulator of stem cells and their progeny during growth of the mouse incisor., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Retinoblastoma protein (pRb) phosphorylation plays a central role in mediating cell cycle G1/S stage transition, together with E2F transcription factors. The binding of pRb to E2F is thought to be controlled by the sequential and cumulative phosphorylation of pRb at various amino acids. In addition to well characterized roles as a tumor suppressor, pRb has more recently been implicated in osteoprogenitor and other types of stem cell maintenance, proliferation and differentiation, thereby influencing the morphogenesis of developing organs. In this study, we present data characterizing the expression of pRb and three phosphorylated pRb (ppRb) isoforms-ppRbS780, ppRbS795, ppRbS807/811-in developmentally staged mouse molar and incisor teeth. Our results reveal distinct developmental expression patterns for individual ppRb isoforms in dental epithelial and dental mesenchymal cell differentiation, suggesting discrete functions in tooth development.

Dental pulp, covered with dental hard tissue, is a promising source of mesenchymal stem cells and osteoprogenitor cells for regenerative medicine. Our previous studies showed that 73% of dental pulp cells isolated from magnetically cryopreserved teeth where their viability, morphology, and expression of stem cell surface markers were similar to the cells isolated from fresh teeth, suggesting that magnetic cryopreservation is an applicable method for intact tooth as well as dental pulp tissue banking. However, the cryoprotectant, concentration, contact surface, and equilibration time for magnetic cryopreservation of dental pulp require optimization. In addition, the integrity and viability of post-thawed dental pulp with and without dental hard tissue covering after magnetic cryopreservation were investigated. Lower concentration of the cryoprotectant (5% dimethyl sulfoxide [DMSO]) and shorter preequilibration time are required for magnetic cryopreservation compared with the conventional cryopreservation method. The structure of at least 33% of post-thawed pulp with dental hard tissue from the open end remained intact where >80% of cells were viable. The addition of the cryoprotectant additive trehalose did not replace or improve DMSO's efficacy for magnetic cryopreservation of dental pulp or intact tooth. Tooth banking for transplantation provides an alternative treatment to replace missing teeth. The optimized cryoprotectant conditions for dental pulp tissue during magnetic cryopreservation should lead to more satisfactory outcomes in clinical applications such as autotransplantation and the isolation and expansion of dental pulp stem cells for tissue repair.

The localization of the planar cell polarity proteins Vang12, frizzled-3, Vang11, and Celsr1 in the rat incisors was examined using immunocytochemistry. The results showed that Vang12 was localized at two regions of the Tomes' processes of inner enamel-secretory ameloblasts in rat incisors: a proximal and a distal region. In contrast, frizzled-3 was localized at adherens junctions of the proximal and distal areas of inner enamel- and outer enamel-secretory ameloblasts, where N-cadherin and β-catenin were localized. frizzled-3 was also localized in differentiating inner enamel epithelial cells. Vang11 was localized sparsely in differentiating preameloblasts and extensively at the cell boundary of stratum intermedium. Celsr1 was not localized in ameloblasts but localized in odontoblasts extensively. These results suggest the involvement of planar cell polarity proteins in odontogenesis.

Differentiated odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. Preodontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated odontoblasts. In conclusion, nestin is valuable as a differentiation marker for odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.

Odontogenic ameloblast-associated (ODAM) and amelotin (AMTN) are secreted by maturation stage ameloblasts and accumulate at the interface with enamel where an atypical basal lamina (BL) is present. This study aimed at determining and quantifying the ultrastructural distribution of ODAM and AMTN at the cell-tooth interface. Ultrathin sections of enamel organs from the early to mid- and late maturation stage of amelogenesis were processed for immunogold labeling with antibodies against ODAM, AMTN or with the lectins wheat germ agglutinin, Helix pomatia agglutinin (HPA) and Ricinus communis I agglutinin. Immunolabeling showed that both ODAM and AMTN localized to the BL. Quantitative analyses indicated that at the beginning of maturation there is a concentration of ODAM on the cell side of the BL while AMTN appears more concentrated on the enamel side. In the late maturation stage, such differential distribution is no longer apparent. All three lectins are bound to the BL. Competitive incubation with native lectins did not affect the binding efficiency of ODAM; however, AMTN binding was significantly reduced after incubation with HPA. In conclusion, ODAM and AMTN are bona fide components of the BL associated with maturation stage ameloblasts and they organize into different subdomains during the early maturation stage. The data also suggest that the BL is a dynamic structure that rearranges its organization as enamel maturation advances. Finally, the abrogation of AMTN antibody labeling by HPA supports the presence of O-linked sugars in the molecule and/or its close association with other O-glycosylated molecules.

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization., (Copyright © 2011 S. Karger AG, Basel.)

MT1-MMP (membrane type matrix metalloproteinase-1) has been considered an important membrane-type matrix metalloproteinase involved in the remodeling process in tissue and organ development, including the processes of the tooth and root growth and dental eruption. Therefore, the aims of this study were to evaluate MT1-MMP expression in the odontogenic region, as well as the eruption rate and morphology of the lower-left rat incisor, where the eruption process was interrupted for 14 days by a steel wire attached from the center of the incisor labial face and braced to the first molar. In the interrupted eruption group, the eruption rate was significantly reduced, producing drastic morphological alterations in the tooth germ and socket area. The MT1-MMP expression was widespread in the dental follicle, in both groups studied (normal and interrupted eruption groups); however a significant decrease in immunostaining was observed in the interrupted eruption group. Results indicate that MT1-MMP may have an important role in the process of dental eruption.

Objective: Our goal was to evaluate the expression patterns for voltage gated sodium channels in odontoblasts of developing and mature rat teeth., Design: We analysed immunoreactivity (IR) of the alpha subunit for all nine voltage gated sodium channels (Nav1.1-1.9) in teeth of immature (4 weeks), young adult (7 weeks), fully mature adult (3 months), and old rats (6-12 months). We were interested in developmental changes, crown/root differences, tetrodotoxin sensitivity or resistance, co-localization with nerve regions, occurrence in periodontium, and coincidence with other expression patterns by odontoblasts such as for transient receptor potential A1 (TRPA1)., Results: We found that Nav1.1-1.9-IR each had unique odontoblast patterns in mature molars that all differed from developmental stages and from incisors. Nav1.4- and Nav1.7-IR were intense in immature odontoblasts, becoming limited to specific zones in adults. Crown odontoblasts lost Nav1.7-IR and gained Nav1.8-IR where dentine became innervated. Odontoblast staining for Nav1.1- and Nav1.5-IR increased in crown with age but decreased in roots. Nav1.9-IR was especially intense in regularly scattered odontoblasts. Two tetrodotoxin-resistant isoforms (Nav1.5, Nav1.8) had strong expression in odontoblasts near dentinal innervation zones. Nav1.6-IR was concentrated at intercusp and cervical odontoblasts in adults as was TRPA1-IR. Nav1.3-IR gradually became intense in all odontoblasts during development except where dentinal innervation was dense., Conclusions: All nine voltage-gated sodium channels could be expressed by odontoblasts, depending on intradental location and tooth maturity. Our data reveal much greater complexity and niche-specific specialization for odontoblasts than previously demonstrated, with implications for tooth sensitivity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Rodent incisors grow continuously throughout life, and epithelial progenitor cells are supplied from stem cells in the cervical loop. We report that epithelial Runx genes are involved in the maintenance of epithelial stem cells and their subsequent continuous differentiation and therefore growth of the incisors. Core binding factor β (Cbfb) acts as a binding partner for all Runx proteins, and targeted inactivation of this molecule abrogates the activity of all Runx complexes. Mice deficient in epithelial Cbfb produce short incisors and display marked underdevelopment of the cervical loop and suppressed epithelial Fgf9 expression and mesenchymal Fgf3 and Fgf10 expression in the cervical loop. In culture, FGF9 protein rescues these phenotypes. These findings indicate that epithelial Runx functions to maintain epithelial stem cells and that Fgf9 may be a target gene of Runx signaling. Cbfb mutants also lack enamel formation and display downregulated Shh mRNA expression in cells differentiating into ameloblasts. Furthermore, Fgf9 deficiency results in a proximal shift of the Shh expressing cell population and ectopic FGF9 protein suppresses Shh expression. These findings indicate that Shh as well as Fgf9 expression is maintained by Runx/Cbfb but that Fgf9 antagonizes Shh expression. The present results provide the first genetic evidence that Runx/Cbfb genes function in the maintenance of stem cells in developing incisors by activating Fgf signaling loops between the epithelium and mesenchyme. In addition, Runx genes also orchestrate continuous proliferation and differentiation by maintaining the expression of Fgf9 and Shh mRNA., (Copyright © 2011 AlphaMed Press.)

Aims: Dental tissue has been the focus of attention as an easily accessible postnatal tissue source of high-quality stem cells. Since the first report on the dental pulp stem cells (DPSCs) from permanent third molar teeth, stem cells from human exfoliated deciduous teeth (SHED) were identified as a population distinct from DPSCs. In this study, we compared DPSCs from supernumerary teeth and SHED in three age- and sex-matched patients., Patients & Methods: Dental samples were obtained from the three patients, who were 6 years old and male, with the parental consent of the three donors, and then isolated cells from dental pulp for comparative analysis between supernumerary DPSCs and SHED., Results: Colony-forming unit fibroblast levels and the proliferation rate of supernumerary DPSCs were slightly lower than that of SHED. The expression of cell surface antigens in supernumerary DPSCs and SHED were almost identical. Cells were mainly expressing endogenous mesodermal and ectodermal lineage markers. Differentiation capacity to osteogenic, adipogenic and chondrogenic lineage was similar in the SHED and supernumerary DPSCs. Migration assay revealed that both supernumerary DPSCs and SHED rapidly migrated toward wounded areas. Supernumerary DPSCs were altered in cell growth after storage for 2 years. Specially, the population doubling time of supernumerary DPSCs increased while that of SHED remained nearly unchanged., Conclusion: Both supernumerary teeth and deciduous teeth share many characteristics, such as highly proliferative clonogenic cells with a similar immunophenotype to that of mesenchymal stem cells, although they are inferior to SHED for long-term banking. Our findings suggest that supernumerary teeth are also easily accessible and noninvasive sources of postnatal stem cells with multipotency and regenerative capacity.

During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel-secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho-associated coiled-coil-containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E-cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E-cadherin, and β-catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E-cadherin-mediated cell-cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis., (Copyright © 2010 Wiley-Liss, Inc.)

Objective: To investigate the effects of fluoride on Fas expression, caspase-3 and caspase-8 activity and apoptosis in rat incisor cells., Methods: Forty male SD rats were divided into 4 groups randomly and provided with distilled water containing NaF at the doses of 0, 10, 50 and 100 mg/L respectively. Each group had 10 animals. Five animals were sacrificed at 60 and 90 days respectively. Fas expression was measured with immunohistochemistry, and colorimetric assay was used to examine caspase-3 and caspase-8 activity with enzyme-labelled meter. The apoptosis was detected by flow cytometry in mandibular incisor cells., Results: NaF at the doses of 10, 50 and 100 mg/L for 60 d and 90 d caused Fas overexpression, promoted activity of caspase-3 and caspase-8, increased apoptosis rate in mandibular incisor cells. At 60 days, the value of Fas expression was 0.1819 ± 0.0025 for control, 0.2120 ± 0.0084 for 10 mg/L NaF group, 0.2283 ± 0.0183 for 50 mg/L NaF group, 0.2818 ± 0.0233 for 100 mg/L NaF group. At 90 days, the value of Fas expression was 0.2077 ± 0.0289 for control, 0.2216 ± 0.0105 for 10 mg/L NaF group, 0.2377 ± 0.0059 for 50 mg/L NaF group, 0.2775 ± 0.0088 for 100 mg/L NaF group. Statistics analysis yielded close relationship between the dose of NaF in water and the Fas expression, and also between the dose of NaF in water and caspase-3 activities, and the relative coefficient was 0.9728 (60 d, P < 0.01) and 0.9889 (90 d, P < 0.01) for Fas expression, 0.9533 (60 d, P < 0.01) and 0.9849 (90 d, P < 0.01) for caspase-3 activity respectively. Apoptosis rate and caspase-8 activity also had close relationship with the NaF doses, and the relative coefficient was 0.9733 (90 d, P < 0.01) for apoptosis, 0.9928 (90 d, P < 0.01) for caspase-8. At the doses of 10, 50 and 100 mg/L NaF for 60 d and 90 d, obvious relationship was found between Fas expression and caspase-3 activity, and the relative coefficient was 0.9619 (60 d, P < 0.01) and 0.9912 (90 d, P < 0.01). Obvious relationship between Fas expression and apoptosis, between Fas expression and caspase-8 activity was found in groups for 90 d, and the relative coefficient was 0.9841 (P < 0.01) for apoptosis, 0.9767 (P < 0.01) for caspase-8., Conclusions: Fluoride could induce Fas overexpression and mediate caspase activation and apoptosis at the doses of 10, 50 and 100 mg/L for 60 d and 90 d in rat mandibular incisor cells.

Mouse incisors grow continuously throughout life. This growth is supported by the division of dental epithelial stem cells that reside in the cervical loop region. Little is known about the maintenance and regulatory mechanisms of dental epithelial stem cells. In the present study, we investigated how transforming growth factor β (TGF-β) signaling-mediated mesenchymal-epithelial cell interactions control dental epithelial stem cells. We designed two approaches using incisor organ culture and bromodeoxyuridine (BrdU) pulse-chase experiments to identify and evaluate stem cell functions. We show that the loss of the TGF-β type I receptor (Alk5) in the cranial neural crest-derived dental mesenchyme severely affects the proliferation of TA (transit-amplifying) cells and the maintenance of dental epithelial stem cells. Incisors of Wnt1-Cre; Alk5(fl/fl) mice lost their ability to continue to grow in vitro. The number of BrdU label-retaining cells (LRCs) was dramatically reduced in Alk5 mutant mice. Fgf10, Fgf3, and Fgf9 signals in the dental mesenchyme were downregulated in Wnt1-Cre; Alk5(fl/fl) incisors. Strikingly, the addition of exogenous fibroblast growth factor 10 (FGF10) into cultured incisors rescued dental epithelial stem cells in Wnt1-Cre; Alk5(fl/fl) mice. Therefore, we propose that Alk5 functions upstream of Fgf10 to regulate TA cell proliferation and stem cell maintenance and that this signaling mechanism is crucial for stem cell-mediated tooth regeneration.

Background and Aim: Maintaining an intact enamel surface is an essential aspect of orthodontic therapy; however, various therapeutic measures can affect this surface. The aim of our study was to evaluate roughness of the enamel surface after different conditioning and polishing procedures., Materials and Methods: 42 bovine incisors were submitted to conventional abrasion (using 37% phosphoric acid), to air abrasion, and a combination of the two. Brackets were put in place and then debonded, and the remaining adhesive removed with a carbide bur or via air abrasion. The enamel surface's roughness was assessed using a confocal laser scanning microscope (CLSM)., Results: Mean roughness (R(a)) was 33.1. There were no statistically significant differences among the six groups, or in R(q) values. Under CLSM, the roughness after polishing via air abrasion appeared even. Although it was macroscopically smoother after polishing with a carbide bur, the surface showed a wave-like pattern., Conclusion: The method of enamel conditioning revealed no significant effect on the enamel surface after debonding. Neither polishing via air abrasion nor carbide bur resulted in differences in superficial roughness. However, the carbide bur left a wave-like pattern on the enamel surface.

Introduction: Sclerostin is the product of the SOST gene. Loss-of-function mutations in the SOST gene result in a high bone mass phenotype, thus confirming that sclerostin is a negative regulator of bone mass. SOST knockdown in humans also causes oral and dental malformations. However, the relationship between sclerostin and tooth development is unclear., Methods: Using immunohistochemical techniques, we investigated sclerostin expression during fetal mouse tooth development and adult mouse tooth morphogenesis., Results: Sclerostin was expressed in the secretory odontoblasts located along the ameloblasts of fetal mouse tooth germ and adult incisor. Sclerostin expression was also observed in the fetal and adult osteocytes in the jaw bone., Conclusion: These results suggest that sclerostin, one of the important regulatory factors of differentiated odontoblast function, may usable in vital pulp therapy., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The Notch pathway regulates the renewal and fate decisions of stem cells in multiple tissues. Notch1, -2, as well as the Notch target gene Hes1 are expressed in the putative stem cells in the continuously growing mouse incisors, but so far there has not been any evidence for a function of the Notch pathway in the regulation of the incisor stem cells. We have analysed the effects of the Notch pathway inhibitor DAPT on the maintenance, proliferation, and differentiation of the epithelial stem cells in explant cultures of the mouse incisor. The proximal part of the incisor containing the cervical loop stem cell niche was dissected from newborn mice and cultured for 2-6 days in vitro. DAPT inhibited the expression of Notch target gene Hes1 in the cervical loop indicating that Notch signalling was inhibited in the putative stem cells. The most striking effect of DAPT was a significant reduction in the size of the cervical loop. DAPT caused a marked but partially reversible decrease in cell proliferation, as well as massive apoptosis in the epithelial stem cell niche. Interestingly, restricted apoptosis was detected within the Notch expressing putative stem cells also in the control cultures as well as in incisors in vivo, suggesting that apoptosis may be a mechanism regulating the size of the epithelial stem cell pool in the incisor. The differentiation of the epithelial cells into enamel-forming ameloblasts was not affected by DAPT but the number of preameloblasts was progressively decreased during culture period reflecting the depletion of stem and progenitor cells. Our results indicate that Notch signalling is required for epithelial stem cell survival and enamel formation in the continuously growing mouse incisor., (Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Rodent incisors regenerate throughout the lifetime of the animal owing to the presence of epithelial and mesenchymal stem cells in the proximal region of the tooth. Enamel, the hardest component of the tooth, is continuously deposited by stem cell-derived ameloblasts exclusively on the labial, or outer, surface of the tooth. The epithelial stem cells that are the ameloblast progenitors reside in structures called cervical loops at the base of the incisors. Previous studies have suggested that FGF10, acting mainly through fibroblast growth factor receptor 2b (FGFR2b), is crucial for development of the epithelial stem cell population in mouse incisors. To explore the role of FGFR2b signaling during development and adult life, we used an rtTA transactivator/tetracycline promoter approach that allows inducible and reversible attenuation of FGFR2b signaling. Downregulation of FGFR2b signaling during embryonic stages led to abnormal development of the labial cervical loop and of the inner enamel epithelial layer. In addition, postnatal attenuation of signaling resulted in impaired incisor growth, characterized by failure of enamel formation and degradation of the incisors. At a cellular level, these changes were accompanied by decreased proliferation of the transit-amplifying cells that are progenitors of the ameloblasts. Upon release of the signaling blockade, the incisors resumed growth and reformed an enamel layer, demonstrating that survival of the stem cells was not compromised by transient postnatal attenuation of FGFR2b signaling. Taken together, our results demonstrate that FGFR2b signaling regulates both the establishment of the incisor stem cell niches in the embryo and the regenerative capacity of incisors in the adult.

In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells.

The continuous growth of rodent incisors requires the presence of stem cells capable of generating ameloblasts and odontoblasts. While epithelial stem cells giving rise to ameloblasts have been well-characterized, cells giving rise to the odontoblasts in incisors have not been fully characterized. The goal of this study was to gain insight into the potential population in dental pulps of unerupted and erupted incisors that give rise to odontoblasts. We show that pulps from unerupted incisors contain a significant mesenchymal-stem-cell (MSC)-like population (cells expressing CD90+/CD45-, CD117+/CD45-, Sca-1+/CD45-) and few CD45+ cells. Our in vitro studies showed that these cells displayed extensive osteo-dentinogenic potential, but were unable to differentiate into chondrocytes and adipocytes. Dental pulps from erupted incisors displayed increased percentages of CD45+ and decreased percentages of cells expressing markers of an MSC-like population. Despite these differences, pulps from erupted incisors also displayed extensive osteo-dentinogenic potential and inability to differentiate into chondrocytes and adipocytes. These results provide evidence that continuous generation of odontoblasts and dentin on the labial and lingual sides of unerupted and erupted incisors is supported by a progenitor population and not multipotent MSCs in the dental pulp.

HCl-demineralized murine lower incisors were implanted intramuscularly into syngeneic BALB/c mice to induce heterotopic osteogenesis. Implants were exposed at the early, preosteogenic stage (4), or at the later, osteogenic stage (12) to the Moloney sarcoma virus (MSV), which within 3-4 days results in a sarcoma. The yield of bone induction was determined by weight of dry bone mass following NaOH hydrolysis of soft tissues. To verify the effect of this sarcoma on orthotopic local femoral bone, the dry mass of the tumor-exposed femora was measured and compared with the weight of MSV-unexposed contralateral controls. MSV-sarcoma or cells involved with their spontaneous rejection have a stimulatory effect on the periosteal membrane of the tumor-adjacent femoral bones, increasing their dry mass on average by 18%. No stimulatory effect on heterotopic bone induction was observed when the MSV sarcoma grew during the early, preosteogenic stage (4 onward), but when the tooth matrix had been exposed to such tumor at the already bone-forming stage, (12 onward), the yield of bone induction was enhanced. Thus, it is postulated that lesions induced by MSV during the early, preosteogenic stage inhibit recruitment of osteoprogenitor cells or degrade Bone Morphogenetic Proteins (BMPs) released by matrix resorbing inflammatory cells, whereas when acting on already existing bone they have a stimulatory effect.

beta-Catenin signaling is required for embryonic tooth morphogenesis and promotes continuous tooth development when activated in embryos. To determine whether activation of this pathway in the adult oral cavity could promote tooth development, we induced mutation of epithelial beta-catenin to a stabilized form in adult mice. This caused increased proliferation of the incisor tooth cervical loop, outpouching of incisor epithelium, abnormal morphology of the epithelial-mesenchymal junction, and enhanced expression of genes associated with embryonic tooth development. Ectopic dental-like structures were formed from the incisor region following implantation into immunodeficient mice. Thus, forced activation of beta-catenin signaling can initiate an embryonic-like program of tooth development in adult rodent incisor teeth.

Tight junctions (TJs) function primarily as a barrier against paracellular transport between epithelial cells and are composed mainly of occludin (OLD) and claudins (CLDs). The CLD family consists of 24 members that show tissue- or cell-specific expression. Ameloblasts, which originate from the oral epithelium, form enamel, and enamel proteins and minerals are transported across the ameloblastic layer during amelogenesis. We immunohistochemically examined the distribution patterns of TJs in ameloblasts by observing the expression patterns of OLD and CLDs (CLD-1 to CLD-10). Secretory ameloblasts contained OLD and CLD-1, -8, and -9 at the distal end of the cell. In mature ameloblasts, OLD and CLD-1, -6, -7, -8, -9, and -10 were present mainly at both the distal and proximal ends of the cell, regardless of whether the ameloblasts were ruffle-ended or smooth-ended. Mature ameloblasts in which only the proximal ends were stained for OLD and CLDs were also found. These results indicate that the expression patterns of CLDs and the distribution patterns of TJs change drastically between the secretory and mature ameloblast stages, suggesting that these patterns reflect the different functions of these cells, specifically in the transport of proteins and ions for enamel formation.

A multi-scale strategy is presented for simulating organogenesis that uses a single cell response function to define the behavior of individual cells in an organ-scale simulation of a large cell population. The response function summarizes detailed information about the behavior of individual cells in a sufficiently economical way that the organ-scale model can be commensurate with the entire organ. The first application demonstrates the effects of strain stimulus on the migration of ameloblasts during enamel formation. Ameloblasts are an attractive study case because mineralization preserves a complete record of their migratory paths. The response function in this case specifies the motions of cells responding to strain stimuli that propagate through the population. The strain stimuli are related to the curvature of the surface from which the ameloblasts migrate (the dentin-enamel junction or DEJ). A single unknown rate parameter is calibrated by an independent datum from the human tooth. With no remaining adjustable parameters, the theory correctly predicts aspects of the fracture-resistant, wavy microstructure of enamel in the human molar, including wavelength variations and the rate of wave amplitude damping. At a critical value of curvature of the DEJ, a transition in the ordering of cells occurs, from invariant order over the whole population to self-assembly of the population into groups or gangs. The prediction of an ordering transition and the predicted critical curvature are consistent with gnarled enamel in the cusps of the human molar. The calibration of the model using human data also predicts waves in the mouse incisor and an ordering transition at the chimpanzee cingulum. Widespread compressive strain is predicted late in the migration for both the human molar and mouse incisor, providing a possible signal for the termination of amelogenesis.

The Wnt pathway is crucial for tooth development as shown by dental defects caused by impaired Wnt signaling in mouse and human. We investigated Wnt signaling in continuously growing mouse incisors focusing on epithelial stem cells. Ten Wnt ligands were expressed both in the dental epithelium and mesenchyme, and were associated mainly with odontoblast and ameloblast differentiation. Wnt/beta-catenin activity was detected in mesenchyme in BATgal and TOPgal reporter mice while Axin2, also a reporter of Wnt/beta-catenin signaling, was expressed additionally in the epithelium. Axin2 was, however, excluded from the epithelial stem cells in the cervical loop. Interestingly, these cells expressed specifically Lgr5, a Wnt target gene and stem cell marker in the intestine, suggesting that Lgr5 is a marker of incisor stem cells but is not regulated by Wnt signaling in the incisor. We conclude that epithelial stem cells in the mouse incisors are not regulated directly by Wnt/beta-catenin signaling., ((c) 2009 Wiley-Liss, Inc.)

It is well-known that limb muscle reflexes are modulated during human movements. However, little is known about the existence of equivalent masticatory muscle reflex modulation. We hypothesized that masticatory reflexes would be modulated during chewing so that smooth masticatory movements occur. To examine this hypothesis, we studied the modulation of inhibitory reflexes evoked by periodontal mechanoreceptor activation and of excitatory reflexes evoked by muscle spindle activation during simulated mastication. In 28 participants, 1- and 2-N mechanical taps were delivered to the incisor. Reflex responses to these taps were examined in the average masseteric electromyogram. To differentiate between periodontal mechanoreceptor- and muscle-spindle-mediated reflex components, we performed experiments prior to, and in the presence of, periodontal anesthesia. Both periodontal mechanoreceptor and muscle spindle reflexes were reduced during simulated masticatory movements.

Unlabelled: The odontoblast is the secretory cell responsible for primary, secondary and tertiary reactionary dentinogenesis. We provide evidence that the changes in secretory activity of odontoblasts reflect differential transcriptional control and that common regulatory processes may exist between dentine and bone., Introduction: Based on the hypothesis that differential dentine secretion (primary and secondary dentinogenesis) is associated with changes in the transcriptional control within the cell, we have investigated the transcriptome of odontoblasts at young and mature stages and subsequently used this information to identify key regulatory intracellular pathways involved in this process., Materials and Methods: We used microarray analysis to compare the transcriptome of early stage (primary dentinogenesis) and late stage (secondary dentinogenesis) odontoblasts from 30 month old bovine teeth. Secondarily, we used post-array sqRT-PCR to confirm the differential expression of 23 genes in both populations of odontoblasts. Finally, immunohistochemistry was performed on bovine and murine tissues with antibodies to DMP1 and anti-phospho p38 proteins., Results: DMP-1 and osteocalcin gene expression were up-regulated in the mature odontoblasts, whereas collagen I, DSPP, TGF-beta1 and TGF-beta1R gene expression were down-regulated. Microarray analysis highlighted 574 differentially regulated genes (fold change>2 - p<0.05). This study supports further existing similarities between pulp cells and bone cells. Using post-array Sq-RT-PCR we characterized transcript levels of genes involved in the p38 MAP kinase pathway (PTPRR, NTRKK2, MAPK13, MAP2K6, MKK3). Differential p38 gene activation was confirmed by immunohistochemistry for p38 protein in murine teeth. Finally, immunohistochemistry for DMP1 indicated that odontoblasts involved in primary and secondary dentinogenesis may coexist in the same tooth., Conclusion: As established in bone cells, the transcriptome of the odontoblast was shown here to evolve with their stage and functional maturity. Identification of the involved signalling pathways, as highlighted for p38, will enable the deciphering of physiology and pathology of mineralised tissue formation.

Considering tooth crown engineering, three main parameters have to be taken into account: (1) the relationship between crown morphology and tooth functionality, (2) the growth of the organ, which is hardly compatible with the use of preformed scaffolds, and (3) the need for easily available nondental competent cell sources. In vitro reassociation experiments using either dental tissues or bone marrow-derived cells (BMDC) have been designed to get information about the mechanisms to be preserved in order to allow crown engineering. As the primary enamel knot (PEK) is involved in signaling crown morphogenesis, the formation and fate of this structure was investigated (1) in heterotopic reassociations between embryonic day 14 (ED14) incisor and molar enamel organs and mesenchymes, and (2) in reassociations between ED14 molar enamel organs and BMDC. A PEK formed in cultured heterotopic dental tissue reassociations. The mesenchyme controls the fate of the EK cells, incisor or molar-specific using apoptosis as criterion, and functionality to drive single/multiple cusps tooth development. Although previous investigations showed that they might differentiate as odontoblast- or ameloblast-like cells, BMDC reassociated to an enamel organ could not support the development of multicusp teeth. These cells apparently could neither maintain nor stimulate the formation of a PEK., ((c) 2009 Wiley-Liss, Inc.)

The enamel-related periodontium (ERP) in rat incisors is related to bone resorption. In these teeth the face of the socket related to the enamel is continuously removed at the inner side and newly formed at the outer side. CSF-1, RANKL and OPG are regulatory molecules essential for osteoclastogenesis. To verify the effects of impeded eruption on bone remodeling, the tooth eruption was prevented by immobilization of lower rat incisor and CSF-1, RANKL and OPG distribution in the ERP was analyzed after 18 days of immobilization and in normal eruption. The region of the alveolar crest of the rat incisor was used. Immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) were performed. The immunostaining of the dental follicle was quantified using Leica QWin software. Positive-TRAP osteoclasts were counted, and both groups were compared. In the normal incisor, the number of osteoclasts was significantly greater than in the immobilized tooth. In the dental follicle, there was no significant difference in the immunostaining intensity for CSF-1 and OPG between the groups (p > 0.05), but for RANKL the immobilized incisor group showed immunostaining intensity smaller than the normal incisor group (p < 0.01). These findings suggest that changes in the ERP, in the immobilized incisor, modify the RANKL/OPG ratio, in the presence of CSF-1, altering the metabolism of cells that participate in the bone remodeling.