Tooth Tissue Engineering: Optimal Dental Stem Cell Harvest Based on Tooth Development

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.

MMP1 and MMP20 contribute to tooth agenesis in humans

Objective: Variations in genes that are critical for tooth formation may contribute to the tooth agenesis. MMPs are potential candidate genes for dental alterations based on the roles they play during embryogenesis. The aim of this study was to investigate the possible association between MMP1, MMP3, and MMP20 and tooth agenesis. Methods: One hundred sixty-seven nuclear families from two different populations were analysed, 116 from Brazil and 51 from Turkey. Probands had at least one congenitally missing tooth. DNA samples were obtained from blood or saliva samples and genotyping was performed using TagMan chemistry. In addition, Mmp20 was selected for quantitative real-time polymerase chain reaction analysis with SYBR Green I Dye in mouse tooth development. Results: Associations between tooth agenesis and MMP1 (p = 0.007), and MMP20 (p = 0.03) were found in Brazilian families. In the total dataset, MMP20 continued to be associated with tooth agenesis (p = 0.01). Mmp20 was not expressed during the initial stages of tooth development. Conclusion: Our findings provide evidence that MMP1 and MMP20 play a role in human tooth agenesis. (C) 2010 Elsevier Ltd. All rights reserved.

Edar/Eda interactions regulate enamel knot formation in tooth morphogenesis.

tabby and downless mutant mice have apparently identical defects in teeth, hair and sweat glands. Recently, genes responsible for these spontaneous mutations have been identified. downless (Dl) encodes Edar, a novel member of the tumour necrosis factor (TNF) receptor family, containing the characteristic extracellular cysteine rich fold, a single transmembrane region and a death homology domain close to the C terminus. tabby (Ta) encodes ectodysplasin-A (Eda) a type II membrane protein of the TNF ligand family containing an internal collagen-like domain. As predicted by the similarity in adult mutant phenotype and the structure of the proteins, we demonstrate that Eda and Edar specifically interact in vitro. We have compared the expression pattern of Dl and Ta in mouse development, taking the tooth as our model system, and find that they are not expressed in adjacent cells as would have been expected. Teeth develop by a well recorded series of epithelial-mesenchymal interactions, similar to those in hair follicle and sweat gland development, the structures found to be defective in tabby and downless mice. We have analysed the downless mutant teeth in detail, and have traced the defect in cusp morphology back to initial defects in the structure of the tooth enamel knot at E13. Significantly, the defect is distinct from that of the tabby mutant. In the tabby mutant, there is a recognisable but small enamel knot, whereas in the downless mutant the knot is absent, but enamel knot cells are organised into a different shape, the enamel rope, showing altered expression of signalling factors (Shh, Fgf4, Bmp4 and Wnt10b). By adding a soluble form of Edar to tooth germs, we were able to mimic the tabby enamel knot phenotype, demonstrating the involvement of endogenous Eda in tooth development. We could not, however, reproduce the downless phenotype, suggesting the existence of yet another ligand or receptor, or of ligand-independent activation mechanisms for Edar. Changes in the structure of the enamel knot signalling centre in downless tooth germs provide functional data directly linking the enamel knot with tooth cusp morphogenesis. We also show that the Lef1 pathway, thought to be involved in these mutants, functions independently in a parallel pathway.

Exclusion of known gene for enamel development in two Brazilian families with amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a genetically heterogeneous group of diseases that result in defective development of tooth enamel. Mutations in several enamel proteins and proteinases have been associated with AI. The object of this study was to evaluate evidence of etiology for the six major candidate gene loci in two Brazilian families with AI. Genomic DMA was obtained from family members and all exons and exon-intron boundaries of the ENAM, AMBN, AMELX, MMP20, KLK4 and Amelotin gene were amplified and sequenced. Each family was also evaluated for linkage to chromosome regions known to contain genes important in enamel development. The present study indicates that the AI in these two families is not caused by any of the known loci for AI or any of the major candidate genes proposed in the literature. These findings indicate extensive genetic heterogeneity for non-syndromic AI.

Novel molecular insights into human tooth agenesis

The development of dentition is a fascinating process that involves a complex series of epithelial-mesenchymel signaling interactions. That such a precise process frequently goes awry is not surprising. Indeed, tooth agenesis is one of the most commonly inherited disorders in humans that affects up to twenty percent of the population and imposes significant functional, emotional and financial burdens on patients. Mutations in the paired box domain containing transcription factor PAX9 result in autosomal dominant tooth agenesis that primarily involves posterior dentition. Despite these advances, little is known about how PAX9 mediates key signaling actions in tooth development and how aberrations in PAX9 functions lead to tooth agenesis. As an initial step towards providing evidence for the pathogenic role of mutant PAX9 proteins, I performed a series of molecular genetic analyses aimed at resolving the structural and functional defects produced by a number of PAX9 mutations causing non-syndromic posterior tooth agenesis. It is likely that the pathogenic mechanism underlying tooth agenesis for the first two mutations studied (219InsG and IIe87Phe) is haploinsufficiency. For the six paired domain missense mutations studied, the lack of functional defects observed for three of the mutant proteins suggests that these mutations altered PAX9 function through alternate mechanisms. Next, I explored further the nature of the partnership between Pax9 and the Msx1 homeoprotein and their role in the expression of a downstream effector molecule, Bmp4. When viewed in the context of events occurring in dental mesenchyme, the results of these studies indicate that the Pax9-Msx1 protein interaction involves the localized up-regulation of Bmp4 activity that is mediated by synergistic interactions between the two transcription factors. Importantly, these assays corroborate in vivo data from mouse genetic studies and support reports of Pax9-dependent expression of Bmp4 in dental mesenchyme. Taken together, these results suggest that PAX9 mutations cause an early developmental defect due to an inability to maintain the inductive potential of dental mesenchyme through involvement in a pathway involving Msx1 and Bmp4. ^

Incremental lines of von Ebner in dinosaurs and the assessment of tooth replacement rates using growth line counts

Dinosaur dentine exhibits growth lines that are tens of micrometers in width. These laminations are homologous to incremental lines of von Ebner found in extant mammal and crocodilian teeth (i.e., those of amniotes). The lines likely reflect daily dentine formation, and they were used to infer tooth development and replacement rates. In general, dinosaur tooth formation rates negatively correlated with tooth size. Theropod tooth replacement rates negatively correlated with tooth size, which was due to limitations in the dentine formation rates of their odontoblasts. Derived ceratopsian and hadrosaurian dinosaurs retained relatively rapid tooth replacement rates through ontogeny. The evolution of dental batteries in hadrosaurs and ceratopsians can be explained by dentine formation constraints and rapid tooth wear. In combination with counts of shed dinosaur teeth, tooth replacement rate data can be used to assess population demographics of Mesozoic ecosystems. Finally, it is of historic importance to note that Richard Owen appears to have been the first to observe incremental lines of von Ebner in dinosaurs more than 150 years ago.

Tooth Viability Following Distraction Osteogenesis in Patients with Maxillary Hypoplasia

Thesis (Master's)--University of Washington, 2016-06

Medidas lineares dos estágios de desenvolvimento da dentição permanente humana usando Tomografia Computadorizada de Feixe Cônico: um estudo preliminar

OBJETIVO: determinar as medidas lineares dos estágios de desenvolvimento da dentição permanente humana, usando tomografia computadorizada de feixe cônico (TCFC). MÉTODOS: este estudo foi desenvolvido a partir de bancos de dados de clínicas radiológicas privadas, envolvendo 18 pacientes (13 do sexo masculino, 5 do sexo feminino, com idades variando entre 3 e 20 anos). As imagens das TCFC foram obtidas por meio do sistema i-CAT e medidas com uma função específica do programa desse mesmo sistema. Duzentos e trinta e oito dentes foram analisados, em diferentes estágios de desenvolvimento, nos planos coronal e sagital. O método foi baseado na delimitação e mensuração das distâncias entre pontos anatômicos correspondentes ao desenvolvimento das coroas e raízes dentárias. A partir dos valores obtidos, pôde-se desenvolver um modelo quantitativo para se avaliar os estágios inicial e final de desenvolvimento para todos os grupos dentários. RESULTADOS E CONCLUSÕES: as medidas obtidas dos diferentes grupos dentários estão de acordo com as estimativas das investigações publicadas previamente. As imagens por TCFC dos diferentes estágios de desenvolvimento podem contribuir no diagnóstico, planejamento e resultado dos tratamentos em diversas especialidades odontológicas. As dimensões das coroas e das raízes dentárias podem ter importantes aplicações clínicas e em pesquisas, constituindo uma técnica não invasiva que contribui com estudos in vivo. Entretanto, mais estudos são recomendados a fim de minimizar possíveis variáveis metodológicas.

Genetic Variation among Major Human Geographic Groups Supports a Peculiar Evolutionary Trend in PAX9

A total of 172 persons from nine South Amerindian, three African and one Eskimo populations were studied in relation to the Paired box gene 9 (PAX9) exon 3 (138 base pairs) as well as its 5' and 3' flanking intronic segments (232 bp and 220 bp, respectively) and integrated with the information available for the same genetic region from individuals of different geographical origins. Nine mutations were scored in exon 3 and six in its flanking regions; four of them are new South American tribe-specific singletons. Exon3 nucleotide diversity is several orders of magnitude higher than its intronic regions. Additionally, a set of variants in the PAX9 and 101 other genes related with dentition can define at least some dental morphological differences between Sub-Saharan Africans and non-Africans, probably associated with adaptations after the modern human exodus from Africa. Exon 3 of PAX9 could be a good molecular example of how evolvability works.

Dual origin of mesenchymal stem cells contributing to organ growth and repair

In many adult tissues, mesenchymal stem cells (MSCs) are closely associated with perivascular niches and coexpress many markers in common with pericytes. The ability of pericytes to act as MSCs, however, remains controversial. By using genetic lineage tracing, we show that some pericytes differentiate into specialized tooth mesenchyme-derived cells-odontoblasts-during tooth growth and in response to damage in vivo. As the pericyte-derived mesenchymal cell contribution to odontoblast differentiation does not account for all cell differentiation, we identify an additional source of cells with MSC-like properties that are stimulated to migrate toward areas of tissue damage and differentiate into odontoblasts. Thus, although pericytes are capable of acting as a source of MSCs and differentiating into cells of mesenchymal origin, they do so alongside other MSCs of a nonpericyte origin. This study identifies a dual origin of MSCs in a single tissue and suggests that the pericyte contribution to MSC-derived mesenchymal cells in any given tissue is variable and possibly dependent on the extent of the vascularity.

Expression of proteins in the extracellular matrix of pulp tissue in human primary teeth during physiologic root resorption

Objectives: To analyze the expression of tenascin, fibronectin, collagens I and III, osteonectin, and bone morphogenetic protein 4 (BMP4) in the extracellular matrix of pulp tissue in primary teeth during physiologic root resorption. Method and Materials: Eighteen teeth were decalcified and equally distributed into 3 groups (group I, teeth with two-thirds root length; group II, teeth with one-third root length; and group III, teeth lacking the root). Results: Immunohistochemical analysis showed that all the proteins were expressed. Tenascin, collagen I, and osteonectin showed strong and broad reactivity in group I, with weaker and rare reactivity in groups II and III. The expression of fibronectin, collagen III, and BMP4 did not vary with root resorption phase. Conclusion: The expression of tenascin, collagen I, and osteonectin was reduced in the extracellular matrix and odontoblasts during root resorption. This fact may be related to the decreasing pulp response to damage and treatment during the progression of root resorption. (Quintessence Int 2009; 40: 553-558)

Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: Immunolocalization according to the odontogenesis phases

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunobistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3 were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

Nuclear factor I-C regulates TGF-{beta}-dependent hair follicle cycling.

Skin appendages such as teeth and hair share several common signaling pathways. The nuclear factor I C (NFI-C) transcription factor has been implicated in tooth development, but a potential role in hair growth had not been assessed. In this study we found that NFI-C regulates the onset of the hair growth cycle. NFI-C(-/-) mice were delayed in the transition from the telogen to anagen phase of the hair follicle cycle after either experimental depilation or spontaneous hair loss. Lack of NFI-C resulted in delayed induction of the sonic hedgehog, Wnt5a, and Lef1 gene expression, which are key regulators of the hair follicle growth initiation. NFI-C(-/-) mice also showed elevated levels of transforming growth factor β1 (TGF-β1), an inhibitor of keratinocyte proliferation, and of the cell cycle inhibitor p21 at telogen. Reduced expression of Ki67, a marker of cell proliferation, was noted at the onset of anagen, indicating impaired activation of the hair progenitor cells. These findings implicate NFI-C in the repression of TGF-β1 signaling during telogen stage, resulting in the delay of progenitor cell proliferation and hair follicle regeneration in NFI-C-deficient mice. Taken together with prior observations, these findings also designate NFI-C as a regulator of adult progenitor cell proliferation and of postnatal tissue growth or regeneration.

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.

Dens invaginatus type III: report of a case and 10-year radiographic follow-up

Aim the purpose of this article is to report the 10-year follow-up of a right mandibular central incisor with 'dens invaginatus' that was root filled.Summary 'Dens invaginatus ' is a rare malformation of teeth, probably resulting from an infolding of the dental papilla during tooth development. It has alternatively been called 'dens in dente' and 'dilated composite odontome'. Radiographic examination may clearly demonstrate this feature, although no signs may be recognized clinically. If no entrance to the invagination can be detected and there are no signs of pulp pathosis, then no treatment is required other than fissure sealing of the invagination. In deep invaginations, it is likely that root-canal treatment may be required. Occasionally, when the tooth has an immature root, apexification is necessary. Root-canal treatment of a right mandibular central incisor with 'dens invaginatus ' is described along with 10-year follow-up.Key learning pointsBoth clinical and radiographic examinations are necessary to determine morphological features of teeth before root-canal treatment.Sensibility testing to determine the pulp condition is critical prior to treatment.