Tooth development results from sequential and reciprocal interactions between the oral epithelium and the underlying neural crest-derived mesenchyme. The generation of dental structures and/or entire teeth in the laboratory depends upon the manipulation of stem cells and requires a synergy of all cellular and molecular events that finally lead to the formation of tooth-specific hard tissues, dentin and enamel. Although mesenchymal stem cells from different origins have been extensively studied in their capacity to form dentin in vitro, information is not yet available concerning the use of epithelial stem cells. The odontogenic potential resides in the oral epithelium and thus epithelial stem cells are necessary for both the initiation of tooth formation and enamel matrix production. This review focuses on the different sources of stem cells that have been used for making teeth in vitro and their relative efficiency. Embryonic, post-natal or even adult stem cells were assessed and proved to possess an enormous regenerative potential, but their application in dental practice is still problematic and limited due to various parameters that are not yet under control such as the high risk of rejection, cell behaviour, long tooth eruption period, appropriate crown morphology and suitable colour. Nevertheless, the development of biological approaches for dental reconstruction using stem cells is promising and remains one of the greatest challenges in the dental field for the years to come.

Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis-associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification.Introduction: During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra-and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered. Materials and Methods:The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH 2 PO 4 /Na 2 HPO 4 and CaCl 2 , respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase-polymerase chain reaction [RT-PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer-1 (EthD-1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl-2/Bax mRNA ratio (RT-PCR analysis). Results: We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl-2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase-3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD-fmk reduces -induced mineralization. Conclusions: These findings suggest that Pi regulates chondrocyte maturation and apoptosis-associated mineralization, highlighting a possible role for Pi in the control of skeletal development.

Regenerative dental pulp strategies require the identification of precursors able to differentiate into odontoblast-like cells that secrete reparative dentin after injury. Pericytes have the ability to give rise to osteoblasts, chondrocytes, and adipocytes, a feature that has led to the suggestion that odontoblast-like cells could derive from these perivascular cells. In order to gain new insights into this hypothesis, we investigated the effects of dexamethasone (Dex), a synthetic glucocorticoid employed to induce osteogenic differentiation in vitro, in a previously reported model of human dental pulp cultures containing pericytes as identified by their expression of smooth muscle actin (SMA) and their specific ultrastructural morphology. Our data indicated that Dex (10(-8) M) significantly inhibited cell proliferation and markedly reduced the proportion of SMA-positive cells. Conversely, Dex strongly stimulated alkaline phosphatase (ALP) activity and induced the expression of the transcript encoding the major odontoblastic marker, dentin sialophosphoprotein. Nevertheless, parathyroid hormone/parathyroid hormone-related peptide receptor, core-binding factor a1/osf 2, osteonectin, and lipoprotein lipase mRNA levels were not modified by Dex treatment. Dex also increased the proportion of cells expressing STRO-1, a marker of multipotential mesenchymal progenitor cells. These observations indicate that glucocorticoids regulate the commitment of progenitors derived from dental pulp cells to form odontoblast-like cells, while reducing the proportion of SMA-positive cells. These results provide new perspectives in deciphering the cellular and molecular mechanisms leading to reparative dentinogenesis.

The Notch signalling pathway is an evolutionarily conserved intercellular signalling mechanism that is essential for cell fate specification and proper embryonic development. We have analysed the expression, regulation and function of the jagged 2 (Jag2) gene, which encodes a ligand for the Notch family of receptors, in developing mouse teeth. Jag2 is expressed in epithelial cells that give rise to the enamel-producing ameloblasts from the earliest stages of tooth development. Tissue recombination experiments showed that its expression in epithelium is regulated by mesenchyme-derived signals. In dental explants cultured in vitro, the local application of fibroblast growth factors upregulated Jag2 expression, whereas bone morphogenetic proteins provoked the opposite effect. Mice homozygous for a deletion in the Notch-interaction domain of Jag2 presented a variety of severe dental abnormalities. In molars, the crown morphology was misshapen, with additional cusps being formed. This was due to alterations in the enamel knot, an epithelial signalling structure involved in molar crown morphogenesis, in which Bmp4 expression and apoptosis were altered. In incisors, cytodifferentiation and enamel matrix deposition were inhibited. The expression of Tbx1 in ameloblast progenitors, which is a hallmark for ameloblast differentiation and enamel formation, was dramatically reduced in Jag2(-/-) teeth. Together, these results demonstrate that Notch signalling mediated by Jag2 is indispensable for normal tooth development.