Stem cells for tooth engineering.

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.

Culture medium modulates the behaviour of human dental pulp-derived cells: technical note.

In vitro approaches have extensively been developed to study reparative dentinogenesis. While dental pulp is a source of unidentified progenitors able to differentiate into odontoblast-like cells, we investigated the effect of two media; MEM (1.8 mM Ca and 1 mM Pi) and RPMI 1640 (0.8 mM Ca and 5 mM Pi) on the behaviour of human dental pulp cells. Our data indicate that MEM significantly increased cell proliferation and markedly enhanced the proportion of alpha-smooth muscle actin positive cells, which represent a putative source of progenitors able to give rise to odontoblast-like cells. In addition, MEM strongly stimulated alkaline phosphatase activity and was found to induce expression of transcripts encoding dentin sialophosphoprotein, an odontoblastic marker, without affecting that of parathyroid hormone/parathyroid hormone related protein-receptor and osteonectin. In conclusion, these observations demonstrate that not only proliferation but also differentiation into odontoblast-like cells was induced by rich calcium and poor phosphate medium (MEM) as compared to RPMI 1640. This study provides important data for the determination of the optimal culture conditions allowing odontoblast-like differentiation in human pulp cell culture.

Development of an odontoblast in vitro model to study dentin mineralization.

The aim of the present work was to characterize the odontoblastic proliferation, differentiation, and matrix mineralization in culture of the recently established M2H4 rat cell line. Proliferation was assessed by cell counts, differentiation by RT-PCR analysis, and mineralization by alizarin red staining, atomic absorption spectrometry, and FTIR microspectroscopy. The results showed that M2H4 cell behavior closely mimics in vivo odontoblast differentiation, with, in particular, temporally regulated expression of DMP-1 and DSPP. Moreover, the mineral phase formed by M2H4 cells was similar to that in dentin from rat incisors. Finally, because in mice, transforming growth factor (TGF)-beta1 over-expression in vivo leads to an hypomineralization similar to that observed in dentinogenesis imperfecta type II, effects of TGF-beta1 on mineralization in M2H4 cell culture were studied. Treatment with TGF-beta1 dramatically reduced mineralization, whereas positive control treatment with bone morphogenetic protein-4 enhanced it, suggesting that M2H4 cell line is a promising tool to explore the mineralization mechanisms in physiopathologic conditions.

Phosphate is a specific signal for ATDC5 chondrocyte maturation and apoptosis-associated mineralization: possible implication of apoptosis in the regulation of endochondral ossification.

UNLABELLED: 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 NaH2PO4/Na2HPO4 and CaCl2, 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-(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 pi-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.

Differential gene expression analysis in a rabbit model of osteoarthritis induced by anterior cruciate ligament (ACL) section.

Osteoarthritis (OA) is the most common of all joint diseases to affect mankind and is characterized by the degradation of articular cartilage. The low availability of normal and pathologic human cartilage and the inability to study the early stages of the disease in humans has led to the development of numerous animal models of OA. The aim of our study was to establish gene expression profiles during the progression of a rabbit model of OA induced by anterior cruciate ligament (ACL) section. Semiquantitative RT-PCR was used to follow expression of several relevant molecules (type II and X collagens, aggrecan, osteonectin, betaig-h3, BiP, TIMP-1, MMP-1, -3, -13, aggrecanase-1, -2) during development of OA in articular cartilage. In parallel, we monitored the activities of collagenase, caseinase, phospholipase A2 and glycosyltransferases (xylosyl-, galactosyl-, glucuronyl- and N-acetyl-galactosaminyl-transferase). Novel cDNA clones for rabbit type X collagen, aggrecanase-1 and -2, osteonectin and BiP were constructed to obtain species-specific primers. Ours result show that MMP-13 (collagenase-3) gene expression increased dramatically early after ACL surgery and remained high thereafter. An increase in MMP-1 (collagenase-1) and MMP-3 expression was also noted with an absence of variation for TIMP-1 expression. In addition, the global MMPs activities paralleled the MMP gene expression. These data together characterize at the molecular level the evolution of OA in this rabbit model. Furthermore, we have undertaken a search for identifying differentially expressed genes in normal and OA cartilage in this model, by differential display RT-PCR. We present here preliminary results with the determination of the best technical conditions to obtain reproducible electrophoresis patterns of differential display RT-PCR.

Matrix metalloproteinase-1, -3, -13 and aggrecanase-1 and -2 are differentially expressed in experimental osteoarthritis.

The aim of this study was to characterize the cellular phenotypes of articular cartilage and meniscus in rabbits with experimentally induced osteoarthritis (OA), by histological and molecular biological techniques. OA was induced by severing the anterior cruciate ligament of the knee and rabbits were killed 2, 4 or 9 weeks following surgery. Our histological observations show a progressive destruction of extracellular matrix in both tissues. To determine whether these morphological changes could be related to alterations in the regulation of gene expression for a subset of relevant molecules, levels of mRNA for proteinases and one inhibitor (MMP-1, -3 and -13, aggrecanase-1 and -2 and TIMP-1), matrix molecules and one chaperone (type II and X collagens, aggrecan, osteonectin, betaig-h3 and BiP) were assessed by reverse transcription-polymerase chain reaction. Our results indicate that for most markers expression profiles were similar in both tissues. In particular, matrix protein gene expression remained stable or varied little during progression of OA, suggesting a poor repair capacity of the tissues. MMP gene expression increased rapidly whereas aggrecanase gene expression remained stable. These findings suggest that differential regulation of mRNA levels of MMP-1, -3 and -13 on the one hand and aggrecanase-1 and -2 on the other, occurs during OA.