Immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to BMP2

Bone morphogenetic protein 2 (Bmp2) is essential for odontogensis and dentin mineralization. Generation of floxed Bmp2 dental mesenchymal cell lines is a valuable application for studying the effects of Bmp2 on dental mesenchymal cell differentiation and its signaling pathways during dentinogenesis. Limitation of the primary culture of dental mesenchymal cells has led to the development of cell lines that serve as good surrogate models for the study of dental mesenchymal cell differentiation into odontoblasts and mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines, which were isolated from 1st mouse mandibular molars at postnatal day 1 and immortalized with pSV40 and clonally selected. These transfected cell lines were characterized by RT‐PCR, immunohistochemistry, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iBmp2‐dp, displayed a higher proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth‐specific markers as well as demonstrated the ability to differentiate and form mineralized nodules. In addition, iBmp2‐dp cells were inducible and responded to BMP2 stimulation. Thus, we for the first time described the establishment of an immortalized mouse floxed Bmp2 dental papilla mesenchyma cell line that might be used for studying the mechanisms of dental cell differentiation and dentin mineralization mediated by Bmp2 and other growth factor signaling pathways. J. Cell. Physiol. 225: 132–139, 2010. © 2010 Wiley‐Liss, Inc.     

Odontoblast cells immortalized by telomerase produce mineralized dentin-like tissue both in vitro and in vivo

The formation of dentin provides one well accepted paradigm for studying mineralized tissue formation. For the assembly of dentin, several cellular signaling pathways cooperate to provide neural crest-derived mesenchymal cells with positional information. Further, "cross-talk" between signaling pathways from the mesenchymal derived odontoblast cells and the epithelially derived ameloblasts during development is responsible for the formation of functional odontoblasts. These intercellular signals are tightly regulated, both temporally and spatially. When isolated from the developing tooth germ, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast cell line would be a valuable reproducible tool for studying the modulatory effects involved in odontoblast differentiation as well as the molecular events involved in mineralized dentin formation. In this study an immortalized odontoblast cell line, which has the required biochemical machinery to produce mineralized tissue in vitro, has been generated. These cells were implanted into animal models to determine their in vivo effects on dentin formation. After implantation, we observed a multistep, programmed cascade of gene expression in the exogenous odontoblasts as the dentin formed de novo. Some of the genes expressed include the dentin matrix proteins 1, 2, and 3, which are extracellular matrix molecules responsible for the ultimate formation of mineralized dentin. The biological response was also examined by histology and radiography and confirmed for mineral deposition by von Kossa staining. Thus, a transformed odontoblast cell line was created with high proliferative capacity that might ultimately be used for the regeneration and repair of dentin in vivo.     

Dentin sialoprotein: biosynthesis and developmental appearance in rat tooth germs in comparison with amelogenins,osteocalcin and colagen type-I

A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with ¹⁴C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction.     

Accelerated dentinogenesis by inhibiting the mitochondrial fission factor,dynamin related protein 1

Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth.     

Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ.

The distribution of the matrix protein fibronectin was studied by indirect immunofluorescence in differentiating mouse molars from bud stage to the stage of dentin and enamel secretion, and compared to that of collagenous proteins procollagen type III and collagen type I. Fibronectin was seen in mesenchymal tissue, basement membranes, and predentin. The dental mesenchyme lost fibronectin staining when differentiating into odontoblasts. Fibronectin was not detected in mineralized dentin. Epithelial tissues were negative except for the stellate reticulum within the enamel organ. Particularly intense staining was seen at the epithelio-mesenchymal interface between the dental epithelium and mesenchyme. Fibronectin may here be involved in anchorage of the mesenchymal cells during their differentiation into odontoblasts. Procollagen type III was lost from the dental mesenchyme during odontoblast differentiation but reappeared with advancing vascularization of the dental papilla. Similarly, procollagen type III present in the dental basement membrane during the bud and cap stages disappeared from the cuspal area along with odontoblast differentiation. Weak staining was seen in predentin but not in mineralized dentin. The staining with anti-collagen type I antibodies was weak in dental mesenchyme but intense in predentin as well as in mineralized dentin.     

Changes of mitochondrial respiratory function during odontogenic differentiation of rat dental papilla cells

Dental papilla cells (DPCs) belong to precursor cells differentiating to odontoblasts and play an important role in dentin formation and reproduction. This study aimed to explore the changes and and involvement of mitochondrial respiratory function during odontogenic differentiation. Primary DPCs were obtained from first molar dental papilla of neonatal rats and cultured in odontogenic medium for 7, 14, 21 days. DPCs, which expressed mesenchymal surface markers CD29, CD44 and CD90, had the capacity for self-renewal and multipotent differentiation. Odontoblastic induction increased mineralized matrix formation in a time-dependent manner, which was accompanied by elevated alkaline phosphatase (ALP), dentin sialophosphoprotein and dentin matrix protein 1 expression at mRNA and protein levels. Notably, odontogenic medium led to an increase in adenosine-5′-triphosphate content and mitochondrial membrane potential, whereas a decrease in intercellular reactive oxygen species production and NAD⁺/NADH ratio. Furthermore, odontogenic differentiation was significantly suppressed by treatment with rotenone, an inhibitor of mitochondrial respiratory chain. These results demonstrate that enhanced mitochondrial function is crucial for odontogenic differentiation of DPCs.     

Possible involvement of maspin in tooth development

Maspin is a 42 kDa serine protease inhibitor that possesses tumor suppressive and anti-angiogenic activities. Despite of a huge amount of data concerning the expression pattern of maspin in various tissues and its relevance to the biological properties of a variety of human cancer cells, little is known on the maspin expression in skeletal and tooth tissues. Recently, we reported that maspin may play an important role in extracellular matrix formation in bone by enhancing the accumulation of latent TGF-β in the extracellular matrix. This study was performed to elucidate the possible role of maspin in tooth development. First, an immunohistochemical analysis for human tooth germs at the late bell stage showed the expression of maspin by active ameloblasts and odontoblasts that were forming enamel and dentin, respectively. During rat tooth development, maspin expression was observed for the first time in inner and outer enamel epithelial cells and dental papilla cells at early bell stage. The neutralizing anti-maspin antibody inhibited the proper dental tissue formation in organ cultures of mandibular first molars obtained from 21-day-old rat embryos. In addition, the proliferation of HAT-7 cells, a rat odontogenic epithelial cell line, and human dental papilla cells were suppressed in a dose-dependent manner with anti-maspin antibody. Moreover, RT-PCR analysis showed that the expression of mRNA for tooth-related genes including dentin matrix protein 1, dentin sialophosphoprotein and osteopontin in human dental papilla cells was inhibited when treated with anti-maspin antibody. These findings suggest that maspin expressed in ameloblasts and odontoblasts plays an important physiological role in tooth development through the regulation of matrix formation in dental tissues.     

Changes in the distribution of tenascin during tooth development

Tenascin is an extracellular matrix molecule that was earlier shown to be enriched in embryonic mesenchyme surrounding the budding epithelium in various organs including the tooth. In the present study tenascin was localized by immunohistology throughout the course of tooth development in the mouse and rat using polyclonal antibodies against chick tenascin. The results indicate that tenascin is expressed by the lineage of dental mesenchymal cells throughout tooth ontogeny. The intensity of staining with tenascin antibodies in the dental papilla mesenchyme was temporarily reduced at cap stage when the tooth grows rapidly and undergoes extensive morphogenetic changes. During the bell stage of morphogenesis, the staining intensity increased and tenascin was accumulated in the dental pulp even after completion of crown development and eruption. Tenascin was present in the dental basement membrane at the time of odontoblast differentiation. The dental papilla cells ceased to express tenascin upon differentiation into odontoblasts and tenascin was completely absent from dentin. It can be speculated that the remarkable expression of tenascin in the dental mesenchymal cells as compared to other connective tissues is associated with their capacity to differentiate into hard-tissue-forming cells.     

Cell pellets from dental papillae can reexhibit dental morphogenesis and dentinogenesis

We isolated dental papilla mesenchymal cells (DPMCs) from different rat incisor germs at the late bell stage and incubated them as cell pellets in polypropylene tubes. In vitro pellet culture of DPMCs presented several crucial characteristics of odontoblasts, as indicated by accelerated mineralization, positive immunostaining for dentin sialophosphoprotein and dentin matrix protein 1, and expression of dentin sialophosphoprotein mRNA. The allotransplantation of these pellets into renal capsules was also performed. Despite the absence of dental epithelial components, dissociated DPMCs with a complete loss of positional information rapidly underwent dentinogenesis and morphogenesis, and formed a cusp-like dentin-pulp complex containing distinctive odontoblasts, predentin, dentin, and dentinal tubules. These results imply that DPMCs at the late bell stage can reexhibit the dental morphogenesis and dentinogenesis by themselves, and epithelial-mesenchymal interactions at this stage may not be indispensable. Furthermore, different DPMC populations from the similar stage may keep the same developmental pattern.     

Fate map of the dental mesenchyme: dynamic development of the dental papilla and follicle

At the bud stage of tooth development the neural crest derived mesenchyme condenses around the dental epithelium. As the tooth germ develops and proceeds to the cap stage, the epithelial cervical loops grow and appear to wrap around the condensed mesenchyme, enclosing the cells of the forming dental papilla. We have fate mapped the dental mesenchyme, using in vitro tissue culture combined with vital cell labelling and tissue grafting, and show that the dental mesenchyme is a much more dynamic population then previously suggested. At the bud stage the mesenchymal cells adjacent to the tip of the bud form both the dental papilla and dental follicle. At the early cap stage a small population of highly proliferative mesenchymal cells in close proximity to the inner dental epithelium and primary enamel knot provide the major contribution to the dental papilla. These cells are located between the cervical loops, within a region we have called the body of the enamel organ, and proliferate in concert with the epithelium to create the dental papilla. The condensed dental mesenchymal cells that are not located between the body of the enamel organ, and therefore are at a distance from the primary enamel knot, contribute to the dental follicle, and also the apical part of the papilla, where the roots will ultimately develop. Some cells in the presumptive dental papilla at the cap stage contribute to the follicle at the bell stage, indicating that the dental papilla and dental follicle are still not defined populations at this stage. These lineage-tracing experiments highlight the difficulty of targeting the papilla and presumptive odontoblasts at early stages of tooth development. We show that at the cap stage, cells destined to form the follicle are still competent to form dental papilla specific cell types, such as odontoblasts, and produce dentin, if placed in contact with the inner dental epithelium. Cell fate of the dental mesenchyme at this stage is therefore determined by the epithelium.     

Neutral morphogenetic activity of epithelium in heterologous tissue recombinations

To assess the existence of specific and nonspecific epithelial instructions for mesenchymal cell differentiation we compared homospecific and heterospecific mouse and quail tissue recombinations. In heterospecific recombinants between trypsin-dissociated mouse molar mesenchyme and quail epithelia neither odontoblasts nor chondrocytes differentiated. Cartilage appeared if the quail epithelium was contaminated with homologous limb mesenchyme and odontoblasts differentiated if the mouse dental epithelium was contaminated with dental papilla cells.     

Neutral morphogenetic activity of epithelium in heterologous tissue recombinations

Abstract. To assess the existence of specific and nonspecific epithelial instructions for mesenchymal cell differentiation we compared homospecific and heterospecific mouse and quail tissue recombinations. In heterospecific recombinants between trypsin-dissociated mouse molar mesenchyme and quail epithelia neither odontoblasts nor chondrocytes differentiated. Cartilage appeared if the quail epithelium was contaminated with homologous limb mesenchyme and odontoblasts differentiated if the mouse dental epithelium was contaminated with dental papilla cells.     

Spatial and temporal expression of histone demethylase,Kdm2a,during murine molar development

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.     

Production of monoclonal antibodies against mouse molar papilla cells

To develop markers for the analysis of the molecular mechanisms of dental papilla cells differentiation, 10 monoclonal antibodies were produced against trypsin-isolated mouse molar dental papilla cells. These antibodies identify matrix components, cell membrane associated antigens and intracellular-constituents. Changes of the staining patterns were correlated with a typological hierarchy of dental papilla cells and with terminal differentiation of odontoblasts.     

Role of epithelial-stem cell interactions during dental cell differentiation

Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin. We also found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies.     

Gestational diabetes mellitus affects odontoblastic differentiation of dental papilla cells via Toll-like receptor 4 signaling in offspring

Gestational diabetes mellitus (GDM) is an important factor involved in the pathogenesis of organ development in the offspring. Here, we analyzed the effects of GDM on odontoblastic differentiation of dental papilla cells (DPCs) and dentin formation in offspring and investigated their underlying mechanisms. A GDM rat model was induced by intraperitoneal injection of streptozotocin and offspring were collected. The results showed that GDM significantly affected odontoblast differentiation and dentin formation in offspring tooth. GDM activated the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-ĸB) signaling pathway and inhibited SMAD1/5/9 signaling to modulate the odontoblastic differentiation of DPCs in offspring. Inhibition of TLR4 signaling by treated with TAK-242 significantly reverses the suppression of odonto-differentiation of DPCs in diabetic offspring. Taken together, these data indicate GDM activated the offspring DPCs TLR4/NF-ĸB signaling, which suppressed the SMAD1/5/9 phosphorylation and then inhibited odontoblasts differentiation and dentin formation.