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.     

Epithelial-Directed Mesenchyme Differentiation in vitro

To assess the requirement for specific or possibly non-specific epithelial instructions for mesenchymal cell differentiation, we designed studies to evaluate and compare homotypic with heterotypic tissue recombinations across vertebrate species. These studies further tested the hypothesis that determined dental papilla mesenchyme requires epithelial-derived instructions to differentiate into functional odontoblast cells using a serumless, chemically-defined medium. Theiler stage 25 C57BL/6 or Swiss Webster cap stage mandibular first molar tooth organs or trypsin-dissociated, homotypic epithelial-mesenchymal tissue recombinants resulted in the differentiation of odontoblasts within 3 days. Epithelial differentiation into functional ameloblasts was observed within 7 days. Trypsin-dissociated and isolated mesenchyme did not differentiate into odontoblasts under these experimental conditions. Heterotypic recombinants between quail Hamburger-Hamilton stages 22–26 mandibular epithelium and Theiler stage 25 dental papilla mesenchyme routinely resulted in odontoblast differentiation within 3 days in vitro. Odontoblast differentiation and the production of dentine extracellular matrix continued throughout the 10 days in organ culture. Ultrastructural observations of the interface between quail and mouse tissues indicated the reconstitution of the basal lamina as well as the maintenance of an intact basal lamina during 10 days in vitro. Quail epithelial cells did not differentiate into ameloblasts and no enamel extracellular matrix was observed. These results show that quail mandibular epithelium can provide the required developmental instructions for odontoblast differentiation in the absence of serum or other exogenous humoral factors in a chemically-defined medium. They also suggest the importance of reciprocal epithelial-mesenchymal interactions during epidermal organogenesis.     

Immuno-electron-microscopic localization of laminin and collagen type IV in normal and denervated tooth pulp of the cat

Summary The distribution of laminin-like immunoreactivity in adult normal and denervated cat mandibular tooth pulps was studied by the use of fluorescence microscopy and pre-embedding immunogold electron microscopy. Immunoreactivity to collagen IV was also assessed in order to distinguish basement membranes. In normal pulps, light-microscope laminin-like immunoreactivity was strong along blood vessels and Schwann cell sheaths, and a faint immunoreactivity was seen also in the odontoblast layer. Electron microscopy confirmed the laminin-like immunoreactivity of endothelial and Schwann cell basement membranes at all pulpal levels. In the odontoblast layer and the predentine, nerve-like structures lacking basement membranes but possessing strong membrane laminin-like immunoreactivity were encountered. In addition, a clear-cut laminin-like immunoreactivity of plasma membranes of the somata and processes of odontoblasts was seen. Observations on denervated pulps as well as pulps in which nerve regeneration had taken place did not reveal any changes in the pattern of laminin-immunoreactivity in basement membranes or odontoblasts. Distribution of collagen IV-like immunoreactivity was very similar to laminin-like immunoreactivity in basement membranes of blood vessels and Schwann cells, and appeared unaffected by denervation. The odontoblasts and nerve-like profiles in the odontoblast layer were devoid of collagen IV-like immunoreactivity. We propose that odontoblast-associated laminin could be of significance as guidance for regenerating terminal pulpal nerve fibers to appropriate targets.     

Effects of TGF-beta 1 on interleukin profile of human dental pulp and odontoblasts

Transforming growth factor-beta 1 (TGF-beta1) is the most extensively studied growth factor in dentin-pulp complex, with pleiotropic effects on pulp response and healing. Our main objective was to analyze the expression profile of pulp tissue and odontoblasts, and the effects of TGF-beta1 on these profiles in cultured human pulp and odontoblasts with a specific interest in the anti- and pro-inflammatory cytokines. For that purpose, pulps and odontoblasts were cultured for different time periods, and microarray was performed to both cultured and native samples. Of cytokines, various interleukins (IL) were confirmed by RT-PCR, and in +/- TGF-beta1 treated pulps also by antibody array. Pro-inflammatory IL-7, -12alpha and -16 mRNAs were detected in native pulp. The expression levels of pro-inflammatory IL-1alpha, -1beta, -6 and -8 were clearly induced after TGF-beta1 treatment, while no anti-inflammatory cytokines were induced. Of all pulpal interleukins analyzed IL-6 and -8 were present at the highest levels in conditioned pulp tissue media. In native odontoblasts pro-inflammatory IL-6 and -7 mRNAs were detected, and in cultured odontoblasts pro-inflammatory IL-8 mRNA showed over 20-fold transient induction after TGF-beta1 treatment. Our results demonstrate that TGF-beta1 is a potent regulator of pro-inflammatory responses and defensive reactions in dentin-pulp complex.     

Patterns of nerve growth factor (NGF), proNGF, and p75 NGF receptor expression in the rat incisor: comparison with expression in the molar

Abstract. Nerve growth factor (NGF), a target-derived neurotrophic substance, may have broader biological functions in various types of nons-neuronal differentiating cells. The effects of NGF are dependent on initial binding of NGF to specific cell-surface receptors (p75^NGFR and p140^prototrk) on responsive cells. The continously growing rat incisor offers an excellent model demonstrating defined territories of differentiation of specific cell populations. We used immunohistochemistry to determine sites of NGF. proNGF and p75^NGFR accumulation in the rat incisor, whereas NGF mRNA expression was visualized by in situ hybridization in the developing rat molar and incisor. Strictly similar patterns of NGF mRNA, proNGF and NGF expression were observed in differentiating cells responsible for the production of the main structural matrices of the tooth. Thus, proNGF-like and NGF-like immunoreactivity, as well as the NGF mRNA signal were observed in preameloblasts and young ameloblasts of the dental epithelium and in polarizing odontoblasts of the dental mesenchyme. In contrast, the distribution of p75^NGFR was correlated with differentiation events only in dental mesenchyme: polarizing odontoblasts expressed p75^NGFR whereas the molecule was absent in functional odontoblasts. In dental epithelium, the restricted expression of p75^NGFR in ameloblast precursor cells was correlated with proliferative phenomena. The patterns of proNGF, NGF and p75^NGFR expression in epithelium and mesenchyme implicate both an autocrine and paracrine mode of action of the NGF molecule in dental tissues. The findings reported here are important for understanding NGF action in specific dental cell populations and suggest that this molecule is involved in the cascade of events that directs tooth development.     

Basement Membrane Formation in Transfilter Tooth Culture and Its Relation to Odontoblast Differentiation

The mesenchymal cells of the developing tooth differentiate into odontoblasts as a result of an epithelio-mesenchymal interaction. Odontoblast differentiation was studied in vitro by cultivating dental mesenchyme and epithelium with interposed filters. Separation of the two components by enzyme treatment resulted in removal of the basement membrane. When the epithelium was grown alone, or transfilter from killed lens capsule, the basement membrane was not restored. Transfilter cultivation with dental mesenchyme resulted in basement membrane formation, but only if the filter pores allowed penetration of cytoplasmic processes. Hence, a close association between the epithelial and the mesenchymal cells seems to be a prerequisite for the restoration of the basement membrane. Differentiation of odontoblasts took place only in explants in which a basement membrane was formed. Differentiation did not occur when contact of the mesenchymal cells with the basement membrane was prevented by small pore size filters. Further experiments demonstrating an intact basement membrane suggested that membrane contacts between the epithelial and the mesenchymal cells are not needed for odontoblast differentiation. Hence, we suggest that differentiation of odontoblasts is triggered via contact of the mesenchymal cells with the basement membrane.     

Role of dental pulp in the histogenesis of the enamel organ]

The histological study of in vitro cultured associations between dental pulps and outer dental epithelium showed that the predontoblasts initiated the histogenesis of the enamel organ and particularly the differentiation of the inner dental epithelium.     

Change of microtubular arrangement around centrioles in rat incisor odontoblasts during cell differentiation

Three stages during cell differentiation of rat incisor odontoblasts were classified, and change of microtubular arrangement around centrioles in the odontoblasts was examined with three-dimensional analyses using serial ultrathin sections. In the undifferentiated odontoblasts, microtubules were observed to radiate from the pericentriolar area, whereas, in the differentiating odontoblasts, some microtubules became poorly related to the centrioles. In the differentiated odontoblasts, arrangement of most microtubules appeared to have a poor relationship to the centrioles. Throughout the differentiation of the odontoblasts, one of the centriolar pair was ciliated, and Golgi apparatus was invariably observed near the centrioles. The present study suggests that a pericentriolar area, or a centrosome, could function as a microtubule-organizing center (MTOC) in the undifferentiated odontoblasts, but their function might be attenuated during cell differentiation.     

35S]autoradiographic study of sulfated GAG accumulation and turnover in embryonic mouse tooth germs

The accumulation of sulfated GAG in embryonic mouse molars before, during, and after terminal differentiation of odontoblasts was localized by [35S]autoradiography combined with the use of chondroitin ABC lyase. Much more sulfated GAG were accumulated in the dental papilla than in the dental epithelium. High incorporation of [35S]sulfate occurred at the epithelio-mesenchymal junction, which is the site of dental basement membrane and predentin. Before terminal differentiation of odontoblasts, the distribution of sulfated GAG was uniform at the basement membrane. After the onset of terminal differentiation of odontoblasts, much more sulfated GAG accumulated at the tip of principal cusps than at the apical (inferior) parts of cusps, and sulfated GAG were then found to be degraded more rapidly at the epithelio-mesenchymal junction than at other parts of the tooth germ. Thus regional variation in the rate of degradation of GAG exists in the tooth germs. Trypsin-isolated dental epithelia cultured in vitro synthesized a new basement membrane that could be labeled with [3H]glucosamine but not with 35SO4(-2). The epithelial-derived basal lamina contains little or no sulfatated GAG.     

Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

The effect of colchicine on protein secretion by differentiating odontoblasts and ameloblasts in the hamster tooth in vitro as shown by radioautography with 3H-proline

Summary We have examined radioautographically the protein synthetic and secretory activity of differentiating odontoblasts and ameloblasts, exposed for 9 h in vitro to various concentrations of colchicine in the presence of ³H-proline. Colchicine impairs the cytodifferentiation of the dental epithelium into ameloblasts and of the dental mesenchyme into odontoblasts; the effects depend on the dose. However, denial epithelial cells are more sensitive to the drug than dental mesenchymal cells. In stages prior to odontoblast differentiation, colchicine enhances the number of radioautographic grains over the dental epithelium without changing the grain counts over the dental basement membrane area: This suggests that in vitro the dental epithelium synthesizes and secretes proline-containing components that are not constituents of the dental basement membrane. Also, during the subsequent stages of ameloblast differentiation colchicine increases the number of radioautographic grains over the preameloblasts. The present data suggest that the primary in vitro target of colchicine is not the dental mesenchyme, but the dental epithelium. The data also indicate that differentiating ameloblasts synthesize and secrete significant amounts of proteins in vitro prior to the first deposition of enamel.     

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry detecting NOS in healthy and chronically inflamed pulp

The aim of this study was to examine the expression of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in human dental pulps and determine whether there are changes of the activity in chronically inflamed pulp tissue. Nineteen pulps with clinical diagnosis of chronic pulpitis were collected during endodontic treatment. The healthy controls were obtained from teeth extracted for orthodontic therapy. The clinical diagnosis was confirmed by histological analysis. Healthy pulps showed stratified odontoblasts in peripheral parts, while in central area there was normal connective tissue. Chronically inflamed pulps showed less expressed stratification of odontoblasts and infiltration of lymphocytes, polymorphonuclear leukocytes, plasma cells and mastocytes. NADPH-d granular reactivity was assessed semi quantitatively under the light microscope by a single observer and scored on an intensity scale from negative reaction to very strong reaction. In healthy human pulps, NADPH-d activity was strong to very strong in odontoblastic layer. Endothelial cells and Schwann cells showed strong NADPH-d reactivity, while the other parts of central area were weakly positive. Similar distribution of reactivity was expressed also in chronically inflamed pulp; moderate to strong reaction was observed in stromal area as result of positive reaction in inflammatory cells and endothelial cells of abundant newly formed capillaries.     

Sonic hedgehog regulates growth and morphogenesis of the tooth

During mammalian tooth development, the oral ectoderm and mesenchyme coordinate their growth and differentiation to give rise to organs with precise shapes, sizes and functions. The initial ingrowth of the dental epithelium and its associated dental mesenchyme gives rise to the tooth bud. Next, the epithelial component folds to give the tooth its shape. Coincident with this process, adjacent epithelial and mesenchymal cells differentiate into enamel-secreting ameloblasts and dentin-secreting odontoblasts, respectively. Growth, morphogenesis and differentiation of the epithelium and mesenchyme are coordinated by secreted signaling proteins. Sonic hedgehog (Shh) encodes a signaling peptide which is present in the oral epithelium prior to invagination and in the tooth epithelium throughout its development. We have addressed the role of Shh in the developing tooth in mouse by using a conditional allele to remove Shh activity shortly after ingrowth of the dental epithelium. Reduction and then loss of Shh function results in a cap stage tooth rudiment in which the morphology is severely disrupted. The overall size of the tooth is reduced and both the lingual epithelial invagination and the dental cord are absent. However, the enamel knot, a putative organizer of crown formation, is present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in the wild type. At birth, the size and the shape of the teeth are severely affected and the polarity and organization of the ameloblast and odontoblast layers is disrupted. However, both dentin- and enamel-specific markers are expressed and a large amount of tooth-specific extracellular matrix is produced. This observation was confirmed by grafting studies in which tooth rudiments were cultured for several days under kidney capsules. Under these conditions, both enamel and dentin were deposited even though the enamel and dentin layers remained disorganized. These studies demonstrate that Shh regulates growth and determines the shape of the tooth. However, Shh signaling is not essential for differentiation of ameloblasts or odontoblasts.     

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 in the distribution of type IV collagen,laminin, proteoglycan,and fibronectin during mouse tooth development

The distribution of certain basement membrane (BM) components including type IV collagen, laminin, BM proteoglycan, and fibronectin was studied in developing mouse molar teeth, using antibodies or antisera specific for these substances in indirect immunofluorescence. At the onset of cuspal morphogenesis, type IV collagen, laminin, and BM proteoglycan were found to be present throughout the basement membranes of the tooth. Fibronectin was abundant under the inner enamel epithelium at the region of differentiating odontoblasts and also in the mesenchymal tissues. After the first layer of predentin had been secreted by the odontoblasts at the epithelial-mesenchymal interface, laminin remained in close association with the epithelial cells whereas type IV collagen, BM proteoglycan, and fibronectin were distributed uniformly throughout this area. Later when dentin had been produced and the epithelial cells had differentiated into ameloblasts, basement membrane components disappeared from the cuspal area. These matrix components were not detected in dentin while BM proteoglycan and fibronectin were present in predentin. The observed changes in the collagenous and noncollagenous glycoproteins and the proteoglycan appear to be closely associated with cell differentiation and matrix secretion in the developing tooth.     

Localization of Carbonic Anhydrase in the Plumula of the Tooth of Lytechinus variegatus (Echinodermata: Echinoidea)

Labeled inhibitor autoradiography showed that carbonic anhydrase (CA) occurs in (1) the epithelium, (2) free odontoblasts and fibroblasts, (3) row odontoblasts and their membranes surrounding the calcareous parts and (4) extracellular areas. Extracellular CA occurs in areas where fibroblasts and collagen fibers are abundant. The localization of CA suggests that CA facilitates the movement of CO₂ through the membrane and/or extracellular spaces to promote calcification.     

A substractive PCR-based cDNA library from human odontoblast cells: identification of novel genes expressed in tooth forming cells.

Odontoblasts are highly specialized cells aligned at the edge of the dental pulp. As a step towards understanding the complex mechanisms underlying their terminal differentiation, the gene expression pattern was examined in human cultured odontoblast cells. Suppression substractive hybridization (SSH) was used to establish a substracted cDNA library specific for human odontoblasts. For this purpose, cDNAs from human cultured fibroblastic pulp cells were substracted to cDNA from human cultured odontoblasts. The nucleotide sequence of 154 substracted cDNA clones was determined. We identified 130 preferentially expressed gene fragments in odontoblasts as compared with the fibroblastic pulp cells. Ten of them were already identified in odontoblasts such as DSPP, BSP, enamelysin and Col1A1. We confirmed their overexpression by RT-PCR on the cultured cells and in vivo by in situ hybridization on human molars. Another 64 clones corresponded to known genes. Among them, two clones were of particular interest: reelin, which was first detected in the brain and osteoadherin, which was first located in bone. Fifty-six clones were unknown genes even though 82% matched expressed sequence tags or genomic clones. A reverse Northern dot blot showed that 96% of them were overexpressed at different rates in cultured odontoblasts. These latest results indicate that there are still unknown genes that are associated with the control of the odontoblast phenotype. Thus, cloning of odontoblast differentiation-associated genes not only opens up new methods of elucidating the normal development but also the recruitment of odontoblasts when required to initiate repair of dentin.     

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.