Sequence of protein expression of bone sialoprotein and osteopontin at the developing interface between repair cementum and dentin in human deciduous teeth

Experimental periodontal regeneration studies have revealed the weak binding of repair cementum to the root surface, whereas attachment of cementum to dentin preconditioned by odontoclasts appears to be superior. The aim of this study has been, therefore, to analyze the structural and partial biochemical nature of the interface that develops between resorbed dentin and repair cementum by using human deciduous teeth as a model. Aldehyde-fixed and decalcified tooth samples were embedded in acrylic or epoxy resins and sectioned for light and transmission electron microscopy. Antibodies against bone sialoprotein (BSP) and osteopontin (OPN), two noncollagenous proteins accumulating at hard tissue interfaces in bone and teeth, were used for protein A-gold immunocytochemistry. Light microscopy revealed a gradually increasing staining intensity of the external dentin matrix starting after the withdrawal of the odontoclast. Labeling for both BSP and OPN was first detected among the exposed collagen fibrils and in the intratubular dentin matrix when odontoclasts had withdrawn but mesenchymal cells were present. Subsequently, collagen fibrils of the repair cementum were deposited concomitantly with the appearance of labeling for BSP and OPN over the intratubular, intertubular, and peritubular dentin matrix. Labeled mineralization foci indicated the advancing mineralization front, and the collagenous repair matrix became integrated in an electron-dense organic material that showed labeling for BSP and OPN. Thus, no distinct planar interfacial matrix layer lies between the resorbed dentin and the repair cementum. The results suggest that odontoclasts precondition the dentin matrix such that the repair cementum becomes firmly attached.This study was supported by the Clinical Research Foundation (CRF) for the Promotion of Oral Health, University of Berne, Berne, Switzerland.     

The NH2-terminal and COOH-terminal fragments of dentin matrix protein 1 (DMP1) localize differently in the compartments of dentin and growth plate of bone

Multiple studies have shown that dentin matrix protein 1 (DMP1) is essential for bone and dentin mineralization. After post-translational proteolytic cleavage, DMP1 exists within the extracellular matrix of bone and dentin as an NH2-terminal fragment, a COOH-terminal fragment, and the proteoglycan form of the NH2-terminal fragment (DMP1-PG). To begin to assess the biological function of each fragment, we evaluated the distribution of both fragments in the rat tooth and bone using antibodies specific to the NH2-terminal and COOH-terminal regions of DMP1 and confocal microscopy. In rat first molar organs, the NH2-terminal fragment localized to predentin, whereas the COOH-terminal fragment was mainly restricted to mineralized dentin. In the growth plate of bone, the NH2-terminal fragment appeared in the proliferation and hypertrophic zones, whereas the COOH-terminal fragment occupied the ossification zone. Forster resonance energy transfer analysis showed colocalization of both fragments of DMP1 in odontoblasts and predentin, as well as hypertrophic chondrocytes within the growth plates of bone. The biochemical analysis of bovine teeth showed that predentin is rich in DMP1-PG, whereas mineralized dentin primarily contains the COOH-terminal fragment. We conclude that the differential patterns of expression of NH2-terminal and COOH-terminal fragments of DMP1 reflect their potentially distinct roles in the biomineralization of dentin and bone matrices.     

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.     

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.     

Developmental appearance of dentin matrix protein 1 during the early dentinogenesis in rat molars as identified by high-resolution immunocytochemistry

Dentin matrix protein 1 (DMP 1) is an acidic phosphoprotein that has been postulated to play an important role in mineralized tissue formation. We have examined rat molar tooth germs by applying a high-resolution immunocytochemical approach with the purpose to identify the temporal and spatial localization of DMP 1 at the onset of dentinogenesis. Upper molar tooth germs of 2- to 3-day-old Wistar rats were fixed in a cacodylate-buffered 0.1% glutaraldehyde + 4% formaldehyde fixative, left unosmicated and embedded in LR White resin. The sections were incubated with a polyclonal DMP 1 antibody for postembedding colloidal gold immunolabeling and examined in a Jeol 1010 transmission electron microscope. The earliest localization of DMP 1 was in the Golgi region as well as in the nucleus of differentiating odontoblasts. When mineralization spread from matrix vesicles to the surrounding matrix, DMP 1 was extracellularly detected around the mineralizing globules. In the regions of fully mineralized mantle dentin, it was present in the mineralized regions, mainly around the peritubular dentin. The appearance of DMP 1 during early dentinogenesis implies a direct role for this protein in both odontoblast differentiation and matrix mineralization.     

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.     

Formation of bone-like tissue by dental follicle cells co-cultured with dental papilla cells

During tooth root formation, dental follicle cells (DFCs) differentiate into osteoblasts/cementoblasts when they are in contact with pre-existing dentin. Since some factors of dentin matrix were also produced by dental papilla cells (DPCs) and could induce DFCs differentiation, we hypothesized that DPCs can directly promote DFCs differentiation and that differentiation could occur in a co-culture model. To test this hypothesis, we investigated the characteristics of DFCs that are influenced by DPCs in an in vitro co-culture and in vivo heterotopic transplant model. One week into the co-culture, there were significant increases in the mRNA level of bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), bone sialoprotein (BSP) and osteocalcin (OCN), and a decrease of the receptor activator of nuclear factor κB ligand (RANKL). Additionally, the number of BMP2-, OPG-, BSP- and OCN-positive DFCs increased whereas RANKL-positive DFCs decreased. Three weeks after co-culture, DFCs produced calcified nodules, accompanied with increased sub-cellular organelles for protein synthesis and secretion. In the heterotopic transplant model, the adult male rats were used as hosts, DFCs were transplanted into the omentum. In vivo 5-week growth of DFCs in the presence of DPCs led to the formation of bone-like tissues, positive for BSP, OCN and BMP2. In contrast, DFCs alone led to fibrous-like tissues. These results indicated that in the absence of pre-existing dentin, DPCs can stimulate osteogenesis and inhibit osteoclastogenesis in DFCs and suggested a novel strategy to promote DFCs differentiation.     

Immunohistochemistry of the intercellular matrix components and the epithelio-mesenchymal junction of the human tooth germ

Summary The immunohistochemical localization of heparan sulphate, collagen type I, III and IV, laminin, tenascin, plasma- and cellular fibronectin was studied in tooth germs from human fetuses. The lamina basalis ameloblastica or membrana preformativa, which separates the pre-ameloblasts from the pre-dentin and dentin, contained heparan sulphate, collagen type IV, laminin and fibronectin. Enamel reacted with antifibronectin, but the reaction varied depending on the type of fibronectin and the source of antibody. In early pre-dentin, collagen type I, laminin, tenascin and fibronectin were present. In late pre-dentin and dentin collagen type I was found in intertubular dentin and in the zone between enamel and dentin. The close relationship between collagen type I in dentin and fibronectin in immature enamel is interesting, as it may contribute to the stabilization of the amelodentinal interface. In dental pulp, collagen type IV and laminin were found in the endothelial basement membranes. Collagen type I and III, tenascin and fibronectin were localized to the mesenchymal intercellular matrix.The results of this study have supported the assumption that the lamina basalis ameloblastica is a basement membrane, and have lead to the suggestion that ameloblasts are producers of fibronectin or a fibronectin-like substance.     

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.     

Expression of FAM20C in the Osteogenesis and Odontogenesis of Mouse

Mutations in FAM20C were recently identified as the cause of lethal osteosclerotic bone dysplasia, which highlighted the important role of this molecule in biomineralization. No systematic studies have been performed to evaluate the expression pattern of this relatively new molecule in the developmental processes of bone and tooth. In the present study, we analyzed in detail the expression profile of FAM20C during osteogenesis and odontogenesis using ISH and IHC approaches. The specimens analyzed were mouse tissues spanning embryonic day 13.5 (E13.5) to postnatal 8 weeks. The earliest presence of FAM20C was observed at E14.5. During osteogenesis, FAM20C mRNA was detected in the chondrocytes and osteoblasts of the long bone, whereas its protein was observed in the extracellular matrix (ECM) of bone and in the cytoplasm of the chondrocytes, osteoblasts, and osteocytes. During odontogenesis, FAM20C mRNA was detected in the ameloblasts, odontoblasts, cementoblasts, and periodontal ligament fibroblasts, whereas its protein was observed in the matrices of dentin, enamel, and alveolar bone and in the cytoplasm of the aforementioned cells. The temporospatial expression profile revealed in this study indicates that FAM20C is an ECM protein that may play an important role in controlling the mineralization of bone and tooth. (J Histochem Cytochem 58:957–967, 2010)     

Expression of cathepsin K mRNA and protein in odontoclasts after experimental tooth movement in the mouse maxilla by in situ hybridization and immunoelectron microscopy

This study demonstrated the simultaneous expression of cathepsin K (CK) mRNA by in situ hybridization and CK protein by immunoelectron microscopy in odontoclasts in mouse maxillae after experimental tooth movement. On the pressure side (the area under pressure during tooth movement), CK mRNA was detected in odontoclasts in resorption lacunae in the tooth root, in osteoclasts in bone resorption lacuane, and in fibroblasts in the periodontal ligament. Using electron microscopy, CK protein was detected at the apex of odontoclasts, intracellularly in vesicles and granules, and extracellularly in irregularly shaped vacuoles (extracellular spaces), on the plasma membrane of the ruffled border, and on and between typical striated type I collagen fibrils in the lacunae. These vesicles and granules appeared to fuse with irregular vacuoles containing CK-positive fragmented fibril-like structures close to the ruffled border. In the basolateral portion of odontoclasts, small amounts of CK-positive rough endoplasmic reticulum (ER) were found. CK-positive intracellular vacuoles (not extracellular spaces) also appeared to fuse with the vesicles and granules. However, these fused organelles rarely contained fragmented fibril-like structures. They are probably endolysosomes. The distribution of CK in odontoclasts was similar to that previously seen in osteoclasts. Furthermore, CK-positive fibril-like structures were found in the vacuoles of fibroblasts. These results indicated that during tooth movement CK is synthesized in odontoclasts on the pressure side and secreted into the tooth resorption lacunae. Therefore, CK may take part in the degradation of the dentin matrix (type I collagen fibrils and non-collagenous protein) of the tooth root, and in the subsequent intracellular degradation of endocytosed fragmented fibril-like structures in endolysosomes.     

Fine structure of the secretory and non-secretory ameloblasts in the frog. II. Fine structure of the non-secretory ameloblast.

The non-secretory ameloblasts present at the enamel-free surfaces of maxillary teeth in the frog Rana pipiens were examined by electron microscopy at different stages of tooth development. Their main fine structural features seem to reflect a transport function. During early tooth development, the non-secretory ameloblasts adjacent to odontoblasts and predentin exhibit extensive lateral surface specializations and numerous cytoplasmic vesicles. During late tooth development, the non-secretory ameloblasts adjacent to mineralizing dentin show numerous cellular junctions, well-developed intercellular channels with numerous interdigitating processes and labyrinthine configurations at their distal surfaces. An intact basal lamina is present between the non-secretory ameloblasts and the dentin surface until the dentin becomes fully mineralized. At this stage the adjacent cells no longer exhibit surface specializations. It is suggested that the non-secretory ameloblasts may participate in the mineralization of adjacent dentin at the enamel-free surfaces. This surface dentin becomes fully mineralized at a later stage of development than the underlying dentin.     

Inhibition of chondroitin sulfate glycosaminoglycans incorporation affected odontoblast differentiation in cultured embryonic mouse molars

Chondroitin sulfate proteoglycan (CSPG) is an important component of extracellular matrix (ECM), it is composed of a core protein and one or more chondroitin sulfate glycosaminoglycan side chains (CS-GAGs). To investigate the roles of its CS-GAGs in dentinogenesis, the mouse mandibular first molar tooth germs at early bell stage were cultivated with or without β-xyloside. As expected, the CS-GAGs were inhibited on their incorporation to CSPGs by β-xyloside, accompanied by the change of morphology of the cultured tooth germs. The histological results and the transmission electron microscopy (TEM) investigation indicated that β-xyloside exhibited obvious inhibiting effects on odontoblasts differentiation compared with the control group. Meanwhile the results of immunohistochemistry, in situ hybridization and quantitative RT-PCR for type I collagen, dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein, the products of differentiated odontoblasts, further proved that odontoblasts differentiation was inhibited. Collagen fibers detected in TEM decreased and arranged in disorder as well. Thus we conclude that the inhibition of CS-GAGs incorporation to CSPGs can affect odontoblast differentiation in cultured embryonic mouse molars.     

Colocalization of dentin matrix protein 1 and dentin sialoprotein at late stages of rat molar development.

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are acidic proteins found in the extracellular matrices of bones and teeth. Recent data from gene knockouts, along with those of gene mutations, indicate that these two phosphoproteins are critical for bone and tooth development and/or maintenance. However, the precise functions of the two proteins have not been elucidated. In order to gain insights into their functions in tooth formation, we performed systematic, comparative investigations on the immunolocalization of DMP1 and dentin sialoprotein (DSP, a cleaved fragment of DSPP), using the rat first molar at different developmental stages as a model. Immunohistochemistry (IHC) was performed with specific, monoclonal antibodies against the COOH-terminal fragments of DMP1 and against DSP. In 1-day- and 1-week-old rats, weak immunoreactions for DMP1 were observed in dentinal tubules while stronger reactions for DSP were seen in the tubules and predentin. In rats older than 2 weeks, immunoreactions for DMP1 were found in dentinal tubules, predentin and odontoblasts. In 5-week- and 8-week-old rats, strong immunoreactions for DMP1 were widely distributed in odontoblasts and predentin. The distribution pattern of DSP was strikingly similar to that of DMP1 after 2 weeks and the localization of each was distinctly different from that of bone sialoprotein (BSP). The unique colocalization of DMP1 and DSPP in tooth development suggests that the two proteins play complementary and/or synergistic roles in formation and maintenance of healthy teeth.     

Peritubular dentin formation: crystal organization and the macromolecular constituents in human teeth.

Peritubular dentin (PTD) is a relatively dense mineralized tissue that surrounds the tubules of coronal tooth dentin. It is composed mainly of crystals of carbonated apatite together with a small amount of collagen. Its mode of formation has been investigated by studying the relatively dense particles isolated from a powdered preparation. Electron microscopic examination of the PTD particles, including 3-dimensional image reconstruction and electron diffraction, shows that the organization of the crystals of PTD is very similar to that of the adjacent intertubular dentin (ITD). The latter contains relatively large amounts of collagen and the carbonated apatite crystals are closely associated with the collagen matrix. The proteins present in the PTD particles are soluble after decalcification and stain with Stains All. The principal protein has higher molecular weight and a quite different amino acid composition than the phosphophoryns of the intertubular dentin. The interface between the PTD and the ITD shows structural continuity. These data show how two distinct carbonated apatite-based mineralized tissues can be organized and formed contiguously within the same organ by utilizing different sets of matrix proteins.     

Tooth development in a scincid lizard, Chalcides viridanus (Squamata), with particular attention to enamel formation

Comparative analysis of tooth development in the main vertebrate lineages is needed to determine the various evolutionary routes leading to current dentition in living vertebrates. We have used light, scanning and transmission electron microscopy to study tooth morphology and the main stages of tooth development in the scincid lizard, Chalcides viridanus, viz., from late embryos to 6-year-old specimens of a laboratory-bred colony, and from early initiation stages to complete differentiation and attachment, including resorption and enamel formation. In C. viridanus, all teeth of a jaw have a similar morphology but tooth shape, size and orientation change during ontogeny, with a constant number of tooth positions. Tooth morphology changes from a simple smooth cone in the late embryo to the typical adult aspect of two cusps and several ridges via successive tooth replacement at every position. First-generation teeth are initiated by interaction between the oral epithelium and subjacent mesenchyme. The dental lamina of these teeth directly branches from the basal layer of the oral epithelium. On replacement-tooth initiation, the dental lamina spreads from the enamel organ of the previous tooth. The epithelial cell population, at the dental lamina extremity and near the bone support surface, proliferates and differentiates into the enamel organ, the inner (IDE) and outer dental epithelium being separated by stellate reticulum. IDE differentiates into ameloblasts, which produce enamel matrix components. In the region facing differentiating IDE, mesenchymal cells differentiate into dental papilla and give rise to odontoblasts, which first deposit a layer of predentin matrix. The first elements of the enamel matrix are then synthesised by ameloblasts. Matrix mineralisation starts in the upper region of the tooth (dentin then enamel). Enamel maturation begins once the enamel matrix layer is complete. Concomitantly, dental matrices are deposited towards the base of the dentin cone. Maturation of the enamel matrix progresses from top to base; dentin mineralisation proceeds centripetally from the dentin–enamel junction towards the pulp cavity. Tooth attachment is pleurodont and tooth replacement occurs from the lingual side from which the dentin cone of the functional teeth is resorbed. Resorption starts from a deeper region in adults than in juveniles. Our results lead us to conclude that tooth morphogenesis and differentiation in this lizard are similar to those described for mammalian teeth. However, Tomes processes and enamel prisms are absent.     

Vegetation history and agriculture in the cover-sand area west of Breda (province of Noord-Brabant, The Netherlands)

Botanical investigation of archaeological sites situated in the northwest of the region bounded by the rivers Maas, Scheldt and Demer (‘MSD region’), west of the city of Breda, has provided a great deal of evidence about the landscape and its use in the period between 2000 b.c. and a.d. 1500. From pollen analysis, it appears that this cover-sand area gradually lost its woodlands through human activity after the beginning of the Bronze Age (ca. 2000 b.c.). Patches of woodland did survive there, however, until the early Middle Ages. In contrast to the cover-sand area in the vicinity of ’s-Hertogenbosch and Oss-Ussen in the northeast of the MSD region, the first large heathlands in the Breda area did not evolve until the early Middle Ages. In late prehistory, land use in this area was not much different from that in the micro-region of ’s-Hertogenbosch and Oss-Ussen. In the Bronze Age, Hordeum vulgare ssp. vulgare (hulled six-row barley) and Triticum dicoccon (emmer wheat) were grown. During the Iron Age, Panicum miliaceum (common millet) and T. spelta (spelt wheat) were introduced, but these crops disappeared during the Roman period. The Roman period is remarkable because of the lack of any Mediterranean culinary herbs or exotic fruits. Only pollen of Juglans regia (walnut), found around the transition from the Roman period to the early Middle Ages, indicates the introduction of an exotic tree into the region. From the early Middle Ages onwards, Secale cereale (rye) was the most important cereal, which was grown as a winter crop. In the course of the Middle Ages, arable weeds of the Sclerantho annui-Arnoseridetum plant community appeared, which is associated with the continuous growing of rye.