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.     

High glucose regulates cyclin D1/E of human mesenchymal stem cells through TGF‐β1 expression via Ca2+/PKC/MAPKs and PI3K/Akt/mTOR signal pathways

The elucidation of factors that support human mesenchymal stem cells (hMSCs) growth has remained unresolved partly because of the reliance of many researchers on ill‐defined, proprietary medium formulation. Thus, we investigated the effects of high glucose (D ‐glucose, 25 mM) on hMSCs proliferation. High glucose significantly increased [³H]‐thymidine incorporation and cell‐cycle regulatory protein expression levels compared with 5 mM D ‐glucose or 25 mM L ‐glucose. In addition, high glucose increased transforming growth factor‐β1 (TGF‐β₁) mRNA and protein expression levels. High glucose‐induced cell‐cycle regulatory protein expression levels and [³H]‐thymidine incorporation, which were inhibited by TGF‐β₁ siRNA transfection and TGF‐β₁ neutralizing antibody treatment. High glucose‐induced phosphorylation of protein kinase C (PKC), p44/42 mitogen‐activated protein kinases (MAPKs), p38 MAPK, Akt, and mammalian target of rapamycin (mTOR) in a time‐dependent manner. Pretreatment of PKC inhibitors (staurosporine, 10^?6 M; bisindolylmaleimide I, 10^?6 M), LY 294002 (PI3 kinase inhibitor, 10^?6 M), Akt inhibitor (10^?5 M), PD 98059 (p44/42 MAPKs inhibitor, 10^?5 M), SB 203580 (p38 MAPK inhibitor, 10^?6 M), and rapamycin (mTOR inhibitor, 10^?8 M) blocked the high glucose‐induced cellular proliferation and TGF‐β₁ protein expression. In conclusion, high glucose stimulated hMSCs proliferation through TGF‐β₁ expression via Ca²⁺/PKC/MAPKs as well as PI3K/Akt/mTOR signal pathways. J. Cell. Physiol. 224:59–70, 2010 © 2010 Wiley‐Liss, Inc.     

Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells

Background information. Although adult bone‐marrow‐derived cell populations have been used to make teeth when recombined with embryonic oral epithelium, the differences between dental and non‐dental stem‐cell‐mediated odontogenesis remain an open question. Results. STRO‐1⁺ (stromal precursor cell marker) DPSCs (dental pulp stem cells) and BMSSCs (bone marrow stromal stem cells) were isolated from rat dental pulp and bone marrow respectively by magnetic‐activated cell‐sorting techniques. Their odontogenic capacity was compared under the same inductive microenvironment produced by ABCs (apical bud cells) from 2‐day‐old rat incisors. Co‐cultured DPSCs/ABCs in vitro showed more active odontogenic differentiation ability than mixed BMSSCs/ABCs, as indicated by the accelerated matrix mineralization, up‐regulated alkaline phosphatase activity, cell‐cycle modification, and the expression of tooth‐specific proteins and genes. After cultured for 14 days in the renal capsules of rat hosts, recombined DPSC/ABC pellets formed typical tooth‐shaped tissues with balanced amelogenesis and dentinogenesis, whereas BMSSC/ABC recombinants developed into atypical dentin—pulp complexes without enamel formation. DPSC and BMSSC pellets in vivo produced osteodentin‐like structures and fibrous connective tissues respectively. Conclusions. DPSCs presented more striking odontogenic capability than BMSSCs under the induction of postnatal ABCs. This report provides critical insights into the selection of candidate cells for tooth regeneration between dental and non‐dental stem cell populations.     

Effect of budesonide on TGF‐β1‐enhanced VEGF production by lung fibroblasts

VEGF (vascular endothelial growth factor) is a potent proangiogenic cytokine, and vascular change is one of the characteristic features of airway remodelling. Since the glucocorticoids have shown antifibrosis properties, we sought to investigate whether budesonide, a widely used glucocorticoid in clinical practice, could attenuate TGF‐β1 (transforming growth factor‐β1)‐induced VEGF production by HFL‐1 (human lung fibroblasts). HFL‐1 fibroblasts were treated with various concentrations of budesonide (10^?11 M, 10^?9 M and 10^?7 M) in the absence or presence of TGF‐β1. Postculture media were collected for ELISA of VEGF at the indicated times. The cell lysates were subjected to Western blotting analysis to test TGF‐β1/Smad and MAP (mitogen‐activated protein) kinase signalling activation, respectively. The results suggested that budesonide pretreatment reduced the significant increase of VEGF release induced by TGF‐β1 in HFL‐1 fibroblasts in a dose‐dependent manner, and suppressed the increase of phospho‐Smad3 and phosphor‐ERK (extracellular signal‐regulated kinase) protein levels. In conclusion, budesonide may reduce TGF‐β1‐induced VEGF production in the lung, probably through the Smad/ERK signalling pathway and, thus, may provide new sight into the molecular mechanism underlying glucocorticoid therapy for airway inflammatory diseases.     

Odontoblasts: the cells forming and maintaining dentine

Odontoblasts are tall columnar cells located at the periphery of the dental pulp. They derive from ectomesenchymal cells originated by migration of neural crest cells during the early craniofacial development. Odontoblasts form the dentine, a collagen-based mineralized tissue, through secretion of its collagenous and noncollagenous organic matrix components and by control the mineralization process. A conspicuous cell process arises from the cell body of odontoblasts and penetrates into the mineralized dentine. After dentinogenesis, odontoblasts deposit new layers of dentine throughout life and might also form a type of reactionary/reparative dentine in response to dental caries and other external factors may affect teeth.     

Transforming Growth Factor-β Isoform Expression in Mature Human Healthy and Carious Molar Teeth

Transforming growth factor (TGF)-β isoforms have been implicated in cellular signalling during tooth development and repair, but little is known of their cellular localisation or distribution within the dental tissues in the mature tooth. This study investigated the presence of TGF-β1, β2 and β3 isoforms in tissues of sound and carious human molar teeth, to understand better the expression of TGF-βs during health and disease. In healthy tissues, odontoblasts, cells of the cell rich layer, pulpal fibroblasts and endothelial cells were stained to varying degrees for all isoforms, with TGF-β3 showing the greatest intensity and TGF-β1 the weakest intensity. Similar patterns of staining were observed in carious teeth; however, TGF-β1 showed significantly increased staining intensity within odontoblasts and pulpal cells of carious teeth (p<0.001). Biochemical analysis showed greater amounts of TGF-β1 in tertiary dentine than in primary dentine samples. The expression of TGF-βs in odontoblasts and the increased presence of TGF-β1 in tertiary dentine suggest that these isoforms may be important in odontoblast behaviour and the modulation of the tissue response to injury.     

Reduced expression of dentin sialophosphoprotein is associated with dysplastic dentin in mice overexpressing transforming growth factor-beta 1 in teeth

Transforming growth factor (TGF)-beta1 is expressed in developing tooth from the initiation stage through adulthood. Odontoblast-specific expression of TGF-beta1 in the tooth continues throughout life; however, the precise biological functions of this growth factor in the odontoblasts are not clearly understood. Herein, we describe the generation of transgenic mice that overexpress active TGF-beta1 predominantly in the odontoblasts. Teeth of these mice show a significant reduction in the tooth mineralization, defective dentin formation, and a relatively high branching of dentinal tubules. Dentin extracellular matrix components such as type I and III collagens are increased and deposited abnormally in the dental pulp, similar to the hereditary human tooth disorders such as dentin dysplasia and dentinogenesis imperfecta. Calcium, one of the crucial inorganic components of mineralization, is also apparently increased in the transgenic mouse teeth. Most importantly, the expression of dentin sialophosphoprotein (dspp), a candidate gene implicated in dentinogenesis imperfecta II (MIM 125420), is significantly down-regulated in the transgenic teeth. Our results provide in vivo evidence suggesting that TGF-beta1 mediated expression of dspp is crucial for dentin mineralization. These findings also provide for the first time a direct experimental evidence indicating that decreased dspp gene expression along with the other cellular changes in odontoblasts may result in human hereditary dental disorders like dentinogenesis imperfecta II (MIM 125420) and dentin dysplasia (MIM 125400 and 125420).     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.     

Differentiation of odontoblasts is negatively regulated by MEPE via its C-terminal fragment

Matrix extracellular phosphoglycoprotein (MEPE) is an extracellular matrix protein that is mainly expressed in mineralizing tissues, including the dental pulp. The purposes of this study were to clarify the localization of MEPE in the tooth germ and to investigate the roles of MEPE in the differentiation of odontoblasts. The immunohistochemical staining in the tooth germ of the upper first molars of male Wistar rats (postnatal day 3) revealed that MEPE was mainly localized in odontoblasts during dentinogenesis. Stable MEPE-overexpressing and MEPE-knockdown cell lines, which were established in odontoblast-lineage cells (OLCs), showed lower and higher differentiation capabilities, respectively. Eukaryotic proteins of the N-terminal fragment of MEPE produced in HEK cells had no effect on the differentiation of OLCs, whereas the C-terminal fragment containing an RGD sequence inhibited their differentiation. These results indicated that the C-terminal fragment of MEPE containing an RGD sequence, cleaved in odontoblasts, appeared to be the active form of MEPE, which may play important roles in dentinogenesis and pulpal homeostasis by keeping the odontoblasts in immature condition.     

Developmental expression of a 53 KD dentin sialoprotein in rat tooth organs.

Rat dentin contains a major sialic acid-rich glycoprotein, DSP, with an overall composition similar to that of bone sialoproteins but whose biological role in dentinogenesis is unknown. Using polyclonal affinity-purified antibodies to rat DSP and four immunohistochemical methods of detection, we studied the cell and tissue localization of DSP and the time course of its appearance during odontoblast differentiation. DSP first appeared within young odontoblasts concomitant with early secretion of pre-dentin matrix and before the onset of mineralization but was absent in pre-odontoblasts. DSP immunostaining also localized within secretory odontoblasts and was intense in odontoblastic processes. Early pre-dentin stained positive for DSP, in contrast to more mature pre-dentin, where immunoreactivity was less intense and more restricted to odontoblastic processes. In the zone of mineralized dentin matrix, a moderate and uniform staining pattern was evident. Intense immunostaining was also seen within the cells and matrix of dental pulp during dentinogenesis. Other cells and tissues within the tooth organ and those surrounding it were non-reactive. These findings suggest that DSP is developmentally expressed in cells of the odontoblastic lineage and may be a biochemical marker of odontoblastic activity.     

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.     

Impacts of land‐use change on nitrogen status and mineralization in the Mulga Lands of Southern Queensland

Abstract: In the semiarid Mulga Lands of southern Queensland soil nitrogen (N) levels have declined after clearance of the native mulga (Acacia aneura F. Muell. ex Benth.) and conversion to grazed buffel grass (Cenchrus ciliaris) pasture. At three mulga sites, declines in soil total N ranged from 14% to 28% in the surface 10 cm of soil. In situ net N mineralization from December 2003 until November 2004 in the surface 10 cm was 49.5 kg N ha^?1 year^?1 in the mulga woodland, 48.2 kg N ha^?1 year^?1 in the young (<5 years old) buffel pasture (previously sown to wheat (Triticum aestivum L.) and 34.6 kg N ha^?1 year^?1 in the old buffel pasture (>20 years). Ammonium‐N was the dominant N pool under mulga in the top 30 cm, while nitrate‐N was dominant under the buffel pastures. Although ammonium‐N under mulga was significantly different to that for 21‐year‐old buffel pasture at all depths, nitrification and net N mineralization were not different between the three land uses at any depth or in the entire 90 cm profile. The Soil Nitrogen Availability Predictor model was used to predict field N mineralization rates for the mulga woodland and 21‐year‐old buffel pasture by using a medium‐term (6‐week) laboratory incubation to establish basal rates of N mineralization. The Soil Nitrogen Availability Predictor overestimated annual net N mineralization in the 0–30 cm depth of mulga by 9% and underestimated it by 28% for the old buffel pasture. The Soil Nitrogen Availability Predictor could be modified further to accurately predict net N mineralization for the mulga woodlands.     

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.     

Chondro/osteoblastic and cardiovascular gene modulation in human artery smooth muscle cells that calcify in the presence of phosphate and calcitriol or paricalcitol

Vitamin D sterol administration, a traditional treatment for secondary hyperparathyroidism, may increase serum calcium and phosphorus, and has been associated with increased vascular calcification (VC). In vitro studies suggest that in the presence of uremic concentrations of phosphorus, vitamin D sterols regulate gene expression associated with trans‐differentiation of smooth muscle cells (SMCs) to a chondro/osteoblastic cell type. This study examined effects of vitamin D sterols on gene expression profiles associated with phosphate‐enhanced human coronary artery SMC (CASMC) calcification. Cultured CASMCs were exposed to phosphate‐containing differentiation medium (DM) with and without calcitriol, paricalcitol, or the calcimimetic R‐568 (10^?11–10^?7 M) for 7 days. Calcification of CASMCs, determined using colorimetry following acid extraction, was dose dependently increased (1.6‐ to 1.9‐fold) by vitamin D sterols + DM. In contrast, R‐568 did not increase calcification. Microarray analysis demonstrated that, compared with DM, calcitriol (10^?8 M) + DM or paricalcitol (10^?8 M) + DM similarly and significantly (P < 0.05) regulated genes of various pathways including: metabolism, CYP24A1; mineralization, ENPP1; apoptosis, GIP3; osteo/chondrogenesis, OPG, TGFB2, Dkk1, BMP4, BMP6; cardiovascular, HGF, DSP1, TNC; cell cycle, MAPK13; and ion channels, SLC22A3 KCNK3. R‐568 had no effect on CASMC gene expression. Thus, SMC calcification observed in response to vitamin D sterol + DM may be partially mediated through targeting mineralization, apoptotic, osteo/chondrocytic, and cardiovascular pathway genes, although some gene changes may protect against calcification. Further studies to determine precise roles of these genes in development of, or protection against VC and cardiovascular disease are required. J. Cell. Biochem. 111: 911–921, 2010. © 2010 Wiley‐Liss, Inc.     

Soaking seeds of Lens culinaris with 28-homobrassinolide increased nitrate reductase activity and grain yield in the field in India

Surface‐sterilised seeds of Lens culinaris cv. Pusa‐6 were soaked in 0 M, 10^?6 M, 10^?8 M or 10^?10 M aqueous solutions of 28‐homobrassinolide (HBR) for 4 h, 8 h or 12 h and planted in the field in a sandy loam soil. Plants were sampled 60, 90 and 120 days after sowing (DAS). Soaking with HBR decreased root length and nodule number per plant but increased nitrate reductase activity (E.C.1.6.6.1). Soaking with HBR also increased grain yield at the final harvest 140 DAS. The greatest increase was obtained with an HBR concentration of 10^?8 M.     

The Effect of Environmental Conditions on the Expression of Disease in Cultured Tissues of Brassica napus ssp. oldfera Infected with Leptosphaeria maculans

As a basis for devising an in vitro screening programme, culture conditions were optimized so that tissue cultures from two resistant cultivars of Brassica napus ssp. oleifera (Mikado, Bienvenu) and two susceptible cultivars (Lesira, Ceres) could be differentiated using a disease scoring scheme, when inoculated with Leptosphaeria maculans. Tissues inoculated included thin cell layer explants from soil-grown plants and in vitro-grown shoot cultures and callus tissue formed on such explants. The period of incubation and the incubation temperature were of importance in the development of differential disease reactions. Increasing temperature generally resulted in an increase in infection and too great an incubation period resulted in total overgrowth of the tissue. Increasing concentrations (1 × 10^?6 M-1 ×10^?4 M) of the auxins 1-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and mdole-3-acetic acid (IAA) in the culture medium, resulted in a decrease in disease score of the thin cell layer (TCL) explants from soil-grown plants. The cytokinins examined 6-benzyl-aminopurine (BAP) and 6-4-hydroxy-3-methyl-2-enylaminopurine (zeatin), reduced the extent of infection of the TCL explants when used in combination with the auxin NAA. Medium containing NAA at a concentration of 1 × 10^?6 M in combination with BAP at a concentration of 1× 10^?6 or 1 × 10^?4 M allowed differentiation of the disease reactions of the resistant and susceptible cultivars, when the explants were incubated for 10 days at 20 °C after inoculation. Similar conditions of incubation and the addition of NAA (1 × 10^?6 M) combined with BAP (1 × 10^?6 M) to the medium also allowed the differentiation of the disease reactions on TCL explants from stems of in vitro shoot cultures of the cultivars Mikado and Lesira. Increasing concentrations of the auxin NAA and the cytokinin BAP resulted in a reduction in the mean disease score of the callus tissue produced on TCL explants from soil-grown plants, and NAA (1 × 10^?5 M) combined with BAP (1 × 10^?6 or 1 × 10^?5 M) allowed differentiation of resistance and susceptibility in callus tissues when incubated for 5 days at 20 °C. 2,4-D did not allow differentiation of the cultivars. This was in contrast to the inoculation of callus tissue attached to TCL explants of in vitro shoot cultures, where combinations of 2,4-D and BAP at concentrations of 1 × 10^?6 M allowed differentiation of the resistant and susceptible cultivars. These findings provide a basis for designing selection protocols of value in both traditional as well as in vitro breeding programmes to select lines of oilseed rape with resistance/novel resistance to L. maculans.     

Localization and synthesis of type III collagen and fibronectin in human reparative dentine. Immunoperoxidase and immunogold staining

The injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue. Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine. Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.