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Objective: The aim of the present study was to determine the influence of Notch ligands, Jagged-1 and Dll-1, on osteogenic differentiation by stem cells from human exfoliated deciduous teeth., Design: Notch ligands were immobilized on tissue culture surface using an indirect affinity immobilization technique. Cells from the remaining of dental pulp tissues from human deciduous teeth were isolated and characterized using flow cytometry and differentiation assay. Alkaline phosphatase (ALP) enzymatic activity, osteogenic marker gene expression, and mineralization were determined using ALP assay, real-time polymerase chain reaction, and alizarin red staining, respectively., Results: The isolated cells exhibited CD44, CD90, and CD105 expression but lack of CD45 expression. Further, these cells were able to differentiate toward osteogenic lineage. The upregulation of HES-1 and HEY-1 was observed in those cells on Dll-1 and Jagged-1 coated surface. The significant increase of ALP activity and mineralization was noted in those cells seeded on Jagged-1 surface and these results were attenuated when cells were pretreated with gamma secretase inhibitor. The significant upregulation of ALP and collagen type I gene expression was also observed in those cells seeded on Jagged-1 surface. The inconsistent Dll-1 induced osteogenic differentiation was found and high Dll-1 immobilized dose (50 nM) slightly enhanced alkaline phosphatase enzymatic activity. However, the statistical significant difference was not obtained as compared to the hFc control., Conclusion: The surface immobilization of Notch ligands, Jagged-1 and Dll-1, likely to enhance osteogenic differentiation of SHEDs. However, Jagged-1 had more ability in enhancing osteogenic differentiation than Dll-1 in our model., (Copyright © 2016. Published by Elsevier Ltd.)

Objectives: This study is aimed to test whether glucose-free conditions induce the activation of adenosine monophosphate-activated protein kinase (AMPK) and, to investigate association with AMPK expression and cell viability in human dental pulp cells., Design: Human dental pulp cells were initially maintained in culture medium containing glucose and the medium was subsequently changed to glucose-free medium. To evaluate the expression of AMPK, quantitative real-time RT-PCR, Western blot analysis and immunofluorescence were carried out. Cell viability was evaluated by MTT assay., Results: The expression of AMPK mRNA in glucose free conditions was 2.0-2.5 fold higher than the control at 1, 2 and 3 h (P<0.01). The expression of phosphorylated-AMPK was characterized by Western blot analysis and by immunofluorescence. Compound C-pre-treated group showed a decline of both AMPK expression and cell viability, while AICAR-pre-treated group showed an increase of AMPK and maintain of cell viability at regular level., Conclusions: AMPK plays an important role on fluctuating in accordance with glucose availability and protects cell viability from glucose free condition in human dental pulp cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The two distinct molecular forms of cholinesterase (ChE) are acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Our previous studies have reported that ChE is involved in tooth development. However, further experiments are needed to understand the precise action of ChE in tooth development. This study aimed to localise types of ChE in human tooth germs, and identify their distribution pattern. ChE were localised in frozen sections of jaws which were prepared from dead fetuses, neonates and stillborns who were free from visible abnormalities by Karnovsky and Root method. AChE was identified in the inner and outer enamel epithelia including the cervical loop region, stratum intermedium and preameloblasts of tooth germs at bell stage. Secretory ameloblasts were free from staining. The bud and cap stages of permanent tooth germs showed AChE activity on the lingual aspect and top surface of the epithelial ingrowths, respectively. BuChE activity was localised in the degenerating dental lamina. Our study reported the first evidence of localisation of ChE in human tooth development and identified the possible molecular form of ChE in tooth germs as AChE. Also, our results have provided strong evidence to speculate the action of AChE is on the cells of enamel organ during tooth development.

Unlabelled: Matrix metalloproteinase-13 (MMP-13 or collagenase-3) is a member of the family of matrix metalloproteinases (MMPs) produced in high amounts by cells with mineralising potential. Human dental pulp has been shown to express high levels of MMP-13 RNA., Objective: Since human dental pulp derived cells (HDPC) are known to possess osteoprogenitor properties, we investigated the pattern of expression of MMP-13 in long-term cultures of those cells under conditions that support mineralisation in vitro., Design: Impacted teeth or teeth extracted for orthodontic purposes were used to obtain dental pulp explants and HDPC were cultured for approximately 5 weeks. Pro- and active MMP-13 levels were determined in the cell culture supernatants by means of enzyme-linked immunosorbent assay (ELISA). Cell growth was evaluated through DNA content and osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity and Alizarin Red staining., Results: Mineralising cultures of HDPC produced significantly higher levels of pro-MMP-13 compared to control cultures. Both pro- and active MMP-13 levels displayed a characteristic peak that was found to coincide with the peak in alkaline phosphatase activity and the onset of mineralisation. Once mineralisation was firmly established, MMP-13 expression was significantly reduced., Conclusions: Evidence from this study suggests a role for MMP-13 in the transition of human dental pulp cells to a mature mineralising phenotype and points to MMP-13 as a possible marker in HDPC differentiation.

To examine the potential role of the histamine-forming enzyme, histidine decarboxylase (HDC), in oral inflammation and disease, we studied HDC activity in oral tissue after induction by bacterial agents. Following injection of E. coli-derived lipopolysaccharide (LPS) into mice, we measured the quantitative changes in HDC activity over time in dental pulp and gingiva. Oral tissue taken from individual mice was insufficient for detecting precise HDC activity, thus, we combined dental pulp or gingival tissues from four mice and assayed them over the course of 24 h. Our results indicate that LPS stimulated marked elevations of HDC activity in dental pulp and gingiva. This increase reached a maximum at 6 h after LPS injection and remained detectable at for least 24 h. Since mast cells are known to produce histamine through a difference mechanism than HDC induction, we compared LPS-induced HDC activity in dental pulp and gingiva to that in ear skin (a tissue rich in mast cells) and liver (a tissues lacking in mast cells). LPS also induced a marked increase in the HDC activity in liver and ear skin at 6 h after LPS injection. By contrast, saline injection had no effect on the HDC activity in any of the four tissues, although basal levels of HDC activity in ear skin was markedly higher than basal HDC activity in the other three kinds of tissues. Still, the relative increase in LPS-induced HDC activity in dental pulp and gingiva were much greater than that in ear skin. Since liver are devoid of mast cells and ear skin is considered the tissue richest in mast cells, the differences in HDC activity between tissues indicates that histamine induced by LPS may be produced by cells other than mast cells through another mechanism of action. These results also suggest that histamine produced in oral tissues in response to bacterial agents such as LPS could be involved in development of pulpitis or gingivitis (periodontitis), the most common diseases in the dental clinic, and that efforts to inhibit HDC activity, which elevates histamine levels in oral tissues, might offer the basis for novel treatment strategies.

The semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) from crude homogenates of human dental pulp was shown to catalyse the oxidative deamination of 5-hydroxytryptamine (serotonin; 5-HT) with a K(m) of 318+/-52 microM. In this respect the human enzyme resembles that in pig dental pulp, but differs from SSAO in all other tissues studied, which are inactive towards 5-HT. A method is described for obtaining intact dental pulp in which the anatomical details are preserved. Extracted teeth are frozen in dry ice and later defrosted rapidly before being fractured in a mechanical vice, facilitating pulp removal. Immunohistochemistry showed SSAO in the odontoblast layer, nerve fibres and blood vessels. The presence of SSAO in nerves in dental pulp appears to be unique. Tryptophan hydroxylase, a key enzyme in 5-HT synthesis, was also demonstrated in nerves and the odontoblast layer of human dental pulp.

Retinoic acid is a potent inducer of tissue-non-specific alkaline phosphatase (TNSALP) expression in various osteoblastic and fibroblastic cells, and may be involved in morphogenesis, cellular growth and differentiation. This study investigates the effects of retinoic acid on alkaline phosphatase activity and TNSALP gene expression in human dental pulp cells. Cultured cells were treated with various concentrations of retinoic acid (0, 10(-7), 10(- 6), 10 (-5) M) in 0.5% bovine serum albumin without serum. Alkaline phosphatase activity was determined by the rate of p-nitrophenyl phosphate hydrolysis and was also assayed in the presence of various inhibitors and under thermal inactivation. A set of specific oligonucleotide primers was selected, based on the nucleotide sequences of two human TNSALP mRNA (bone and liver) types, and reverse transcription-polymerase chain reaction (RT-PCR) performed. Inhibitory and thermal inactivation experiments revealed that the elevated alkaline phosphatase activity had properties of the TNSALP type. RT-PCR showed that retinoic acid enhanced the expression of bone-type TNSALP mRNA in pulp cells. However, the liver-type TNSALP mRNA was not detected. These findings suggest that the high alkaline phosphatase activity of retinoic acid-treated dental pulp cells is associated with increased transcription of the bone-type mRNA of the TNSALP gene and not with liver-type.

Findings on the localization and possible roles of the major growth factors, epidermal (EGF), platelet-derived (PDGF) and fibroblast (FGF) in early amelogenesis are contradictory and inconclusive. This study sought to localize immunohistochemically phospholipase (PLCgamma) and the EGF, PDGF and FGF receptors in the cells of the enamel organ during the events leading directly to early enamel formation in rat molars. PLCgamma is an immediate, downstream, signal-transduction pathway effector unique to the three receptors. A whole-head, freeze-dried sectioning method was used to reduce the possibilities of false-negative staining. A modification of the avidin/biotin complex method of immunohistochemical localization was used. Anti-PLCgamma and antibodies to each of EGF, PDGF and FGF receptors colocalized in the preameloblasts of the cervical loop, adjacent to the undifferentiated mesenchymal cells of the dental pulp. This staining disappeared shortly after the beginning of dentine mineralization. Staining for all four antibodies appeared on the proximal ends of the differentiating presecretory ameloblasts at the level of the beginning of predentine matrix deposition and continued in the secretory ameloblasts. It appears that EGF, PDGF and FGF have roles in the differentiation of ameloblasts and in control of cellular functions in presecretory and secretory ameloblasts. Their roles may represent redundancy of the kind seen in highly conserved tissues.

Insulin, insulin-like growth factors (IGF) I and II are considered to play an important part in the growth and differentiation of dental pulp cells. The present study examined the effects of these factors on pulp cells in serum-free culture conditions. The DNA content and alkaline phosphatase (ALPase) activity of dog pulp cells increased when they were cultured in a serum-free medium supplemented with transferrin, yolk lipoprotein and basic fibrobrast growth factor (TYF medium). The pulp cells produced type I collagen but not type III, suggesting that they might proliferate and differentiate into odontoblast-like cells in a serum-free culture. Both IGF-I and IGF-II enhanced the ALPase activity of pulp cells cultured in TYF medium to an equivalent level, but a higher concentration of IGF-II was necessary to produce a similar effect on DNA synthesis to that of IGF-I. Insulin dose-dependently enhanced DNA synthesis and increased ALPase activity, but its effects were weaker than those of the IGFs. These findings suggest that IGF-I might have a primary role in the growth and differentiation of pulp cells.

Fluoride has been used for decades, either systemically or topically, to prevent dental caries. The purpose of this study was to clarify the effects of low concentrations of fluoride on proliferation, differentiation and extracellular-matrix synthesis in normal human dental pulp cells (DP-1 and DP-2) in vitro. The effects were compared with those on a human osteoblastic osteosarcoma cell line, TE-85. Fluoride at micromolar concentrations significantly and dose-dependently stimulated [3H]thymidine incorporation into DNA in DP-1, DP-2 and TE-85 cells, with optimal effects around 50 microM, by 127 +/- 7%, 124 +/- 0.6% and 152 +/- 13.4%, respectively. To assess the potential influence of fluoride on cell differentiation, the effects of mitogenic concentrations on alkaline phosphatase activity were measured. Fluoride significantly increased the enzyme's activity in DP-1 and TE-85 by 177 +/- 12% and 144 +/- 12.3%. To evaluate the effect on extracellular-matrix synthesis, the synthesis of type I collagen was indirectly determined by an assay of procollagen type I c-peptide production. Fluoride significantly increased that production by 150 +/- 8.7% in TE-85, but not in either DP-1 or DP-2. These observations suggest that fluoride, if used at low concentrations, could be a useful therapeutic agent where increased regeneration of dentine is desired, such as after pulp amputation, by stimulating the proliferation and differentiation of the dental pulp cells.

Localization of prostaglandin (PG) I2 synthase immunoreactivity was examined in demineralized sections of rat pulpal, periodontal and skeletal tissues using isn-1, a monoclonal antibody raised against the enzyme. Various calcified tissue-forming cells, i.e. odontoblasts, osteoblasts, osteocytes, cementoblasts, cementocytes and chondrocytes, were similarly immunoreactive for PGI2 synthase, suggesting that they are capable of producing PGI2. In odontoblasts and chondrocytes, the reactivity increased gradually with maturation. Weak immunoreactivity was also observed in endothelial cells and fibroblast-like cells in pulpal and periodontal tissues. However, no reactivity was seen in ameloblasts. These results suggest the possible involvement of PGI2 in the regulation of the metabolism of various calcified tissues. Monoclonal antibodies such as isn-1 may become useful markers of the maturation of calcified tissue-forming cells of mesenchymal origin.

An established pulp cell line (RPC-C2A) was used to study the regulatory effect of insulin on dentinogenesis. Insulin increased alkaline phosphatase activity and the incorporation of [2,3-3H]-proline into collagenase-digestible protein, whereas [3H]-thymidine incorporation by the cells was inhibited by insulin. The enhancing effect of insulin on alkaline phosphatase activity was inhibited by epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta). The stimulatory effect of insulin on collagen synthesis was also inhibited when insulin was combined with EGF, but was accelerated by the addition of TGF-beta. Inhibitory effects of insulin on the [3H]-thymidine incorporation were potentiated by EGF, though EGF alone strongly increased the effect; whereas the addition of TGF-beta had no significant effect on the insulin action. These findings suggest that insulin may be concerned with the differentiation of pulp cells in dentinogenesis and that EGF or TGF-beta regulate the insulin effects.

Platelet-derived growth factor (PDGF), insulin-like growth factor-I and -II (IGF-I and -II), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated [125I]-deoxyuridine incorporation about 13, 6.2-, 4.6-, 3.8-, 3.1- and 1.2-fold, respectively, above control values at a concentration of 50 ng/ml. Transforming growth factor-beta (TGF-beta) decreased incorporation about 30% at the same dose. aFGF, IGF-I, IGF-II, bFGF and TGF-beta increased [35S]-sulphate incorporation 231, 71, 64, 42 and 39%, respectively, in proliferating cells, while EGF, IGF-I, TGF-beta and PDGF decreased incorporation about 30%, and aFGF increased incorporation 80% in stationary-stage culture. TGF-beta, PDGF, aFGF and bFGF caused 65-40% inhibition of alkaline phosphatase activity in proliferating and stationary cultures. These findings suggest that the proliferation of pulp cells may be stimulated mainly by PDGF and IGF-I, and the production of extracellular matrix proteoglycan may be enhanced by aFGF, IGF-I and IGF-II. Furthermore, TGF-beta, PDGF, aFGF and bFGF may regulate the differentiation of pulp cells into odontoblasts.

This activity was demonstrated in blood vessels, fibroblast-like cells, odontoblasts and Schwann cells surrounding non-myelinated axons. It was present on both the luminal and abluminal plasma membrane of endothelial cells. The plasma membrane of fibroblast-like cells and their processes had patches of fine electron-dense end-product corresponding to DPP-IV activity, while the plasma membrane of odontoblasts was loaded with a consistent reaction product. DPP-IV activity in the non-myelin-forming Schwann cells was in the plasma membrane of the free surface in contact with the extracellular matrix as well as in the plasma membrane in close contact with the axolemma. The plasma membrane of Schwann cells producing myelin sheath had no positive reaction for DPP-IV. The DPP-IV activity in the plasma membrane of fibroblast-like cells and odontoblasts may be associated with fibronectin-mediated adhesion to collagen, whereas the membrane-bound DPP-IV activity in endothelium and non-myelin-forming Schwann cells may be involved in cleavage of neuropeptides of other biologically active peptides.

These factors influence proliferation and differentiation in various cell types. Their effects on a clonal cell line (RPC-C2A) having high ALPase activity were examined by assay of [3H]-thymidine incorporation and ALPase activity. Neither factor (at a dose of 0.5 ng/ml) altered the shape of the pulp cells. DNA synthesis was not affected by transforming growth factor-beta either in growing cells or in those nearly confluent, but epidermal growth factor, in doses ranging from 0.5 to 10 ng/ml, stimulated the incorporation of [3H]-thymidine in nearly confluent cells. Both factors inhibited ALPase activity in a dose-dependent manner. Indomethacin did not affect this inhibition, suggesting that this effect of growth factors may not be mediated by prostaglandin synthesis. Inhibitory effects of ALPase antagonists (L-phenylalanine, L-homoarginine, levamisole) were not affected by transforming growth factor-beta. Thus epidermal growth factor stimulates DNA synthesis and both transforming growth factor-beta and epidermal growth factor inhibit ALPase activity of clonal rat pulp cells, suggesting that both factors may act as regulators of biological function, including cell differentiation, in pulp cells.

Alkaline phosphatase of rat dental pulp was separated into two forms by anion-exchange chromatography, gel-filtration and electrophoresis. The minor activity (AP I) was eluted at 0.1 M of NaCl from a DEAE-cellulose column, and the major activity (AP II) in the broad range of approx. 0.15-0.5 M. These two enzyme activities gave rise to distinct peaks on a TSK-GEL Toyopearl (Fractogel TSK) HW 55-S column and showed different electrophoretic behaviours on both sodium dodecyl sulphate (SDS) and non-SDS polyacrylamide gels. The molecular weights of AP I and AP II were estimated by gel-filtration to be 130,000-150,000 and above 500,000, respectively. On SDS gel electrophoresis, the molecular weight of AP II was changed to 130,000, while AP I had a molecular weight of 150,000. Judging from the sensitivities to heat and some inhibitors, AP I and AP II are biochemically indistinguishable from each other.

Judging from the mobility after the sodium dodecyl sulphate-gel electrophoresis, the mol. wt of the dental pulp enzyme was estimated to be 125,000-130,000. The electrophoretic behaviour of this enzyme was identical with that of calvarial phosphatase but not with those of kidney and intestinal enzymes. These results suggest that the dental pulp enzyme has a similar molecular structure to bone alkaline phosphatase.

By ammonium-sulphate fractionation, phosphocellulose column chromatography, and isoelectric focusing, pyruvate kinase (EC 2.7.1.40) was purified from pig dental pulps to yield a homogeneous preparation with a single protein band of approx. 63,000 daltons upon SDS-polyacrylamide gel electrophoresis. As the molecular weight of the enzyme was estimated to be 250,000 by Sephadex G-200 gel filtration, a tetrameric structure is indicated. The isoelectric point of the purified enzyme was 8.0; the optimal pH, pH 7.2. At this pH, there was no substrate inhibition of the enzyme by either phosphoenolpyruvate (PEP) or ADP, and the relationship between reaction velocity and each substrate concentration was well-explained by the Michaelis-Menten equation. The Km values were 53 and 286 microM for PEP and ADP, respectively. The enzyme had different catalytic properties at pH 8.0: with 0.4 mM ADP, the kinetic behaviour gave a sigmoidal curve with respect to PEP and fructose-1,6-diphosphate acted as an allosteric activator. With 2.0 mM ADP, a hyperbolic curve resulted with PEP.

Phosphofructokinase (EC 2.7.1.11) was partially purified 19-fold from dental pulps of rat incisors, by ammonium-sulphate fractionation and phenyl-Sepharose chromatography with a recovery of about 95 per cent. With a 0.05 M tris-HCl buffer, the pH optimum of the enzyme was determined to be 8.4. At this pH, substrate inhibition of the enzyme by either fructose-6-phosphate (F6P) or ATP was not observed, and the relationship between reaction velocity and each substrate concentration was well explained by the Michaelis-Menten equation. The Km values were determined to be 5 X 10(-5) and 1.8 X 10(-4) M for ATP and F6P, respectively. The enzyme, however, showed different catalytic properties at pH 7.6, i.e. the kinetic behaviour was sigmoidal with respect to F6P and it was inhibited by high concentrations of ATP. The Hill coefficient for F6P was determined graphically to be 1.4. At pH 7.6, the enzyme activity was inhibited by citrate and phosphoenolpyruvate (PEP), neither of which showed any inhibitory effect on the enzyme at pH 8.4.

Alkaline phosphatase (E.C.3.1.3.1.) from unerupted bovine pulp was extracted from the microsomal fraction with eta-butanol and purified 77-fold, using DEAE-cellulose chromatography, Sephadex G-200 gel-filtration and concanavalin-A affinity chromatography, to a final specific activity of 92.3 units/mg protein. Affinity chromatography confirmed the glycoprotein nature of the enzyme. The pH optimum for the purified enzyme was 10.0 with rho-nitrophenylphosphate, and 8.7 with phosphoserine. The apparent Km was estimated to be 0.7 mM, using rho-nitrophenylphosphate in glycine-NaOH buffer, pH 10.0. The enzyme was markedly inhibited by EDTA, bromotetramisole and homoarginine but was insensitive to phenylalanine, and therefore resembled the alkaline phosphatase of liver and bone, but not that of intestine and placenta. No protein phosphatase activity towards dentine phosphoprotein and phosvitin was observed.

Collagen concentration was fairly low in young bovine 3rd premolar pulps, about 9 per cent by dry weight of pulp in stage I, but it increased consistently up to more than 25 per cent at stage V as the pulp matured. The ratio of type III to type I collagen similarly increased from 13 per cent at stage I to 32 per cent at stage V. Both collagenase and possibly collagenolytic neutral peptidases showed significantly high activity in the early stage of pulp development where the rate of collagen synthesis was also elevated. Higher rates of both collagen synthesis and its degradation might explain the low concentration and content in the early stage of pulp development.

The sensitivity of the dental pulp enzyme to heat was similar to that of enzymes from bone and kidney; alkaline phosphatases from liver and intestine were more stable to heat. L-Homoarginine strongly inhibited the enzyme activities from pulp, bone, kidney and liver but did not affect intestinal enzyme activity. Increasing the molarity of carbonate buffer or glycine buffer in the assay solution decreased intestinal alkaline phosphatase activity more markedly than enzyme activities of other tissues. In 100 mM glycine-NaOH buffer, the effects of Zn2+, Mg2+ and Ca2+ on pulp alkaline phosphatase activity were similar to those on the enzyme activities of bone, kidney and liver. The electrophoretic pattern of the pulp enzyme on sodium dodecyl sulphate-gels was identical with that of bone enzyme and differed from the patterns of enzymes from other tissues. These results suggest that the dental pulp alkaline phosphatase may be the same as bone enzyme.

Maxillary incisor pulp cells from male Wistar rats (7 weeks old) were cultured. After the 52nd subculture, cloning was performed twice and a clonal cell line (RPC-C2A) with high alkaline phosphatase (ALP) activity was established. The population doubling time of RPC-C2A cells was 13.7 h, and the mode of the chromosome number was 42. The heat stability of ALP, the effect of the ALP inhibitors, and the ALP polyacrylamide gel electrophoresis pattern in RPC-C2A cells was the same as for the ALP of the isolated dental pulp.

Odontoblasts of incisor and premolar teeth showed high prolyl-hydroxylase (EC 1.14.11.2; proline, 2-oxoglutarate dioxygenase) activity, but the activities in other regions of these dental pulps were low. These results suggest that these odontoblasts are concerned with the biosynthesis and processing of dentine collagen.