Effect of neurotrophins on differentiation, calcification and proliferation in cultures of human pulp cells

Cell Biology International 31 (2007) 1462e1469 www.elsevier.com/locate/cellbi

Effect of neurotrophins on differentiation, calcification and proliferation in cultures of human pulp cells Noriyoshi Mizuno*, Hideki Shiba, Wan-peng Xu, Takafumi Inui, Tsuyoshi Fujita, Mikihito Kajiya, Katsuhiro Takeda, Naohiko Hasegawa, Hiroyuki Kawaguchi, Hidemi Kurihara Department of Periodontal Medicine, Division of Frontier Medical Science, Programs for Biomedical Research, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan Received 28 June 2006; revised 30 March 2007; accepted 26 June 2007

Abstract Neurotrophins (NTs) are expressed during tooth development. However, little is known about a role of NTs in differentiation of pulp cells into mineralizing cells. In this study, mRNA expressions of hard tissue-related proteins, calcification and proliferation are examined in cultures of human pulp (HP) cells. Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5 increased the mRNA levels of dentin sialophsphoprotein, alkaline phosphatase, osteopontin, type I collagen and bone morphogenetic protein-2 and mineral deposition in cultures of HP cells. The increased levels and manners varied, depending on the concentrations of NTs and hard-tissue related protein tested. On the other hand, only NGF significantly stimulated DNA synthesis in cultures of HP cells. These findings suggest that NTs characteristically regulate hard-tissue related protein expression, calcification and proliferation in pulp cells. NTs may accelerate pulp cell differentiation. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Neurotrophins; Human pulp cells; Hard tissue-related proteins

1. Introduction Neurotrophins (NTs) in mammals consist of a family of four polypeptide growth factors: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5. NTs exert their biological functions by binding high affinity receptors and a low affinity receptor. High affinity receptors are tyrosine kinase receptor (trk) A, trkB and trkC and the low-affinity receptor is 75-kDa glycoprotein receptor (p75) (McInnes and Sykes, 1997; Dechant, 2001; Patapoutian and Reichardt, 2001). NGF, BDNF and NT-4/5, and NT-3 bind to trkA, trkB and trkC, respectively

* Corresponding author. Tel.: þ81 82 257 5663; fax: þ81 82 257 5664. E-mail address: [email protected] (N. Mizuno).

(Kaplan et al., 1991; Klein et al., 1991; Lamballe et al., 1991). NT-3 also has some cross-binding to both trkA and trkB (Kaplan et al., 1991; Klein et al., 1991). On the other hand, all the NTs bind to the p75 (Johnson et al., 1986). The biological function of NTs was originally recognized as regulating the differentiation, maintenance, survival and cell death of neurons in the central and peripheral nervous systems (Chao, 1992; Barrett, 2000). Cellular responses of various non-neuronal cells are also regulated by NTs. For example, BDNF promotes survival of mouse endothelial cells, and differentiation and survival of Xenopus laevis retinal pigment epithelium (Donovan et al., 2000; Liu et al., 1997). BDNF and NT-3 mediate survival and proliferation in mouse fibroblasts (Glass et al., 1991). NGF regulates migration and proliferation in human choroidal endothelial cells (Steinle and Granger, 2003).

1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.06.012

N. Mizuno et al. / Cell Biology International 31 (2007) 1462e1469

Pulp tissues possess the system of sensorium and defense against stimulation, such as caries and tooth preparation. Calcium hydroxide that has the potential to stimulate pulp cells to produce reparative dentin is well-known as a pulp capping agents to preserve pulp tissues. However, due to the high alkalinity, it induces necrosis in pulp tissues, that is undesirable phenomenon. Agents that bioactively induce dentin formation without necrosis are required for the preservation of pulp tissues. Earlier work using radioimmunoassay and immunohistochemistry demonstrates that NGF is present in the rat pulp, rat epithelial preameloblasts and rat mesenchymal odontoblasts (Naftel et al., 1992; Mitsiadis et al., 1992, 1993). Furthermore, NTs play a role in tooth development (Nosrat et al., 1998). NTs may modulate the differentiation of odontoblast progenitor cells during tooth development. Therefore, NTs are suggested to be candidates for pulp capping agents which bioactively stimulate regenerative dentin formation to preserve pulp tissues. Dentin sialoprotein and dentin phosphoprotein are expressed in dentin and are the result of a common transcript for dentin sialophsphoprotein (DSPP) (MacDougall et al., 1997; Feng et al., 1998). Alkaline phosphatase (ALPase) activity in odontoblasts and subodontoblasts is higher than that in undifferentiated pulp cells (Nuki and Bonting, 1961). ALPase activity may be needed for differentiation of pulp cells into odontoblasts. Osteopontin (OPN) produced by pulp cells or odontoblasts is thought to play a role in reparative dentin formation (Yokota et al., 1992). Type I collagen constitutes most of the organic portion of dentin (Rabie and Veis, 1995). BMP-2 is involved in both epithelial-mesenchymal interaction and in the differentiation of odontoblast lineage in tooth development (Thesleff et al., 1995). Thus, DSPP, ALPase, OPN, Type I collagen and BMP-2 produced by pulp cells are considered to be markers of mineralizing cells differentiation. (Rashid et al., 2003; Shiba et al., 1995, 2001, 2003; Nakashima et al., 2004). In the present study, to elucidate the role of NTs in differentiation of pulp cells into mineralizing cells, we examined the effect of four NGF, BDNF, NT-3 and NT-4/5 on mRNA expressions of the DSPP, ALPase, OPN, Type I collagen and BMP-2, calcification and the proliferation in cultures of human pulp (HP) cells. 2. Materials and methods

Israel), 100 units/mL penicillin, 100 mg/mL streptomycin and 1 mg/mL amphotericin B (medium A). The cells were seeded in 100 mm diameter plastic culture dishes, and incubated in 5% CO2/95% air at 37  C. When the cells reached confluence, they were harvested and subcultured at a density of 8  105 cells per 100 mm dish in medium A.

2.3. DNA synthesis DNA synthesis was estimated by measuring incorporation of bromodeoxyuridine (BrdU) into DNA using Cell proliferation ELISA system, version 2 (Amersham Pharmacia, Little Chalfont, England). HP cells in cultures at the 6th passage were harvested, seeded at a density of 5  103 cells/well in 96-well plastic culture plate coated with type I collagen, and maintained in 0.1 ml of medium A supplemented with 50 mg/ml ascorbic acid (medium B). After 9 days, each cell was incubated in DMEM supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, 1 mg/ml amphotericin B and 50 mg/ml ascorbic acid (medium C) with or without each NTs for 24 h. BrdU (10 mM) was added 4 h before the end of incubation. Immunodetection of the incorporated BrdU into cells was performed according to the manual supplied with Cell proliferation ELISA system, version 2.

2.4. Isolation of total RNA HP cells in culture at the 6th passage were harvested, seeded at a density of 35  104 cells/60 mm plastic tissue culture dish and maintained in medium B. HP cells were exposed to various concentrations of each NT for 24 h in medium C. Total RNA from each culture was extracted using ISOGENÒ (Wako Pure Chemical Industries, Osaka, Japan) on day 10 and quantified by spectrometry at 260 and 280 nm.

2.5. Real-time PCR Real-time PCR was performed with an ABI 7700 system (Applied Biosystems, Tokyo, Japan). Using a Core Reagent Kit (Applied Biosystems), reactions were carried out according to the manufacturer’s protocol. The TaqMan probe, sense primers and anti-sense primers used for detection of DSPP, ALPase, OPN, Type I collagen and BMP-2 are listed in Table 1. Human commercially available GAPDH (Applied Biosystems) was used for real-time PCR.

2.6. Calcification HP cells in cultures at the 6th passages were harvested and seeded at a density of 3  104 cells/well in 24-well plastic culture plate coated with type I

Table 1 Primers and probes used in real-time PCR Gene

Sequence

DSPP

F: 50 -GCATTCAGGGACAAGTAAGCA-30 R: 50 -CTTGGACAACAGCGACATCCT-30 P: 50 -CATTGACAATGCTGGAGCCACAAACAGA-30 F: 50 -CGAGATACAAGCACTCCCACTTC-30 R: 50 -CTGTTCAGCTCGTACTGCATGTC-30 P: 50 -CCTGGCTCGAAGAGACCCAATAGGTAGTCC-30 F: 50 -CAGGCTGATTCTGGAAGTTCTGA-30 R: 50 -GGCTAGGAGATTCTGCTTCTGAGA-30 P: 50 -CAACAAATACCCAGATGCTGTGGCCA-30 F: 50 -TCTGAAGTCTCTCAACAACCAGATTG-30 R: 50 -GGGTGGCTGAGTCTCAAGTCA-30 P: 50 -CTCTAGAAAGAACCCAGCTCGCACATGC-30 F: 50 -CCAACACTGTGCGCAGCTT-30 R: 50 -AGAAGAATCTCCGGGTTGTTTTC-30 P: 50 -CACCATGAAGAATCTTTGGAAGAACTACCA-30

2.1. NTs Human recombinant NGF, BDNF, NT-3 and NT-4/5 were purchased from R & D systems (Minneapolis, MN).

ALPase

2.2. Cell culture

OPN

Three periodontally healthy and non-carious upper third molars were collected from three patients. Informed consent was obtained from all the patients according to a protocol approved by ethical authorities at Hiroshima University. HP cells (HP cells-A, -B and -C) were isolated from three pulps and maintained separately as previously described (Rashid et al., 2003). The cell suspension was centrifuged at 800  g for 10 min, and the pellet was suspended in Dulbecco’s modified Eagle’s medium (DMEM, Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS, BEIT HAEMEK,

1463

Type I collagen

BMP-2

F: forward, R: reverse, P: Taqman probe.

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2.8. Statistical analysis

collagen and maintained in 0.5 ml of DMEM with 10% FCS containing 10 mM b-glycerophosphate, 50 mM ascorbic acid and 0.1 mM dexamethasone in the absence or presence of each NT for 21 days. The medium was replaced twice a week. Mineralized formation was identified by von Kossa staining.

Statistical analyses of the data were performed using analysis of variance (ANOVA).

2.7. Cell number

2.9. Measurement of OPN

Cell number in each culture was calculated by CellTiter 96Ò Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI).

HP cells in cultures at the 5th passage were harvested, seeded at a density of 1  104 cells/well in 48-well plastic culture plate, and maintained in 0.2 ml

DSPP 8

6 4

*

* 2

25

5

0

100 (ng/ml)

6

*

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5

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2

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25

100 (ng/ml)

8

6

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100 (ng/ml)

ALPase BDNF

NGF

4

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NT-4/5

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ALPase mRNA/GAPDH mRNA (ratio to control)

ALPase mRNA/GAPDH mRNA (ratio to control)

**

NT-4/5

6

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BDNF

8

NT-3

8

DSPP mRNA/GAPDH mRNA (ratio to control)

0

B

ALPase mRNA/GAPDH mRNA (ratio to control)

DSPP mRNA/GAPDH mRNA (ratio to control)

NGF

ALPase mRNA/GAPDH mRNA (ratio to control)

DSPP mRNA/GAPDH mRNA (ratio to control)

DSPP mRNA/GAPDH mRNA (ratio to control)

A

25

100 (ng/ml)

*

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* 2

1 0

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Fig. 1. Dose-dependent effects of NTs on DSPP and ALPase mRNA expressions in HP cells. HP cells-B were exposed to various concentrations of NGF, BDNF, NT-3 and NT-4/5 for 24 h before the end of incubation on day 10. The mRNA expressions were determined by real-time PCR. The graphs show the ratio of DSPP or ALPase mRNA to GAPDH mRNA. Values are means  S.D. of three cultures. There was a significant difference (**P < 0.05, *P < 0.01) from the control.

N. Mizuno et al. / Cell Biology International 31 (2007) 1462e1469

2.10. ALPase activity

of medium B. After 10 days, cells were washed 3 times with DMEM. Then, HP cells at confluent cultures were exposed to 100 ng/ml NGF, 100 ng/ml BDNF, 25 ng/ml NT-3, and 25 ng/ml NT-4/5 in medium C for 24 h before the end of incubation on day 11. OPN levels in the conditioned media from HP cells cultures were determined using a sandwich ELISA kit (ImmunoBiological Laboratories Co., LTD, Gunma, Japan).

HP cells in cultures at the 5th passage were harvested, seeded at a density of 1  104 cells/well in 24-well plastic culture plate, and maintained in 0.5 ml of medium B. After 10 days, cells were washed 3 times with DMEM. HPL cells at confluent cultures were exposed to 100 ng/ml NGF, 100 ng/ml

A

OPN BDNF

NGF

** *

2 1 0

OPN mRNA/GAPDH mRNA (ratio to control)

OPN mRNA/GAPDH mRNA (ratio to control)

3

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B

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Type I collagen BDNF

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Type I collagen mRNA/GAPDH mRNA (ratio to control)

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Type I collagen mRNA/GAPDH mRNA (ratio to control)

NGF

Type I collagen mRNA/GAPDH mRNA (ratio to control)

100 (ng/ml)

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25

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OPN mRNA/GAPDH mRNA (ratio to control)

OPN mRNA/GAPDH mRNA (ratio to control)

4

Type I collagen mRNA/GAPDH mRNA (ratio to control)

1465

0

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NT-4/5

3

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*

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Fig. 2. Dose-dependent effects of NTs on OPN and Type I collagen mRNA expressions in HP cells. HP cells-B were exposed to various concentrations of NGF, BDNF, NT-3 and NT-4/5 for 24 h before the end of incubation on day 10. The mRNA expressions were determined by real-time PCR. The graphs show the ratio of OPN or Type I collagen mRNA to GAPDH mRNA. Values are means  S.D. of three cultures. There was a significant difference (*P < 0.01, **P < 0.05) from the control.

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BDNF, 25 ng/ml NT-3, and 25 ng/ml NT-4/5 in medium C for 72 h before the end of incubation on day 13 and homogenized at 4  C in 0.5 ml of 10 mM TriseHCl buffer (pH 7.4) containing 2.5 mM MgCl2. ALPase activity was measured by the method of Bessey et al. using p-nitrophenyl phosphate as a substrate. One unit was defined as the amount of enzyme required to hydrolyze 1 nmole p-nitrophenyl phosphate per min.

3. Results Figs. 1e3 show the effects of increasing concentrations of NTs on the mRNA expressions of DSPP (Fig. 1A), ALPase (Fig. 1B), OPN (Fig. 2A), type I collagen (Fig. 2B) and BMP-2 (Fig. 3) in cultures of HP cells-B. The stimulatory effect of NGF, BDNF and NT-3 on DSPP mRNA expression was detected at 25 and 100 ng/ml (Fig. 1A). NGF at 100 ng/ml caused a 1.8-fold increase, BDNF at 100 ng/ml caused a 6.3-fold increase, and NT-3 at 25 ng/ml caused a 3.6-fold increase in DSPP mRNA levels with a maximal effect. NT-4/5 at only 25 ng/ml caused a 2.0-fold increase in DSPP mRNA levels (Fig. 1A). NGF at 100 ng/ml caused a 3.3-fold increase, BDNF at 100 ng/ml caused a 3.0-fold increase, NT-3 at 25 ng/ml caused a 2.6-fold increase and NT-4/5 at 25 ng/ml caused a 2.3-fold increase in ALPase mRNA levels (Fig. 1B). NGF, BDNF and NT-4/5 increased OPN mRNA levels in a dose-dependent manner. NGF at 100 ng/ml caused a 3.2-fold increase, BDNF at 100 ng/ml caused a 2.8-fold increase, and NT-4/5 at 100 ng/ml caused a 2.9-fold increase in OPN mRNA. NT-3 at 25 ng/ml caused a 2.6-fold increase in OPN mRNA with a maximal effect (Fig. 2A). All the NTs tested increased type I collagen mRNA levels in a dose-dependent manner. NGF at 100 ng/ml caused a 2.1-fold increase, BDNF at 100 ng/ml caused a 2.9-fold increase,

NT-4/5 at 100 ng/ml caused a 1.9-fold increase and NT-3 at 25 ng/ml caused a 2.0-fold increase in Type I collagen mRNA levels (Fig. 2B). Increased BMP-2 mRNA levels were seen after the stimulation of NGF, BDNF and NT-4/5 at only 100 ng/ml (Fig. 3A). NT-3 at 5 and 25 ng/ml stimulated BMP-2 mRNA expression. All the NTs tested increased mRNA levels of DSPP, ALPase, OPN, Type I collagen and BMP-2. Thus, the increased levels and manners varied, depending on the gene examined and the concentrations of NTs. Similar results were obtained with HP cells-A and HP cells-C (Table 2). Fig. 4 shows the effect of NTs on calcification in cultures of HP cells-A. Calcification was identified by von Kossa staining. Von Kossa positive substances were observed in cultures of HP cells exposed to all the NTs. Similar results were obtained with HP cells-B and HP cells-C (data not shown). Fig. 5 shows the effect of NTs on the incorpotarion of BrdU into DNA in HP cells-B. NGF at 25 ng/ml significantly increased BrdU incorporation in HP cells. On the other hand, BDNF, NT-3 and NT-4/5 did not influence the incorporation of BrdU into DNA in HP cells. Similar results were obtained with HP cells-A and HP cells-C (data not shown). Fig. 6 shows the effect of NTs on the secreted levels of OPN (A) and ALPase activity (B). All the NTs stimulated the OPN secretion and ALPase activity. 4. Discussion Pulp cells have been reported to consist of cell populations, such as pulp fibroblasts and dental pulp stem cells. The dental pulp stem cells are able to differentiate into odontoblasts and

2

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BMP-2 mRNA/GAPDH mRNA (ratio to control)

BMP-2 NGF

BDNF

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NT-4/5

3

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Fig. 3. Dose-dependent effects of NTs on BMP-2 mRNA expressions in HP cells. HP cells-B were exposed to various concentrations of NGF, BDNF, NT-3 and NT-4/5 for 24 h before the end of incubation on day 10. The mRNA expressions were determined by real-time PCR. The graphs show the ratio BMP-2 mRNA to GAPDH mRNA. Values are means  S.D. of three cultures. There was a significant difference (**P < 0.05) from the control.

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Table 2 Effect of NTs on DSPP, ALPase, OPN, Type I collagen and BMP-2 mRNA expressions in HP cells-A, -B and -C DSPP mRNA/GAPDH mRNA (ratio to control, means  S.D.) HP cells

NGF (100 ng/ml)

BDNF (100 ng/ml)

A 1.89  0.06 4.78  0.30 B 1.76  0.03 6.26  0.18 C 2.48  0.22 6.11  0.15 ALPase mRNA/GAPDH mRNA (ratio to control, means  S.D.) HP cells NGF (100 ng/ml) BDNF (100 ng/ml) A 2.54  0.09 2.57  0.20 B 3.23  0.06 2.96  0.23 C 3.93  0.17 2.52  0.17 OPN mRNA/GAPDH mRNA (ratio to control, means  S.D.) HP cells NGF (100 ng/ml) BDNF (100 ng/ml) A 2.70  0.09 2.07  0.11 B 3.16  0.17 2.75  0.12 C 4.03  0.39 3.14  0.09 Type I collagen mRNA/GAPDH mRNA (ratio to control, means  S.D.) HP cells NGF (100 ng/ml) BDNF (100 ng/ml) A 2.30  0.10 2.86  0.04 B 2.09  0.11 2.90  0.18 C 2.03  0.11 2.31  0.05 BMP-2 mRNA/GAPDH mRNA (ratio to control, means  S.D.) HP cells NGF (100 ng/ml) BDNF (100 ng/ml) A 2.10  0.10 2.07  0.09 B 1.89  0.07 1.80  0.13 C 2.57  0.15 2.11  0.08

osteoblasts (Papaccio et al., 2006). The differentiated odontoblasts produce DSPP (Papagerakis et al., 2002), although this protein is not expressed in undifferentiated pulp cells. Recently, DSPP has also been found to be expressed in osteoblasts. Therefore, DSPP is a phenotypic marker of osteoblasts as well as odontoblasts. The present study showed that NTs increases DSPP mRNA levels in cultures of HP cells. Especially, BDNF and NT-3 remarkably enhanced the DSPP mRNA expression. Furthermore, NTs also increased ALPase, OPN, OC, BMP-2 and type I collagen mRNA levels and calcified substances in cultures of HP cells. The increased levels and manners varied, depending on the hard-tissue related gene examined and the concentration of NTs. NTs and their receptors were expressed in cultures of HP cells (unpublished data). These findings suggest that NTs have the capacity to differentiate pulp cells into mineralizing cells. Further study is necessary to elucidate what cells stimulation of NTs differentiates the stem cells into. The tyrosine kinase receptors (trk) are essential components of neurotrophin high-affinity binding sites that trigger cell survival, growth, and differentiation. The major trk signaling pathways are correlated with MAP kinase and serine/threonine kinase Akt (Segal and Greenberg, 1996; Casaccia-Bonnefil et al., 1999; Patapoutian and Reichardt, 2001). It has been reported that there are at least 2 parallel and distinct p75 signalling pathways; NFkB and JNK (Carter et al., 1996; Bothwell, 1996; Carter and Lewin, 1997). The mechanism whereby NTs act on pulp cells through their receptors and signaling needs to be researched in order to elucidate the detached role of NTs in reparative dentin formation.

NT-3 (25 ng/ml)

NT-4/5 (25 ng/ml)

3.66  0.14 3.57  0.24 4.75  0.09

1.80  0.11 1.96  0.14 2.04  0.03

NT-3 (25 ng/ml) 2.36  0.17 2.58  0.21 2.20  0.08

NT-4/5 (25 ng/ml) 2.47  0.22 2.25  0.03 2.65  0.18

NT-3 (25 ng/ml) 2.01  0.05 2.56  0.22 2.47  0.28

NT-4/5 (100 ng/ml) 2.19  0.06 2.86  0.04 3.05  0.13

NT-3 (25 ng/ml) 2.15  0.15 1.99  0.22 1.76  0.09

NT-4/5 (25 ng/ml) 1.85  0.12 1.89  0.03 2.21  0.12

NT-3 (5 ng/ml) 2.22  0.13 1.62  0.05 1.68  0.02

NT-4/5 (100 ng/ml) 1.98  0.13 2.26  0.13 2.23  0.01

NGF stimulated DNA synthesis in cultures of HP cells. On the other hand, the proliferative activity of BDNF, NT-3 and NT-4/5 was not observed in cultures of HP cells. Thus, BDNF, NT-3 and NT-4/5 may function as a differentiation

NGF

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NT-4

0

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(ng/ml) Fig. 4. Effect of increasing concentrations of NTs on calcification in HP cells cultures. HP cells-A were seeded at 3  104 cells/well in 24 well plastic culture plate, grown in the medium for calcification and fixed. Mineral deposition was stained with von Kossa technique.

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DNA synthesis BDNF

* BrdU incorporation/cell ( of control)

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Fig. 5. Effect of increasing concentrations of NTs on DNA synthesis in cultures of HP cells. HP cells-B were exposed to various concentrations of NGF, BDNF, NT-3 and NT-4/5 for 24 h on day 10. DNA synthesis was determined by incorporation of BrdU into DNA using an ELISA kit. Values are means  S.D. of three cultures. There was a significant difference (*P < 0.05) from the control.

factor of HP cells rather than as a proliferation factor. On the other hand, NGF may work an HP cells differentiation during both proliferative and differentiative stages. Cell proliferation is fundamental process during tissue regeneration, since the

Secreted OPN (ng/ml)

A

*

50

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30 20 10

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ALPase activity (ratio to control)

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NGF (100 ng/ml)

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BDNF NGF (100 ng/ml) (100 ng/ml)

NT-3 (25 ng/ml)

NT-4/5 (25 ng/ml)

Fig. 6. Effect of NTs on secreted levels of OPN and ALPase activity in cultures of HP cells. HP cells were exposed to 100 ng/ml NGF, 100 ng/ml BDNF, 25 ng/ml NT-3, and 100 ng/ml NT-4/5 for 24 and 72 h before the end of incubation, for the determination of the OPN levels (A) and ALPase activity (B), respectively. Values are means  S.D. of three cultures. There was a significant difference (**P < 0.05) from the control.

number of cells due to be involved in mineralizing cells differentiation is increased. The outstanding stimulation of calcification by NGF in HP cells may result from cell proliferative activity NGF possesses. BMP-2 accelerates DSPP mRNA expression, but it does not influence cell proliferation in human pulp cells (Saito et al., 2004). Thus, BMP-2 is suggested to promote the differentiation of human pulp cells into mineralizing cells. In the present study, NTs increased BMP-2 mRNA levels in HP cells. Therefore BMP-2 produced by NTs may be partially involved in the stimulation of DSPP expression. We examined the effects of NTs in cultures of HP cells in order to clarify whether these peptides have a potential of HP cells differentiation. The present study, for the first time, demonstrates that NTs accelerate DSPP, ALPase, OPN, type I collagen and BMP-2 mRNA expressions and calcified substances formation in cultures of HP cells. In addition, NGF stimulated cell proliferation in cultures of HP cells. These findings suggest that a new role of NTs in differentiating pulp cells into mineralizing cells in addition to the reported functioning of the peptides. In wound healing of pulp tissue, suitable biological molecules are needed for reparative dentin formation. NTs may be candidates of capping agents, which biologically stimulate reparative dentin formation. Acknowledgements This work was supported in part by Grant-in-Aid for Scientific Research (A) (No. 14207082) and Grant-in-Aid for Encouragement of Young Scientists (B) (No. 16791314) from the Japan Society for the Promotion of Science, Japan.

N. Mizuno et al. / Cell Biology International 31 (2007) 1462e1469

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