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Nitric Oxide in Pulp Cell Growth, Differentiation, and Mineralization

¹ Department of Biochemistry and
² Department of Clinical Cariology and Endodontology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

^* corresponding author, yoichim{at}dent.showa-u.ac.jp

	ABSTRACT

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Dental preparation sometimes causes transient congestion, edema, and necrosis of the pulp. We hypothesized that nitric oxide (NO) is involved in the pathophysiological changes in pulp after preparation. The mRNA and protein expression of the inducible isoform of NO synthase (iNOS) was examined in murine pulp after dental preparation. The effects of NO on the proliferation, mineralization, and apoptosis of pulp cells were also studied in vitro. We found that not only iNOS, but also mRNAs for alkaline phosphatase and plasma membrane glycoprotein-1, were expressed in the pulp after preparation. NOC-18, an NO donor, suppressed the proliferation of pulp cells without inducing cell death, whereas it promoted the mineralization of cells cultured in the presence of ß-glycerophosphate, ascorbic acid, dexamethasone, and KH₂PO₄. Under these conditions, NOC-18 induced the apoptosis of pulp cells. These results suggest that NO regulates the growth, apoptosis, and mineralization of pulp cells.

KEY WORDS: dental pulp • nitric oxide • proliferation • mineralization • apoptosis

	INTRODUCTION

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Elimination of infected lesions by tooth preparation, followed by restoration of the defects with artificial materials, is one of the common treatments for dental caries. Tooth preparation sometimes causes transient congestion, edema, and necrosis of the pulp (Law et al., 1999; Hirata et al., 2005). It is believed that mechanical stimuli, including drilling, are transmitted to the pulp via dentinal tubules (Murray et al., 2003). However, the molecular mechanisms by which such changes are produced in the pulp have not been fully understood.

Nitric oxide (NO), a gaseous free radical produced by the inducible NO synthase (iNOS), is regarded as one of the mediators of inflammation in various tissues (Moncada et al., 1991; Moilanen and Vapaatalo, 1995). It has been reported that iNOS is expressed in human pulp with suppurative pulpitis (Di Nardo Di Maio et al., 2004), murine pulp exposed to lipopolysaccharide (Kawashima et al., 2005), and inflamed rat pulp after dental preparation and demineralization (Law et al., 1999). Recently, soluble guanylate cyclase (sGC), the receptor for NO, as well as 3 isotypes of NO synthases, including iNOS, were detected in rat odontoblasts (Korkmaz et al., 2005). These observations indicate the involvement of NO in the onset and/or progression of pulpitis.

Formation of reparative dentin is another phenomenon observed after tooth preparation. Pre-existing and/or newly developed odontoblasts in the pulp are regarded as responsible for the mineralization of reparative dentin matrix. NO mediates not only pro-inflammatory activities, but also the signals for the regulation of cell growth and differentiation (Teixeira et al., 2005). Therefore, it is plausible that NO serves as one of the modulators of odontoblast-like cell differentiation. It has been reported that NO is also involved in the mineralization of chondrocytes and osteoblasts (Inoue et al., 1995; Hikiji et al., 1997; Johnson et al., 2001), indicating its possible role in the production of tertiary dentin by odontoblasts.

The aim of this study was to clarify the role of NO in the responses of pulp cells after tooth preparation. We first examined the expression of iNOS in mouse pulp after tooth preparation, and then studied the effects of NO on the growth, differentiation, and mineralization of pulp cells in vitro.

	MATERIALS & METHODS

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Reagents
NOC-18, an NO donor, which liberates 2 NO molecules with a half-life of 21 hrs, was purchased from Dojindo Laboratories (Kumamoto, Japan). An NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human bone morphogenetic protein-2 (BMP-2) was kindly donated by Astellas Pharmaceuticals (Tokyo, Japan). Recombinant human transforming growth factor-ß1 (TGF-ß1) and fibroblast growth factor-2 (FGF-2) were obtained from R&D Systems (Minneapolis, MN, USA).

Tooth Preparation and Pulp-cell Culture
Male C57BL/6 mice were used according to the protocol approved by the Ethical Board for Animal Experiments at Showa University. Tooth preparation was performed as previously described (Hirata et al., 2005). Briefly, we abraded the apices of lower incisors of five-week-old mice under ether anesthesia, to reduce their length by 3 mm, using a carborundum point saw at 30,000 rpm. The mice were killed by decapitation, and pulp tissues were collected at 0.05, 1, and 3 hrs after tooth preparation.

Pulp was removed from the lower incisors of seven-day-old C57BL/6 mice, and cultured for 1 wk in MEM containing 20% fetal calf serum (FCS) (Trace Bioscience, Castel Hill, Australia) to allow for cell outgrowth (Nakao et al., 2004). The cells were detached with 0.1% collagenase A (Roche Diagnostic, Mannheim, Germany) and used for the experiments. To assess proliferation, we cultured the cells on the plates with or without type IA collagen coating (Nitta Gelatin, Osaka, Japan), as described previously (Nakao et al., 2004), in the presence of various concentrations of NOC-18. To examine ALP activity, mineralization, and cell death, we incubated cells with or without NOC-18 in MEM plus 20% FCS (normal medium) or in the same medium supplemented with 10 mM ß-glycerophosphate, 0.1 mM L-ascorbate, 10 nM dexamethasone, and 1.8 mM KH₂PO₄ (Narayanan et al., 2001), referred to as the ’mineralization-promoting (MP) medium’.

RT-PCR
Total RNA was extracted from pulp tissues and pulp cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RT-PCR analyses for iNOS, dentin sialophosphoprotein (DSPP), dentin matrix protein (DMP), alkaline phosphatase (ALP), and plasma membrane glycoprotein-1 (PC-1) were performed with primers synthesized by Invitrogen (APPENDIX). The primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Clontech Laboratories (Palo Alto, CA, USA).

Immunoblot
The tissue lysates were run on SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated for 1 hr with the primary antibodies for iNOS and dentin sialoprotein (DSP) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as well as with that for ß-actin (Sigma-Aldrich), followed by incubation with a horseradish-peroxidase-conjugated anti-rabbit IgG (GE Healthcare, Piscataway, NJ, USA). Immunoreactive bands were visualized by the enhanced chemiluminescence reaction with ECL reagent (GE Healthcare).

Trypan Blue Dye Exclusion Assay and Bromodeoxyuridine (BrdU) Labeling
The total and viable cell numbers were determined by a trypan blue dye exclusion assay. Cell growth was assessed by BrdU incorporation, with the BrdU Labeling and Detection Kit I (Roche Diagnostics). The BrdU incorporated by the cells was observed under a fluorescence microscope, and counted by flow cytometry (FACSCaliber, Becton Dickinson, San Jose, CA, USA). The flow cytometry data were analyzed with CellQuest (BDIS) software (Verity Software House, Topsham, ME, USA).

Alkaline Phosphatase (ALP) Activity and Alizarin Red Staining
To visualize ALP activity, we incubated the cells (fixed with 10% formalin in PBS) for 20 min with a mixture of 0.1 mg/mL naphthol AS-MX phosphate (Sigma-Aldrich), 0.6 mg/mL Fast Blue BB salt (Sigma-Aldrich), 2 mM MgCl₂, and 0.5% N,N-dimethylformamide in 0.1 M Tris-HCl, pH 8.5 (Takada et al., 2003). For quantification of ALP activity, the cells were disrupted by sonication on ice in 50 mM Tris-HCl (pH 7.5) containing 0.1% Triton X-100. ALP activity in the lysates was determined by incubation for 30 min at 37°C with the substrate, p-nitrophenylphosphate (Wako, Osaka, Japan) in the buffer (pH 10) containing 0.1 M 2-amino-2-methyl-1- propanol and 2 mM MgCl₂ (Takada et al., 2003).

The cells fixed with 95% methanol were stained with 1% Alizarin red for 5 min, washed with PBS, and observed under a microscope. Alizarin red associated with the cells was solubilized in 10% cetylpyridium chloride and determined by reading of the absorbance at 570 nm (Hessle et al., 2002).

Propidium Iodide (PI) Staining and TUNEL Staining
Cell death was assessed by flow cytometry after cells were stained for 5 min with 10 µg/mL PI as described above. We used a TUNEL assay with the Mebstatin Apoptosis Kit Direct 2 (MBL, Nagoya, Japan) to detect apoptosis. After cells were counterstained with Hoechst 33258, the fluorescence of FITC-labeled nick ends and Hoechst 33258 was observed under a fluorescence microscope.

Statistical Analysis
Data were expressed as the mean ± SD. Student’s t test was used for statistical analyses, and p values < 0.05 were considered significant.

	RESULTS

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Tooth Preparation Induced the Expression of iNOS in the Pulp
The expression of iNOS mRNA in the pulp was induced within 3 min and peaked at 1 hr after tooth preparation in the mandibular incisors of five-week-old mice (Fig. 1A). Increased expression of iNOS protein was confirmed 3 hrs after preparation (Fig. 1B). The expressions of mRNAs for ALP and PC-1, both known as enzymes related to mineralization (Johnson et al., 2001), were also induced in the pulp after abrasion of the teeth. The expression of DSPP and DMP mRNAs (Fig. 1A) and DSP protein (Fig. 1B) indicated that the pulp samples contained odontoblasts (Yamazaki et al., 1999; Narayanan et al., 2001).

View larger version (49K): [in this window] [in a new window]   Figure 1. Expression of mRNAs and proteins for iNOS and the mineralization-related genes in the pulp after tooth preparation. (A) At various time-points after tooth preparation, the pulp was collected, and RT-PCR was performed for iNOS, ALP, PC-1, DSPP, DMP, and GAPDH. The first lane is the control without tooth preparation. The other lanes show mRNA expressions at 0.05, 1, and 6 hrs after tooth preparation. -RT is the negative reverse-transcription control with GAPDH primers. The same results were obtained in 3 separate experiments. (B) Western blot analyses of pulp tissue for iNOS, DSP, and ß-actin were performed before (–) and 3 hrs after tooth preparation (+). NS, non-specific band.

View larger version (45K): [in this window] [in a new window]   Figure 2. Suppression of pulp cell proliferation by NO. (A) The pulp removed from normal one-week-old mice was cultured for 1 wk to allow for the outgrowth of pulp cells, which were plated onto normal plastic dishes or type IA collagen gel-coated dishes and cultured in MEM + 20% FCS in the absence or presence of 50 µM NOC-18. The cells were observed under a phase-contrast microscope at 14-day culture. Note that the pulp cells on the collagen-coated dishes grew faster than those on the uncoated dishes. (B) The number of viable cells was counted at various time-points by trypan-blue dye exclusion assay after culture on collagen-coated dishes in the absence (circle) or presence of 50 µM NOC-18 (square). *The density of cells exposed to NOC-18 was significantly lower than that of cells cultured for the same period without NOC-18 (n = 4). (C) Concentration-dependent suppression of cell growth by NOC-18. *Significantly lower than the control without NOC-18 (n = 4). (D) Incorporation of BrdU (left panels) by pulp cells cultured for 5 days in the absence (upper panels) or presence of 50 µM NOC-18 (lower panels). All nuclei in the same fields were visualized with Hoechst 33258 (right panels). (E) The BrdU incorporation at day 5 was analyzed by a flow-cytometer. The upper and lower panels show the incorporation of BrdU by the control and NOC-18-treated cells, respectively. After incubation with BrdU, the cells were treated with (thick lines) or without (thin lines) the antibody for BrdU. The ratios of BrdU-incorporated cells were 50.63% for the control cells and 32.42% for the NOC-18-treated cells.

NO Augmented Mineralization of Pulp-cell Culture
Pulp cells showed higher ALP activity when cultured in MP medium compared with those in normal medium (Fig. 3A). NOC-18 augmented ALP activity in a concentration-dependent manner in pulp cells cultured in mineralization-promoting (MP) medium, while NOC-18 did not affect the ALP activity of cells in the normal medium (Fig. 3B). It has been reported that several cytokines—including BMP-2, TGF-ß1, and FGF-2—influence the differentiation of pulp cells (Shiba et al., 1998; Nakashima, 2005). In our experimental setting, BMP-2 and FGF-2 increased ALP activity in pulp cells cultured in MP medium, whereas TGF-ß1 lowered it (Fig. 3C). Regardless of whether these cytokines were added, L-NAME suppressed ALP activity to the same extent (Fig. 3C).

View larger version (61K): [in this window] [in a new window]   Figure 3. Increased ALP activity and mineralization of pulp cells after treatment with NO in mineralization-promoting (MP) medium. (A) The pulp cells cultured for 7 days in normal (Normal) and MP medium (MP) in the presence of various concentrations of NOC-18 were stained with ALP activity. (B) ALP activity was measured in cells cultured for 7 days in normal growth medium (circle) or in MP medium (square) in the presence of various concentrations of NOC-18. *Significantly higher than the value obtained in the absence of NOC-18 (n = 4). (C) ALP activity of cells was determined after culture for 7 days without cytokine or with 50 ng/mL BMP-2, 5 ng/mL TGF-ß1, or 10 ng/mL FGF-2 in MP medium in the absence or presence of 5 mM L-NAME. * and indicate that the values are significantly higher and lower, respectively, than the control without cytokine and L-NAME (the column at the far left). #Significant difference. Data are from 4 experiments. (D) The cells were stained with Alizarin red after 14-day culture in MP medium in the absence (Control) or presence of 50 µM NOC-18 (NOC-18). (E) Alizarin red dye associated with the cells was dissolved in 10% cetylpyridium chloride, and quantified by measurement of the absorbance at 570 nm after seven- and 14-day cultures with (filled columns) or without NOC-18 (unfilled columns). *Significant difference (n = 4). (F) Alizarin red binding to the cells cultured for 7 days in MP medium in the absence or presence of 5 mM L-NAME. *Significantly lower than the control without L-NAME (n = 4).

NO Induced Apoptosis in Pulp Cells under Mineralization-promoting Conditions
NOC-18 showed no cytotoxic effect on pulp cells, even though it suppressed their proliferation when cultured in MEM plus 20% FCS (Fig. 2); however, NOC-18 increased the fraction of dead cells when they were cultured in MP medium (Fig. 4A). TUNEL staining of the cells indicated that NOC-18 caused apoptosis of pulp cells under these conditions (Fig. 4B).

View larger version (29K): [in this window] [in a new window]   Figure 4. Apoptotic death of pulp cells induced by NO. (A) Pulp cells were cultured in normal medium (Normal) or mineralization-promoting medium (MP) for 5 days in the absence or presence of 50 µM NOC-18. Cells were detached by collagenase digestion and stained with 10 µg/mL PI. Dead cells stained with PI were analyzed by flow cytometry. *The fraction of PI-positive cells was significantly higher in cells treated with NOC-18 compared with control cells (n = 4). (B) (Left panels) TUNEL staining was performed on the cells cultured for 5 days in MP medium in the absence (Control) or presence of 50 µM NOC-18 (NOC-18). (Right panels) The cells were counter-stained with 5 µg/mL Hoechst 33258.

	DISCUSSION

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iNOS is expressed, and produces a large amount of NO, in response to infection and inflammatory stimuli in various tissues, including dental pulp (Law et al., 1999; van’t Hof and Ralston, 2001; Kawashima et al., 2005). The present results indicate that iNOS expression and subsequent NO production could also be induced in the pulp by abrasion of the tooth. In chondrocytes, iNOS is induced by mechanical compression (Fermor et al., 2001). Therefore, it is plausible that mechanical stimulation by dental preparation caused iNOS expression in the pulp.

In this study, NO suppressed pulp-cell growth and promoted cell mineralization and apoptosis in vitro. NO did not kill pulp cells, but suppressed their proliferation when cultured in normal growth medium, while NO accelerated ALP production and induced apoptosis in pulp cells cultured in MP medium. These results suggest that NO shows various effects on pulp cells, depending on their differentiation stages. There are several reports on the role of NO in growth, differentiation, mineralization, and death of cells from other hard tissues. For instance, NO inhibited the growth and stimulated the differentiation and formation of mineralized nodules by osteoblasts (Inoue et al., 1995; Hikiji et al., 1997; MacPherson et al., 1999). NO induced cell death via apoptotic and necrotic pathways in chondrocytes (Aigner and Kim, 2002; Yasuhara et al., 2005). In growth plate cartilage, it is known that apoptotic cell death of hypertrophic chondrocytes occurs coincidentally with their mineralization. It has been suggested that chondrocyte-derived apoptotic bodies have commonalities with matrix vesicles, and that apoptosis is a prerequisite for mineralization of cartilage (Hashimoto et al., 1998). The release of matrix vesicles from differentiated odontoblasts is also required for the initiation of mineralization (Sela et al., 1981; Hirschfeld et al., 1982; Stratmann et al., 1996; Murray et al., 2003). The relationship between the apoptosis of pulp cells and their mineralization remains to be clarified.

The inhibitory effects of L-NAME on ALP production and mineral deposition by pulp cells suggest that not only NOC-18-derived but also NO produced by NO synthases (Korkmaz et al., 2005) may be involved in pulp cell behavior. While BMP-2, TGF-ß1, and FGF-2 affected ALP production in pulp cells, L-NAME suppressed ALP activity to the same extent as was shown in cells without exposure to these cytokines. It remains to be clarified whether NO and these cytokines are able to regulate pulp cell differentiation independently or interdependently.

It is known that some of biological activities of NO, including the induction of apoptosis, are produced by its metabolites, such as peroxynitrite. Superoxide and nitrated tyrosine were not detected in pulp cells after incubation with NOC-18 in normal or MP medium (not shown), indicating that the contribution of peroxynitrite was not significant in our experimental settings. Direct activation of sGC by NO (Korkmaz et al., 2005) would be one of the possible pathways leading to changes in pulp cells. Further studies are required to clarify the events after NO production.

We found that dental preparation induced the expression of iNOS in pulp, and NO suppressed the growth and accelerated the mineralization and apoptosis of pulp cells. As far as we know, this is the first report on the role of NO in pulp cell behavior. The present study suggests that NO may play a part in odontoblast-like cell differentiation and the subsequent formation of reparative dentin.

	ACKNOWLEDGMENTS

 
This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, a Grant-in-Aid for Scientific Research of Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), a subsidy for the Improvement of Quality of Education and Research in Graduate School, and that for "High-Tech Research Center" Project for Private Universities from MEXT, 2005-2009.

	FOOTNOTES

 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received February 23, 2006; Last revision September 27, 2006; Accepted October 10, 2006

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