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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

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.

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).

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.