The anti-inflammatory effects of matrix metalloproteinase-3 on irreversible pulpitis of mature erupted teeth - PubMed (original) (raw)

The anti-inflammatory effects of matrix metalloproteinase-3 on irreversible pulpitis of mature erupted teeth

Hisanori Eba et al. PLoS One. 2012.

Abstract

Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation and the modulation of cell behavior. These proteinases have also been implicated in tissue repair and regeneration. Our previous studies have demonstrated that MMP-3 elicits stimulatory effects on the proliferation and the migration of endothelial cells as well as anti-apoptotic effects on these cells in vitro. In addition, we found that MMP-3 enhanced the regeneration of lost pulp tissue in a rat incisor pulp injury model. However, continuously erupting rodent incisors exhibit significantly different pulp organization compared with mature erupted teeth. Therefore, we have further extended these studies using a canine irreversible pulpitis model to investigate the effects of MMP-3. In this study, the crowns of the canine mature premolars were removed and the pulp tissues were amputated. The amputated pulp tissues remained exposed for 24 or 72 hours to induce mild or severe irreversible pulpitis, respectively, followed by sealing of the cavities. In both models, the whole pulp tissues became necrotic by day 14. In this mild pulpitis model, the regeneration of pulp tissue with vasculature and nerves was observed until 14 days after sealing with MMP-3, followed by extracellular matrix formation in the regenerated pulp tissues until day 28. The treatment with MMP-3 resulted in a decrease in the number of macrophage and antigen-presenting cells and a significant inhibition of IL-6 expression on day 3. The inhibition of MMP-3 activity abolished these anti-inflammatory effects. Immunofluorescence staining demonstrated that MMP-3 was involved in the modification of serum-derived hyaluronan-associated proteins and hyaluronan (SHAP-HA) complexes possibly through the degradation of versican. These results demonstrate that MMP-3 can act as an anti-inflammatory agent and suggest that MMP-3 might represent a useful therapy for the treatment of mild irreversible pulpitis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The establishment of mild and severe irreversible pulpitis models.

A. Schematic diagrams of the amputation of the dog mature molar pulp tissues and subsequent cavity sealing. The crowns of the upper and lower premolars were removed, and the pulp tissues were amputated using a round burr. The amputated pulp tissues were exposed to allow for infection and treated with solution absorbed in Spongel. After the treatments, the cavity were sealed with phosphate cement and light-cured composite resin. B. The histology of the pulp tissues from dogs with mild irreversible pulpitis. The left panel shows amputated pulp tissue that remained exposed for 24 hours. After 24 hours, the cavity was covered with spongel and sealed. The right panel shows the pulp at 14 days after sealing. C. The histology of the pulp tissues from dogs with severe irreversible pulpitis. The left panel shows amputated pulp tissue that remained exposed for 72 hours. After 72 hours, the cavity was sealed. The right panel shows the pulp tissue at 14 days after sealing. Scale bar, 500 µm.

Figure 2

Figure 2. The pulp tissue regeneration induced by MMP-3 in the irreversible pulpitis model.

A. H&E staining of pulp tissues from dogs with mild pulpitis at 14 and 28 days after MMP-3 or saline treatment, as indicated. The arrows show the imaginary amputated site, with the indicated areas magnified in C. Scale bar, 200 µm. B. H&E staining of pulp tissues from dogs with severe pulpitis at 14 days after MMP-3 or saline treatment, as indicated. The arrows indicate the imaginary amputated site. Scale bar, 200 µm. C. Immunohistochemical analysis for BS-1-lectin, TuJ1, and GAP43 and Masson’s trichrome staining of pulp tissues from dogs with mild pulpitis at 14 or 28 days after MMP-3 treatment, as indicated. The representative staining of the cells is indicated by arrowheads. Scale bar, 50 µm.

Figure 3

Figure 3. Time course of the histological changes of the pulp tissues from dogs with mild pulpitis.

H&E staining of pulp tissues from the dog with mild pulpitis at the indicated number of days after treatment with MMP-3, MMP-3 plus NNGH, NNGH alone or saline alone, as indicated. The arrows indicate the imaginary amputated site. Scale bar, 200 µm.

Figure 4

Figure 4. Immunohistochemical analysis of CD68 and MHC class II in samples from dogs with mild pulpitis.

A. Immunostaining of pulp tissues using anti-CD68 IgG on the indicated days after treatment with MMP-3, MMP-3 plus NNGH, NNGH alone or saline alone, as indicated. Scale bar, 100 µm. B. Quantitative analysis of the CD68-positive cells on the indicated days after treatment with MMP-3, MMP-3 plus NNGH, NNGH alone or saline alone, as indicated. Error bars, ± SEM, **P<0.01. C. Immunostaining of pulp tissues from dogs with mild pulpitis using anti-MHC class II IgG on the indicated days after treatment with MMP-3, MMP-3 plus NNGH, NNGH alone or saline alone, as indicated. Scale bar, 100 µm. D. Quantitative analysis of the MHC class II-positive cells on the indicated days after treatment with MMP-3, MMP-3 plus NNGH, NNGH alone or saline alone, as indicated. Error bars, ± SEM, **P<0.01.

Figure 5

Figure 5. Levels of IL-6 and TNF-α at 3 days after MMP-3 treatment in mild pulpitis model.

Homogenates of pulp tissues were prepared at 3 days after MMP-3 or saline treatment as indicated. A. The average concentration (ng/mg protein) of IL-6 is indicated. B. The average concentration (ng/mg protein) of TNF-α is indicated. Error bars, ± SEM, *P<0.05.

Figure 6

Figure 6. Hyaluronan (HA), SHAP and versican localization in the mild pulpitis model.

Immunofluorescence-stained pulp tissues were prepared 3 days after treatment. A. H&E-stained pulp tissues from dogs with mild pulpitis 3 days after treatment with MMP-3, MMP-3 plus NNGH, NNGH alone or saline as indicated, with the indicated area magnified in B. The arrows indicate the imaginary amputated site. Scale bar, 200 µm. B. Pulpitis tissues stained with biotinylated HABP (HA), anti-SHAP (SHAP) and anti-versican as indicated. Scale bar, 20 µm.

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Grants and funding

This work was supported by the Adaptable and Seamless Technology transfer Program through target-driven R&D (A-STEP) by Japan Science and Technology Agency (JST) (AS232Z01229F to Hiroyuki N), a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (#20390504 to MN, #21390512 to Hiroshi Nakamura, #22592030 to Hiroyuki Nakamura), and by the Research Grant for Longevity Sciences (21A-7) from the Ministry of Health, Labour, and Welfare (MN). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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