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

<p>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.</p

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

<p>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, **<i>P</i><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, **<i>P</i><0.01.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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, *<i>P</i><0.05.</p

Versican A-subdomain is required for its adequate function in dermal development

<p>Versican, a large chondroitin sulfate (CS) proteoglycan, serves as a structural macromolecule of the extracellular matrix (ECM) and regulates cell behavior. We determined the function of versican in dermal development using Vcan^Δ3/Δ3 mutant mice expressing versican with deleted A-subdomain of the N-terminal G1 domain. The mutant versican showed a decreased hyaluronan (HA)-binding ability and failed to accumulate in the ECM. In the early developmental stage, Vcan^Δ3/Δ3 dermis showed a decrease in versican expression as compared with WT. As development proceeded, versican expression further decreased to a barely detectable level, and Vcan^Δ3/Δ3 mice died at the neonatal period (P0). At P0, Vcan^Δ3/Δ3 dermis exhibited an impaired ECM structure and decreased cell density. While the level of collagen deposition was similar in both genotypes, collagen biosynthesis significantly decreased in Vcan^Δ3/Δ3 fibroblasts as compared with that in wild type (WT). Transforming growth factor β (TGFβ) signaling mediated through the Smad2/3-dependent pathway was down-regulated in Vcan^Δ3/Δ3 fibroblasts and a reduced TGFβ storage in the ECM was observed. Microarray analysis revealed a decrease in the expression levels of transcription factors, early growth response (Egr) 2 and 4, which act downstream of TGFβ signaling. Thus, our results suggest that A-subdomain is necessary for adequate versican expression in dermis and that versican is involved in the formation of the ECM and regulation of TGFβ signaling.</p