Figure 1. The immunohistochemical staining of the inflamed pulp tissue with MMP-8-specific antibody.
(A) MMP-8-expressing cells were evenly distributed throughout the whole pulp tissue, but most abundantly (arrows) surrounding the pulp abscess (PA).
(B) PMN cells expressed an intense staining (arrows).
(C) In connection with the dentine chip removed with the pulp tissue, the ?broblast-type cells at the close proximity of the dentine occasionally expressed MMP-8 protein (arrow).
(D) Plasma cells (arrows) with strong expression of MMP-8.
(E) Some endothelial cells in the pulpal vessels expressed fainter, but clearly observable staining with MMP-8.
Scale bars: 10 mm (B, D and E); 25 mm (C);40 mm (A).
Figure 2. MMP-8 protein in root canals in the different time points of the study.
(A) Western blot using MMP-8-specific antibody. Representative samples from the root canals collected in the first, second and third visit of one tooth. Molecular weight standards were used to determine the molecular weight of the proteins detected. The latent and active forms of MMP-8 (pro-MMP-8 and MMP-8, respectively), as well as complexed forms (CF) and low molecular weight truncated forms (TF) are present prior to the root-canal preparation (Visit1). After 2 weeks of Ca(OH)2 treatment,MMP-8 levels are markedly reduced, but still a distinct band corresponding to active form of MMP-8 is present (Visit 2). After another 2 weeks of medication, MMP-8 is virtually absent (Visit 3).
(B) The concentrations of MMP-8 (mean and standard deviation) detected with the IFMA analysis in root canals during the three visits. The decrease in the root-canal samplesMMP-8 is evident, and theMMP-8 level in the last visit is significantly lower when compared to the first visit (Kruskall-Wallis P = 0.02; Mann-Whitney U-test P = 0.0107).
(C) The concentrations of root canalMMP-8 in individual cases during the three visits for endodontic treatment, as detected with the IFMAanalysis.5/11cases presented levels above1000 ng mL_1during the first visit; four of them decrease below detection level after 2 weeks of Ca(OH)2 medication. In one case, MMP-8-levelmarkedly increased after 2 weeks of medication, reaching the value of 14145 ng mL_1 (marked with _ in the figure), and remained high (>1000 ng mL_1) even after another period of Ca(OH)2 medication; the tooth was later found to be fractured. In two cases with initial level of 0 ng mL_1MMP-8, the levels slightly increased in the second measurement, with subsequent decrease after another period of medication.
(D) The relative levels of latent pro-MMP-8 and active MMP-8 in the root-canal samples, as percentage of total amount ofMMP-8 in each sample. The relative proportion of activeMMP-8 in the second and third visit samples increased, but the differences did not reach statistical significance.
Figure 3. Immunohistochemical stainings of the periapical granulomas withMMP-8-specific antibody.(A) PMN cells (short arrow) and macrophage-like cells (long arrow) express distinct staining.
(B, C) The staining of the consecutive sections with anti-MMP-8.
(B) and macrophage-specific marker antibody CD68.
(C) Confirmed the identity of the cells to be macrophages.
Scale bars: 10 mm (A); 25 mm (B and C).
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