The Western blots of the teeth with acute pulpitis demonstrated large amounts ofMMP-8inthepulpal tissue (data not shown). Whilst most of the 70-80 kDa MMP-8 observed in the Western blot most likely to originated from the PMN cells numerously present in the inflamed tissue, the immunohistochemical staining, revealed that other cells can also express MMP-8 in the inflamed human pulpal tissue (Fig. 1).
In the pulp proper, macrophage-like cells (not shown), PMN and plasma cells showed a clear expression of MMP-8, and the most intense MMP-8-positive cell accumulation was observed surrounding the pulpal abscess (Fig. 1A-D). Also, some endothelial cells of the pulp vessels were positive in MMP-8 staining (Fig. 1E). The control sections, incubated without primary antibody or with normal serum, were not stained (not shown).
Figure 1. The immunohistochemical staining of the inflamed pulp tissue with MMP-8-specific antibody.Root-canal exudate samples.
(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).
MMP-8 levels markedly decreased during the root-canal treatment of necrotic teeth (Fig. 2A-C). However, even after removal of the pulpal tissue remnants and debris and a careful mechanical preparation of the root canals accompanied with the local medication with calcium hydroxide paste, MMP-8 was detected occasionally (Fig. 2A-C). Only after the renewal of the local medication for fortnight, MMP-8 was almost completely absent form the canals (Fig. 2A-C). The difference between the first and last visit samples was statistically significant (Kruskall-Wallis P = 0.02; Mann-Whitney U-test P = 0.0107).
The relative proportions of latent and active forms of MMP-8 in the samples were equal in the first visit samples (Fig. 2D). In cases with MMP-8 observed in the subsequent visits, the relative amount of active MMP-8 according to the band scanning intensities was higher (Fig. 2D). However, the differences were not statistically significant, most likely owing to the small number of samples with MMP-8 present.
Only one outof11specimens failed to show decrease in theMMP-8 level below 1000 ng mL in the final periapical exudate sampling (Fig.2C); that tooth was later diagnosed to have a vertical root fracture and was extracted.
Spearman product-moment correlation analysis revealed a statistically significant positive correlation between the Western blot scanning analysis and IFMA (0.4871, P = 0.006). Thus, the scanning analysis of the Western blots confirmed the results obtained with IFMA, and justified the use of IFMA results in statistical analysis.
Immunohistochemical staining of the periapical tissue samples PMN cells and macrophage-like cells expressed the staining with MMP-8-specific antibody, the PMN cells being the predominant cell type to express MMP-8 (Fig. 3A). Staining with CD68 macrophage marker antibody indeed confirmed the nature of the macrophage-like cells staining with the MMP-8 antibody (Fig.3B-C).
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).