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 »  Home  »  Endodontic Articles 11  »  Matrix metalloproteinase-8 (MMP-8) in pulpal and periapical inflammation and periapical root-canal exudates
Matrix metalloproteinase-8 (MMP-8) in pulpal and periapical inflammation and periapical root-canal exudates
Introduction - Materials and methods.



J.Wahlgren, T. Salo, O.Teronen, H. Luoto, T. Sorsa & L.Tjaderhane
Faculty of Medicine and Biomedicum, Oral Pathology Unit/Laboratory Diagnostics, Helsinki University Central Hospital (HUCH), University of Helsinki, Helsinki.
Institute of Dentistry, University of Oulu, Oulu University Hospital, Oulu, Medix Biochemica, Kauniainen, Finland.
Department of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Canada.


Introduction.
Early pulpal and periapical responses to bacteria or their metabolites include the influx and recruitment of polymorphonuclear leucocytes (PMNs) and monocytes/macrophages. In the more chronic states, proliferation of fibroblasts and vascular elements, and the infiltration of macrophages, lymphocytes and plasma cells are characteristic features (Simon 1998). The role of all the factors participating in the spread of inflammation and necrosis in pulp tissue is not completely understood. Since matrix metalloproteinases (MMPs), the host enzymes responsible for the extracellular matrix degradation, have been suggested to be important in other inflammatory conditions such as periodontitis, it is possible that they also participate in the pathogenesis of pulp and periapical inflammation.
MMPs forma family of structurally related but genetically distinct endopeptidases expressed at low levels in normal tissues, but upregulated during inflammation (Birkedal-Hansen 1995). Collagenases (MMP-1 and MMP-8) and gelatinases (MMP-2 and MMP-9) are present in jaw cyst wall extracts and cyst fluids (Teronen et al. 1995a,b), but no data exists of their role in pulpal or periapical inflammation. Previously, MMP-8 has been thought to be uniquely produced by developing PMN cells in bone marrow, and that the MMP-8 activity is dependent on the release of the enzyme from PMN cells by degranulation (reviewed by Mainardi et al. 1991). However, recent findings demonstrated the expression of MMP-8 in mesenchyme derived, non-PMN lineage cells, including dental pulp fibroblasts, odontoblasts, inflamed epithelial cells and plasma cells (Hanemaaijer et al. 1997, Palosaari et al. 2000, Tervahartiala et al. 2000, Wahlgren et al. 2001). The exact role of MMP-8 synthesized by the cells in the pulpo-dentinal complex is not clear (Tjaderhane et al.2001).Therefore, a hypothesis was proposed that MMP-8 could be present in the inflamed or necrotic pulp, and the enzyme level could be related to the activity of tissue destruction of the pulp and periapical tissue. The aim of this study was to evaluate the presence and cellular sources, as well as the levels and molecular forms of MMP-8 in pulpal and periapical inflammation and during the root-canal treatment using immunofluorometric assay (IFMA), Western blot and immunohistochemical staining.

Materials and methods.

Patients.
Ten patients, with noncontributory medical histories and diagnosed to have periapical periodontitis were selected for the study. The diagnosis of apical periodontitis was based mainly on radiographic examination, demonstrating clear bone loss and disappearance of the periodontal ligament space in the periapical region. Occasionally, the patients reported mild spontaneous pain, and percussion sensitivity was observed upon clinical examination. Patients with primary or secondary acute periodontitis were excluded from the study. All teeth (n = 11) had one root canal only, and all were confirmed to be nonvital upon access. The root-canal treatments and the sample collection for the root-canal exudates MMP-8 analysis were performed by one of the authors ( J.Wahlgren) at a private dental office with the approval of Ethical Committee of Helsinki University Faculty of Medicine, and with the patient’s informed consent. The samples were collected during three consecutive appointments within 2-week intervals, during which the root-canal treatment was completed. For MMP-8 IHC stainings, the pulp tissue from vital teeth treated for irreversible pulpitis (n =10) were collected by the same operator.

Sample collection.
Primary access to the pulp was completed with dental burs using rubber dam isolation. Instrumentation of the root canal commenced with size 20 or 25 files. A radiograph was taken to determine the canal length. A sterile paper point was then inserted into the root canal up to the preparation length for 2 min to absorb exudates for the sample. The root canals were then instrumented to a minimum of size 40 with frequent rinsing with 2.5%sodiumhypochlorite (NaOCl). For the final rinsing, 15%ethylenediamine tetraacetic acid (EDTA) and NaOCl were used. The root canals were dried, Ca(OH)2 paste (pH 12.5) (Biocalc,Orion, Helsinki, Finland)was inserted into the canal and the cavity was sealed with Cavit1 (ESPE, Seefeld, Germany) and IRM1 (Dentsply Caulk, Milford, DE, USA).
After 2 weeks, the teeth were isolated with rubber dam and the temporary fillings were removed. The root canals were instrumented gently to facilitate the removal of Ca(OH)2 with sterile saline solution rinsing. After drying of the canals, a sterile paper point was inserted into the canal for 2 min for periapical exudate sample collection. The canal was rinsed with NaOCl, dried and Ca(OH)2 paste was inserted into the canal. After 2 weeks, the procedure for the sample collection was repeated, and the root canals were filled with gutta-percha and sealer using lateral condensation.
After sample collection, the exudate from the absorbent paper points was immediately eluted with 50 mL of50 mm Tris-HCl buffer, pH 7.5, containing 0.15 m NaCl and 1mm CaCl2, placed on a shaker for 3 h and stored at _70 8C for further handling.

MMP-8 analyzes.
The molecular forms of MMP-8were analyzed using the Western blotting method using rabbit polyclonal antibody against MMP-8 in normal fashion, as described previously (Palosaari et al. 2000). Briefly, samples were run on10%SDS-PAGE gels and transferred to nitrocellulose filter. The filters were incubated overnight with MMP-8 antibody, and then with peroxidase-conjugated antirabbit immunoglobulin’s for 1 h. Diaminobenzidine tetrahydrochloride (DAB) was used for the detection. The intensity of molecular forms of MMP-8, both latent and active forms separately (Kiili et al. 2002), were evaluated by scanning the respective bands and analyzing the band intensity with Bio-Rad Model GS-700 Imaging Densitometer using Molecular Analyst1/PC program.
Concentration of MMP-8 in the samples was also determined from the elution buffer by a time-resolved IFMA as described previously (Hanemaaijer et al. 1997). Briefly, two monoclonal MMP-8-specific antibodies were used as catching antibody and tracer antibody, respectively. The tracer antibody was labeled using europium chelate. After adding the enhancement solution, fluorescence was measured using a 1234 Delfia Research Fluorometer (Wallac,Turku, Finland). The specificity of the monoclonal antibodies against MMP-8 corresponded to that of polyclonalMMP-8 (Hanemaaijer et al.1997).
Immunohistochemical staining of the periapical tissue samples Periapical granuloma samples (n ј10) for the immunohistochemical stainings were randomly selected from the files of Department of Oral Pathology, Institute of Dentistry, University of Oulu. Periapical tissue samples were all collected during periapical surgery, undertaken as a part of the normal treatment of patients. In each case a periapical radiolucency was present in an endodontically treated tooth, and the diagnosis of periapical granuloma was confirmed with the histological evaluation.
Immunohistological stainings were performed as described previously (Wahlgren et al. 2001) with polyclonal antibody (diluted 1 : 800 in 1% BSA/PBS) for MMP-8 and monoclonal CD/68Ab-3 (Clone KP1)macrophage- marker (NeoMarkers, Fremont, CA, USA), on formalin fixed paraffin embedded pulp and periapical granuloma tissue sections. Brieffy, sections were incubated with pepsin to reveal the epitopes, and with H2O2 to quench the endogenous peroxidase activity. Nonspecific binding was blocked with 2% normal blocking serum inVectastain1 Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) and incubated with the primary antibody overnight. The control sections were incubated with the buffer alone or nonimmune rabbit serum. For detection, the sections were incubated with biotinylated secondary antibody (antirabbit IgG or antimouse IgG) and with Vectastain1 Elite ABC reagent, and stained with 3-amino-9-ethylcarbazole (Sigma, St Louis, MO, USA), diluted in N,N-dimethylformamide (Merck, Darmstadt, Germany), and counter stained with Mayer’s haematoxylin.

Statistical analysis.
The correlation analysis was performed between the IFMA analysis and respective Western blot scanning results using the Pearson’s product-moment correlation coefficient, to ensure the comparativeness of the two methods and to confirm the use of IFMA results as the primary method for the statistical analysis.
Kruskall-Wallis one-way anova and Mann-Whitney U-test were used to analyze the degree of difference in the MMP-8 levels between the different appointments. The nonparametric tests were used because of the absence of the normal distribution within each sample site groups. Student’s t-test was used to compare the latent and active forms of enzyme in the root-canal exudate.