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Azerbaycan Saytlari

 »  Home  »  Endodontic Articles 5  »  Invasion of vascular cells in vitro by Porphyromonas endodontalis
Invasion of vascular cells in vitro by Porphyromonas endodontalis
Introduction - Materials and methods.



B. R. Dorn, L. J. Harris, C. T. Wujick, F. J. Vertucci & A. Progulske-Fox
Center for Molecular Microbiology, Department of Oral Biology,
Department of Endodontics, College of Dentistry, University of Florida, Gainesville, FL, USA.

Introduction.
There is an emerging paradigm shift from coronary heart disease (CHD) having a purely hereditary/nutritional causation to having an infectious component (Libby et al . 1997, Coles et al . 1998, Nieto 1998). Some potential pathogens include Chlamydia pneumoniae , Helicobacter pylori , Cytomegalovirus, and oral bacteria (Mattila et al . 1998, Epstein et al . 1999). Some epidemiological studies have demonstrated an association between periodontal disease and coronary heart disease (DeStefano et al . 1993, Mattila et al . 1993, Mattila et al . 1995, Beck et al . 1996, Arbes et al . 1999, Morrison et al . 1999). Conversely, several studies have reported no significant associations between oral disease and CHD (Joshipura et al . 1996, Hujoel et al . 2000, Mattila et al . 2000). Two of the latter studies that reported no significant association did find associations between the two diseases in younger age groups. Thus, there may be no overall association, but there may be associations within subsets of the population, for example, younger age groups and specific human genotypes (Beck et al . 2000). Invasion of the cells of the arterial wall by oral bacteria could represent the injury that either initiates and/or more probably exacerbates atherogenesis.
Transient bactaeremias occur in patients after toothbrushing, mastication, extraction of teeth, periodontal therapy, and endodontic treatment (Sconyers et al . 1973, Silver et al . 1977, Carroll & Sebor 1980, Debelian et al . 1995), and this dissemination of oral bacteria may be a possible route of infection to those arterial tissues. Oral bacteria have been found in atherosclerotic plaques (Chiu 1999, Haraszthy et al . 2000). A few periodontal pathogens have been reported to invade oral epithelial tissues. The periodontal pathogens Porphyromonas gingivalis and Prevotella intermedia also invade primary cultures of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) (Dorn et al . 1999).
Anaerobic bacteria are found within root canal infections and may have a role in the pathogenesis of pulpal disease (Assed et al . 1996). Endodontic infections are frequently associated with black-pigmented bacteria (BPB) as a group. Porphyromonas and Prevotella spp., both BPBs, occur within 79% of endodontic lesions (Haapasalo 1989), and P. nigrescens is the most common of the BPBs in the pulp canal (van Winkelhoff et al . 1988, Bae et al . 1997). Several periodontal pathogens, including P . gingivalis and P . intermedia , are frequently found in root canal lesions. Additionally, some microorganisms associated with endodontic lesions ( Porphyromonas endodontalis and Prevotella nigrescens ) are closely related to invasive periodontal pathogens. Therefore, the objective of this study was to determine whether microorganisms associated with endodontic lesions have the ability to invade primary cultures of human coronary artery endothelial cells and coronary artery smooth muscle cells. In addition to the laboratory strains, isolated bacterial strains from root canal lesions from patients at the University of Florida, USA were tested for invasive ability. The bacteria from root canal infections have the potential to be involved in the development of atherosclerosis and CHD since they have access to the systemic circulation.

Materials and methods.

Bacterial and cell culture.
In addition to laboratory strains, black-pigmented bacteria (BPB) were isolated from patients undergoing root canal treatment as described previously (Debelian et al . 1995). Strains of BPBs (Table 1) were grown on tryptic soy agar (Difco Laboratories, Detroit, MI, USA) supplemented with 5.0% sheep blood (Lampire Biological Laboratories, Pipersville, PA, USA), 0.5% yeast extract (Difco), haemin (5 g mL −1 ), and vitamin K (5 g mL −1 ). Overnight cultures of BPB were grown in brain heart infusion (BHI) broth (Difco) supplemented with 0.5% yeast extract, 0.1% cysteine (Sigma Chemical Co., St. Louis, MO, USA), haemin (5 g mL −1 ), and vitamin K (5 g mL −1 ) under anaerobic conditions. E. coli MC1061 (a gift of A. S. Bleiweis) was grown in Luria-Bertani (LB) medium consisting of Bacto Tryptone (10 g L –1 ; Difco), Bacto yeast extract (5 g L –1 ), and NaCl (10 g L –1 ).

Table 1. Bacterial strains used.

Bacterial strains used
a. University of Florida, Gainesville, FL, USA.
b. Universite Laval, Quebec, Canada.
c. State University of New York, Buffalo, NY, USA.
d. Periodontal Disease Research Center, University of Florida, Gainesville, FL, USA.

The HCAEC were maintained in Microvascular Endothelial Growth Medium-2 (EGM-2; Clonetics, Inc., San Diego, CA, USA) consisting of endothelial cell basal medium-2 supplemented with fetal bovine serum, hydrocortisone, human recombinant fibroblast growth factor, vascular endothelial growth factor, recombinant insulin growth factor-1, ascorbic acid, human recombinant epidermal growth factor, gentamicin, and amphotericin (Clonetics). The CASMC (Clonetics) were maintained in smooth muscle growth medium (SmGM-2) consisting of smooth muscle basal medium-2 supplemented with insulin, human recombinant fibroblast growth factor, fetal bovine serum, human recombinant epidermal growth factor, gentamicin, and amphotericin (Clonetics). Cells were cultured in 75-cm 2 flasks at 37 C in a humidified atmosphere of 5% CO 2 . Both the HCAEC and CASMC were obtained cryopreserved third passage and were passaged an additional two or three times before use.

Sampling from the root canal.
The protocol for collection of bacteria from infected root canals was that described by Debelian et al . (1995). Clinical isolates from infected root canals were identified as described by Moore et al . (1994) using the MIDI identification system (Microbial ID, Inc., Newark, DE, USA).

Invasion assay.
For the invasion assays, the bacteria were grown in BHI broth, centrifuged at 3500 g , and resuspended in antibiotic-free media to a concentration of 10 7 cells mL –1 as determined spectrophotometrically (Shimadzu UV- 1201, VWR, Marietta, GA, USA). Approximately 10 5 human cells per well in a 24-well tissue culture plate were washed three times with phosphate-buffered saline (PBS) prior to incubation with 1.0 mL of the bacterial suspension at 37 C aerobically for 90 min. In order to more closely approximate in vivo conditions, the bacteria were not centrifuged onto the cells to promote intimate contact. The media were aspirated off infected cells after 90 min, and the cells were washed three times with PBS. Medium containing gentamicin (300 g mL −1 ) and metronidazole (200 g mL −1 ) was then added to each well to kill any extracellular bacteria, and the plates were incubated for 60 min aerobically at 37 C. Finally, the media were removed, the cells washed three times with PBS, and lysed by a 20-min incubation with 1.0 mL sterile distilled water at 37 C under aerobic conditions. Dilutions of the lysates of cells infected with BPBs were plated in triplicate on Tryptic Soy Agar (Difco) plates supplemented with 5.0% sheep blood, 0.5% yeast extract, haemin (5 g mL −1 ), and vitamin K (5 g mL −1 ) and cultured anaerobically. The dilutions of the lysates of E. coli MC1061 were cultured on LB plates at 37 C aerobically. Colony forming units (CFU) of invasive bacteria were then enumerated. Each assay was performed in duplicate wells and independently at least three times. Viability of vascular cells was examined by trypan blue exclusion.
Control experiments demonstrated that extracellular bacteria were killed at these antibiotic concentrations. Invasive bacteria not protected within human cells had CFUs lower than the non-invasive strains. The CFU of P. gingivalis 381 decreased by four orders of magnitude.

Transmission electron microscopy.
Following the 90 min incubation of eukaryotic cells with bacteria, the cells were washed two times with PBS, fixed in 2% PBS buffered glutaraldehyde at room temperature for 1 h, centrifuged for 5 min at 200 g , and washed with PBS (pH 7.3). Three drops of 3% low-gelling agarose were then added to the pellet and allowed to solidify at 4 C for 10 min. The agarose-embedded pellet was then washed twice for 10 min in PBS, incubated in 1% osmium tetroxide for 1 h, and washed three times in distilled H 2 O. The washed cell pellets were dehydrated in a graded series of ethanol and stained overnight en bloc with 2% uranyl acetate. Finally, the pellets were infiltrated and embedded in EM Bed-812 (Electron Microscopy Sciences, Ft. Washington, PA, USA). Thin sections were cut, poststained with uranyl acetate and lead citrate, and examined in a Hitachi 7000 transmission electron microscope.