Primary mouse calvarial osteoblasts were isolated from 7-day-old CD-1 mice using the method described by Wong and Cohn . Briefly, calvaria were subjected to four sequential 15-minute digestions in an enzyme mixture containing 0.05% trypsin and 0.1% collagenase P at 37°C. Cell fractions 2–4 were pooled and resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, then filtered through a 70 μm cell strainer. Cells were plated at a density of 1 × 104 cells/cm2 and the medium was changed 24 h later. All animal-related experiments were approved by the Center for Laboratory Animal Medicine and Care at the University of Texas Health Science Center at Houston (approved animal protocol number HSC-AWC-10–145).
Bacteria and culture conditions
Porphyromonas gingivalis strain ATCC 33277 was grown anaerobically at 37°C in a Coy anaerobic chamber under an atmosphere of 86% nitrogen, 10% carbon dioxide, 4% hydrogen. The culture medium was Trypticase Soy Broth (TSBY) supplemented with 5% yeast extract, 2% sodium bicarbonate, 7.5 μM hemin and 3 μM menadione. TSB blood agar plates (BAP) were made with the addition of 5% sheep’s blood and 1.5% agarose. The bacteria were inoculated from BAP into 5 ml of TSBY and cultured anaerobically for 18 to 24 h at 37°C, then diluted in TSBY and grown to early log phase. Bacterial cells were harvested by low-speed centrifugation and resuspended in α-MEM (alpha minimum essential medium). Bacteria were then diluted in α-MEM to generate the appropriate MOI (multiplicity of infection) for addition to osteoblast cultures.
To identify the receptors utilized by P. gingivalis during invasion of osteoblasts, P. gingivalis was inoculated into 1-week-old osteoblast cultures at a MOI of 150 for 1 h. To evaluate osteoblast cytoskeleton rearrangement upon P. gingivalis infection, P. gingivalis was inoculated into 1-week-old osteoblast cultures at a MOI of 150 for 30 min, 3 h or 24 h. For signaling pathway and apoptosis assays, bacteria were inoculated at a MOI of 150 for 3 h in 1-week old osteoblast cultures (designated as day 1 on bacterial inoculation), then every other day up to day 21. For all inoculations, the osteoblasts were washed with PBS and then incubated with viable P. gingivalis at 37°C in 5% CO2/95% air for the time periods described above. Osteoblasts were washed with PBS again and cultured in fresh α-MEM until the next inoculation. Controls were subjected to the same media change and wash conditions without the addition of bacteria.
Primary mouse calvarial osteoblasts were isolated and plated in 6-well plates in DMEM supplemented with 10% FBS and antibiotics. After 1 week, the medium was changed to α-MEM supplemented with 10% FBS, 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate to induce the differentiation of osteoblasts. The medium was changed every other day thereafter. On each medium change day, viable P. gingivalis 33277 was inoculated into the cultures at a MOI of 150 for 3 h, and this procedure was carried out for 3 weeks. Protein was extracted from the cultures at the end of each week with ice-cold RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), protease inhibitors (1 μg/ml leupeptin, 0.5 μg/ml pepstatin, 0.7 μg/ml aprotonin, 0.5 mM phenylmethylsulfonyl fluoride), 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 0.004% sodium azide) by shaking at 4°C for 15 min. The homogenates were centrifuged at 10,000 × g for 20 min at 4°C. The supernatant protein concentration was determined by BCA assay. Proteins (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10–20% gels and transferred to nitrocellulose membranes. The blots were incubated with rabbit anti-integrin α5 or β1 polyclonal antibody (1:500; Millipore, Temecula, CA), rabbit anti-large fragment of cleaved caspase-3 polyclonal antibody (1:100; Millipore), rabbit anti-ERK, JNK, or p38 polyclonal antibody (all 1:1000), rabbit anti-mouse phosphorylated (p-) ERK, p-JNK, p-p38 monoclonal antibody (all 1:1000; Cell Signaling Technology, Danvers, MA), and HRP-conjugated goat anti-actin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. The blots were washed and then incubated with goat anti-rabbit HRP conjugated secondary antibody (1:10,000) for 1 h at RT. Protein bands were visualized using an Immun-StarTM HRP substrate kit (BioRad, Hercules, CA). The blots were developed and scanned, and densitometric analysis was performed with Kodak 1D Image Analysis Software (Eastman Kodak, Rochester, NY).
Freshly isolated osteoblasts were plated in 6-well plates in DMEM supplemented with 10% FBS and antibiotics. On day 7, P. gingivalis was inoculated at a MOI of 150 for 1 h. Uninfected osteoblasts were used as controls. Osteoblasts were washed with ice-cold PBS and lysed with ice-cold RIPA buffer containing freshly added protease inhibitors. The soluble fraction was collected by centrifugation at 10,000 × g for 20 min. The cell lysates were pre-cleared by incubation with protein A Sepharose beads at 4°C for 10 min on a rocker. The concentrations of the lysates were determined by BCA assay, and were then diluted to 5 mg/ml with PBS. To 500 μl of cell lysate, rat anti-mouse α5β1 monoclonal antibody (1:25; Millipore) or rabbit anti-rFimA polyclonal antibody (1:100) was added and gently mixed overnight at 4°C on a rocker. The immunocomplexes were captured by adding 100 μl of bead slurry and gently rocking overnight at 4°C. The beads were collected by pulse centrifugation and washed with ice-cold RIPA buffer. The immunocomplexes were dissociated from the beads by boiling in SDS-PAGE sample buffer for 5 min and analyzed by western blotting with rabbit anti-integrin α5 or β1 polyclonal antibody (both 1:500; Millipore) or rabbit anti-FimA polyclonal antibody (1:2000). Crude osteoblast and P. gingivalis extracts were included on the western blots alone as controls to identify the bands for α5, β1, and FimA.
Confocal fluorescence microscopy
To further identify the receptors utilized by P. gingivalis during invasion of osteoblasts, P. gingivalis was inoculated into 7-day-old osteoblast cultures at a MOI of 150 for 1 h. Uninfected osteoblasts were used as controls. The cultures were washed with PBS, fixed in 2% paraformaldehyde (PFA), permeabilized with 0.1% Nonidet P-40, and blocked with 3% BSA and 1% horse serum. The cultures were further incubated with rat anti-mouse integrin α5β1 monoclonal antibody (1:100; Millipore) and rabbit anti-P. gingivalis FimA polyclonal antibody (1:2000) overnight at 4°C, followed by washing and incubation with Alexa Fluor 594 conjugated goat anti-rat and Alexa Fluor 488 conjugated goat anti-rabbit secondary antibodies (both 1:200; Molecular Probes, Invitrogen, Carlsbad, CA) for 1 h at room temperature (RT). Rat and rabbit IgG isotype controls were included to validate the specificity of the staining. Osteoblast nuclei were labeled with DAPI (Molecular Probes). The confocal images were captured with an Olympus FV1000 Laser Confocal microscope using Olympus Fluoview software (Olympus America Inc. Center Valley, PA). The potential binding between osteoblast integrin α5β1 and P. gingivalis fimbriae was indicated by the yellow fluorescence where red (α5β1) and green (fimbriae) fluorescence co-localized.
To determine whether α5β1-fimbriae binding and/or new host protein synthesis were essential for P. gingivalis invasion of osteoblasts, four experimental groups were set up: 1) control, osteoblasts without P. gingivalis inoculation; 2) osteoblasts inoculated with P. gingivalis; 3) osteoblasts treated with a 1:100 dilution of rat anti-mouse integrin α5β1 monoclonal antibody (Millipore) for 1 h at RT prior to bacterial inoculation; 4) osteoblasts pretreated with the protein synthesis inhibitor, cycloheximide (50 μg/ml), 1 h prior to bacterial inoculation. For groups 2, 3 and 4, osteoblasts were inoculated with P. gingivalis at a MOI of 150 for 30 min, 1 h and 3 h. Thereafter, the cultures were washed, fixed, permeabilized and blocked as described above. The cells were incubated with rabbit anti-P. gingivalis polyclonal antibody (1:4000) for 1 hr at RT, followed by washing and incubation with Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (1:200; Molecular Probes) for 1 h at RT. Osteoblast actin and nuclei were labeled with rhodamine phalloidin (Molecular Probes) and DAPI, respectively. The internalization of P. gingivalis into osteoblasts was determined by the localization of the bacteria within the cytoplasmic boundary of osteoblasts, as well as the close proximity of the bacteria to osteoblast nuclei. The number of osteoblasts with bacterial invasion was counted manually and expressed as the percentage of the total number of osteoblasts counted.
To determine whether actin rearrangement is required for P. gingivalis invasion, osteoblasts were inoculated with P. gingivalis at a MOI of 150 for 30 min, 3 h and 24 h with or without the addition of the actin-disrupting agent, cytochalasin D (2.5 μg/ml), for the entire infection period. Uninfected osteoblasts were used as controls. The staining process and confocal image acquisition were performed as described above. The number of osteoblasts with bacterial invasion was counted manually and expressed as the percentage of the total number of osteoblasts counted.
P. gingivalis-infected osteoblast cultures were fixed with 4% PFA in PBS. The TUNEL procedure was performed with the TACS TBL kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Nuclease treatment or exclusion of TdT enzyme was used as the positive or negative control, respectively. Light microscopic examination revealed apoptotic cells as having condensed, blue-stained nuclei. Quantification of apoptotic cells was determined manually and expressed as a percentage of the total number of cells counted.
All experiments were repeated independently three times. Data were analyzed using Student’s t test to determine the significance between groups (P ≤ 0.05).