Browsing by Subject "Porphyromonas gingivalis"
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Item Antimicrobial properties of drug-containing electrospun scaffolds(2012) Jeppson, John; Spolnik, Kenneth Jacob, 1950-; Vail, Mychel Macapagal, 1969-; Erhlich, Ygal; Bottino, Marco C.; Gregory, Richard L.; Legan, Joseph J.; Zunt, Susan L., 1950-Endodontic treatment of the infected immature tooth has undergone a dramatic change. Conventional endodontic treatment can control infection, but root development usually remains impaired. A novel regenerative endodontic procedure, the revascularization method, can now control the infection and enable such teeth to continue root development. This is done by creating a fibrin-matrix scaffold in the antibiotic treated root canal space (RCS). Dental stem cells and growth factors have been able to continue root development in such an environment. The fibrin-matrix scaffold is dependent on the induction of a blood clot into the RCS, and this cannot always be predictably induced. PDS is a biocompatible material that can be electrospun to provide a matrix for cells and growth factors and perhaps improve on the blood clot induced fibrin scaffold by incorporating metronidazole as an adjuvant antimicrobial. A metronidazole containing electrospun PDS scaffold was examined in vitro using a turbidimetric test, the modified direct contact test. This scaffold significantly inhibited growth of an anaerobic primary endodontic pathogen Porphyromonas gingivalis. This scaffold may improve the treatment of the infected immature tooth by providing a designed matrix for root regeneration while serving simultaneously as an antibiotic drug delivery device to disinfect the RCS. The aim of this study is to evaluate in vitro the property of a synthetic scaffold to function as an antibacterial drug delivery device. PDS*II (polydioxanone) suture was obtained from Ethicon, INC. (Somerville, NJ) and was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol, HFIP (Sigma Aldrich). Three different scaffolds were electrospun onto an aluminum foil background; (1) control scaffold with no antibiotic incorporated, (2) scaffold with 5.0-wt % metronidazole incorporated, and (3) 25-wt % metronidazole incorporated. All scaffolds were cut using a 4-mm diameter biopsy punch under aseptic conditions and removed from foil, control scaffold (n = 64), scaffold containing 5.0-wt % metronidazole (n = 32), and scaffold containing 25-wt % metronidazole (n=32). Experimental scaffolds were placed in a 96- well sterile flat bottom microtiter plate. Porphyromonas gingivalis a known primary endodontic pathogen was grown in 5 ml of BHI + YE with 0.25 μl of vitamin K with incubation at 37°C under anaerobic conditions for 48 hours. Microplates were sterilized before inoculation with Pg with 400 μl of 70-percent EtOH for a minimum of 30 minutes then pipetted out. After sterilization the microwells were washed with 400 μl of sterile water and pipetted out. Group 1 (negative control) microwells (n = 8) contained control scaffold and 190 μl of broth only. Group 2 (positive control) microwells (n = 8) contained 190 μl of broth and Pg only. Group 3 microwells (n = 8) contained control scaffold, 190 μl of broth, and 10 μl of Pg inoculum. Group 4 microwells (n = 8) contained scaffold with 5 wt % metronidazole, 190 μl of broth, and 10 μl of Pg inoculum. Group 5 microwells (n = 8) contained scaffold with 25 wt % metronidazole, 190 μl of broth, and 10 μl of Pg inoculum. Group 6 contained 190 μl of uninoculated broth for spectrophotometer calibration. Sterile microplate lids were used to isolate microwells from the surrounding environment. Microplates were incubated at 37°C under anaerobic conditions for 48 hours. After 48 hours the microplates were read by using an endpoint reading in the spectrophotometer. This was repeated four times. Comparisons among the groups for differences in optical density as a measure of bacterial growth were made using mixed-model ANOVA, with a fixed effect for group and a random effect for experimental run. Pair-wise group comparisons were performed using Tukey's multiple comparisons procedure to control the overall significance level at 5 percent. The analyses were performed using the ranks of the data. Broth had significantly lower OD than all other groups (p < 0.0001). Broth+Pg and Broth+Pg+Scaffold had significantly higher OD than 5-wt % Metro (p < 0.0001) and 25-wt % Metro (p < 0.0001), but Broth+Pg and Broth+Pg+Scaffold were not significantly different from each other (p = 0.97). 5-wt % Metro and 25-wt % Metro were not significantly different from each other (p = 0.24). From the results of our study, we concluded that the 5.0-wt % and 25-wt % metronidazole containing scaffolds significantly inhibited bacterial growth and could be effectively utilized for the endodontic regeneration procedure.Item Crosstalk between dihydroceramides produced by Porphyromonas gingivalis and host lysosomal cathepsin B in the promotion of osteoclastogenesis(Wiley, 2022) Duarte, Carolina; Yamada, Chiaki; Garcia, Christopher; Akkaoui, Juliet; Ho, Anny; Nichols, Frank; Movila, Alexandru; Biomedical Sciences and Comprehensive Care, School of DentistryEmerging studies indicate that intracellular eukaryotic ceramide species directly activate cathepsin B (CatB), a lysosomal-cysteine-protease, in the cytoplasm of osteoclast precursors (OCPs) leading to elevated RANKL-mediated osteoclastogenesis and inflammatory osteolysis. However, the possible impact of CatB on osteoclastogenesis elevated by non-eukaryotic ceramides is largely unknown. It was reported that a novel class of phosphoglycerol dihydroceramide (PGDHC), produced by the key periodontal pathogen Porphyromonas gingivalis upregulated RANKL-mediated osteoclastogenesis in vitro and in vivo. Therefore, the aim of this study was to evaluate a crosstalk between host CatB and non-eukaryotic PGDHC on the promotion of osteoclastogenesis. According to a pulldown assay, high affinity between PGDHC and CatB was observed in RANKL-stimulated RAW264.7 cells in vitro. It was also demonstrated that PGDHC promotes enzymatic activity of recombinant CatB protein ex vivo and in RANKL-stimulated osteoclast precursors in vitro. Furthermore, no or little effect of PGDHC on the RANKL-primed osteoclastogenesis was observed in male and female CatB-knock out mice compared with their wild type counterparts. Altogether, these findings demonstrate that bacterial dihydroceramides produced by P. gingivalis elevate RANKL-primed osteoclastogenesis via direct activation of intracellular CatB in OCPs.Item Effect of Curcumin-loaded Photoactivatable Polymeric Nanoparticle on peri-implantitis-related biofilm(2022) Tonon, Caroline Coradi; Panariello, Beatriz; Chorilli, Marlus; Spolidorio, Denise Madalena Palomari; Duarte, Simone; Biomedical and Applied Sciences, School of DentistryCurcumin has been used as a photosensitizer (PS) for antimicrobial photodynamic chemotherapy (PACT). However, its low solubility, instability and poor bioavailability are a challenge for its in vivo application. This study aimed to synthesize curcumin-loaded polymeric nanoparticles (curcumin-NP) and to determine their antimicrobial and cytotoxic effects. Nanoparticles (NP) were synthesized by the nanoprecipitation method using polyprolactone as a polymer. Curcumin-NP was characterized by particle size, polydispersity index and zeta potential, scanning electron microscopy and curcumin encapsulation efficiency (EE). Curcumin-NP was compared to free curcumin solubilized in 10% DMSO as photosensitizers for PACT in single and multi-species Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus oralis biofilms. Chlorhexidine 0.12% (CHX) and ultrapure water were used as positive and negative controls, respectively. The cytotoxic effect of curcumin-NP was evaluated on human periodontal ligament fibroblast cells (HPLF). Data were analyzed by ANOVA (α=0.05). Curcumin-NP exhibited homogeneity and stability in solution, small particle size and 67.5% EE of curcumin. Curcumin-NP presented antibiofilm activity at 500 µg/ml when photoactivated. Curcumin-NP and curcumin with and without photoactivation were not cytotoxic to HPLF cells. Curcumin-NP has antimicrobial and antibiofilm properties, with better effects when associated with blue-light, being a promising therapy for preventing and treating peri-implant diseases.Item THE EFFECTS OF TOBACCO TREATED PORPHYROMONAS GINGIVALIS ON HUMAN EPITHELIAL CELLS(Office of the Vice Chancellor for Research, 2012-04-13) Tursunova, Roziya H.; Al-Shibani, Nouf; Windsor, L. Jack; Gregory, Richard L.Bacteria and tobacco are risk factors for periodontal diseases. Bacteria-host interactions play a critical role in disease development and progression. The effects of tobacco-treated bacteria such as Porphyromonas gingivalis on epithelial cells have not yet been examined. Therefore, P. gingivalis were treated with different tobacco products (nicotine, cigarette smoke conden-sate (CSC), and dissolvable smokeless tobacco (DST) strips) to determine the effects that they have on epithelial cells. P. gingivalis were grown with or without the products for 24 hours at 37◦C. The cells were separated from the supernatant, washed with 0.9% NaCl and incubated at 60◦C to kill the bacte-ria. Protein assays was performed to determine the protein concentration in the cell pellets and supernatants. Lactate dehydrogenase (LDH) assays are being used to measure the cytotoxicity of the cells and supernatants on epi-thelial cells in a dose dependent manner. Non-toxic amounts of the cell pel-lets and supernatants will be used to treat epithelial cells for 72 hours and the media analyzed by cytokine/growth factor protein arrays. The protein assays showed that CSC and nicotine treated P. gingivalis cells had less pro-tein than the others. The total protein in the supernatant for the CSC treated bacteria was less compared to others. The protein data suggests that CSC and nicotine affect protein expression in and by the P. gingivalis cells. To-bacco-treated bacteria are hypothesized to increase the expression of pro-inflammatory cytokines/growth factors by the epithelial cells, thereby con-tributing to the inflammation seen in periodontal diseases. This research was funded by Indiana University-Purdue University Indianapolis, Multidisci-plinary Undergraduate Research Institute (MURI).Item Inhibitory effect of Porphyromonas gingivalis-derived phosphoethanolamine dihydroceramide on acid ceramidase expression in oral squamous cells(Wiley, 2023) Yamada, Chiaki; Ho, Anny; Nusbaum, Amilia; Xu, Ruijuan; Davey, Mary Ellen; Nichols, Frank; Mao, Cungui; Movila, Alexandru; Biomedical Sciences and Comprehensive Care, School of DentistryThe maintenance of diminished acid ceramidase (ASAH1) gene expression leading to the accumulation of antiproliferative intracellular ceramides in oral squamous cell carcinoma (OSCC) has emerged as a prospective oral cancer therapeutic regimen. Our published study demonstrated that the key periodontal pathogen Porphyromonas gingivalis downregulates the expression patterns of ASAH1 mRNA in normal epithelial cells in vitro. Therefore, P. gingivalis may also beneficially diminish the expression of ASAH1 in OSCC. Because a uniquely structured P. gingivalis-derived phosphoethanolamine dihydroceramide (PEDHC) inhibits the proliferation of normal human fibroblasts, this study aimed to test the effect of PEDHC on the survival of human oral squamous OECM-1 cells in vitro. We demonstrated that the P. gingivalis dihydroceramide-null (ΔPG1780) strain upregulates the expression of ASAH1 mRNA and promotes aggressive proliferation and migration of OECM-1 cells compared to the parent P. gingivalis-W83 strain. In addition, the intracellular concentration of ceramides was dramatically elevated in OECM-1 cells exposed to PEDHC in vitro. Furthermore, PEDHC inhibited expression patterns of ASAH1 mRNA as well as some genes associated with degradation of the basement membranes and extracellular matrix, for example, MMP-2, ADAM-17 and IL-6, in OECM-1 cells. Altogether, these data indicated that PEDHC produced by P. gingivalis inhibits acid ceramidase expression, promotes intracellular ceramide accumulation and suppresses the survival and migration of OSCC cells in vitro. Further studies are needed to determine molecular mechanisms of PEDHC-mediated inhibitory effect(s) on OSCC using in vivo models of oral cancer.Item Inhibitory effect of Porphyromonas gingivalis-derived phosphoethanolamine dihydroceramide on acid ceramidase expression in oral squamous cells(Wiley, 2023-05) Yamada, Chiaki; Ho, Anny; Nusbaum, Amilia; Xu, Ruijuan; Davey, Mary Ellen; Nichols, Frank; Mao, Cungui; Movila, Alexandru; Biomedical Sciences and Comprehensive Care, School of DentistryThe maintenance of diminished acid ceramidase (ASAH1) gene expression leading to the accumulation of antiproliferative intracellular ceramides in oral squamous cell carcinoma (OSCC) has emerged as a prospective oral cancer therapeutic regimen. Our published study demonstrated that the key periodontal pathogen Porphyromonas gingivalis downregulates the expression patterns of ASAH1 mRNA in normal epithelial cells in vitro. Therefore, P. gingivalis may also beneficially diminish the expression of ASAH1 in OSCC. Because a uniquely structured P. gingivalis-derived phosphoethanolamine dihydroceramide (PEDHC) inhibits the proliferation of normal human fibroblasts, this study aimed to test the effect of PEDHC on the survival of human oral squamous OECM-1 cells in vitro. We demonstrated that the P. gingivalis dihydroceramide-null (ΔPG1780) strain upregulates the expression of ASAH1 mRNA and promotes aggressive proliferation and migration of OECM-1 cells compared to the parent P. gingivalis-W83 strain. In addition, the intracellular concentration of ceramides was dramatically elevated in OECM-1 cells exposed to PEDHC in vitro. Furthermore, PEDHC inhibited expression patterns of ASAH1 mRNA as well as some genes associated with degradation of the basement membranes and extracellular matrix, for example, MMP-2, ADAM-17 and IL-6, in OECM-1 cells. Altogether, these data indicated that PEDHC produced by P. gingivalis inhibits acid ceramidase expression, promotes intracellular ceramide accumulation and suppresses the survival and migration of OSCC cells in vitro. Further studies are needed to determine molecular mechanisms of PEDHC-mediated inhibitory effect(s) on OSCC using in vivo models of oral cancer.Item Volatile Sulfur Compounds and their Effects on Streptococcus mutans Biofilm(Office of the Vice Chancellor for Research, 2015-04-17) Whitaker, Caitlin; Huang, Ruijie; Gregory, Richard L.Volatile sulfur compounds (VSC) are produced by certain anaerobic bacteria known to cause halitosis in the oral cavity. Porphyromonas gingivalis produces VSC and causes halitosis and periodontal disease. Streptococcus mutans is a facultative anaerobic bacterium that is most commonly known for causing dental caries in the oral cavity. No research has been reported indicating a connection between S. mutans and VSC. An observation was made by Dr. Richard Gregory and Ph.D. student Ruijie Huang that when an S. mutans culture was left in an anaerobic environment with P. gingivalis, the growth of S. mutans appeared to be inhibited. This study explored that observation using not only P. gingivalis culture supernatant containing VSC but also other VSC, such as DTT, and 2ME to demonstrate that VSC inhibit the growth of S. mutans biofilm using total growth and biofilm formation after crystal violet staining. The results were read using a spectrophotometer to read the total growth and biofilm formation. These results indicate that S. mutans total growth and biofilm is significantly inhibited (p<0.05) by the presence of VSC. Results also establish that different VSC inhibit S. mutans depending on the amount of sulfur in each agent; however, each agent greatly reduced the amount of S. mutans biofilm. Due to these results, one can conclude that there is an inhibitory relationship between VSC and S. mutans. A person with a major case of halitosis or a person who has periodontal disease would most likely have little to no evidence of dental caries at that time.