Recovery of putative pathogens from paper point sampling at different depths of periodontal lesions

Nikola Angelov1, Raydolfo M Aprecio1, James Kettering1, Tord Lundgren2, Matt Riggs3, Jan Egelberg11School of Dentistry, Loma Linda University, Loma Linda, CA, USA; 2Department of Periodontology, University of Florida, Gainesville, FL, USA; 3Department of Psychology, California State University, San Bernardino, CA, USABackground: The aim of this study was to compare the recovery of three putative periodontal pathogens from periodontal lesions in samples using paper points inserted to different depths of the lesions.Methods: Twenty 6–8 mm deep periodontal lesions with bleeding on probing were studied. Microbial samples were obtained using paper points inserted to three different depths of the lesions: orifice of lesion; 2 mm into the lesion; and to the base of lesion. Culturing was used for recovery and identification of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia.Results: The recovery of each of the three putative periodontal pathogens was similar following sampling at the various depths of the lesions.Conclusions: The findings may be explained by the fact that the paper points become saturated as they pass through the orifice of the lesion. Absorption of microorganisms will therefore primarily occur at the orifice. It is also conceivable that the pathogens may be present in similar proportions throughout the various depths of the periodontal lesions.Keywords: paper point sampling, P. gingivalis, P. intermedia, A. actinomycetemcomitan

Root canal disinfection comparing conventional irrigation vs photon-induced photoacoustic streaming (PIPS) using a buffered 0.5 % sodium hypochlorite solution

Abstract Background The purpose of this study was to assess the antimicrobial effect of a buffered 0.5 % sodium hypochlorite solution activated by photon-induced photoacoustic streaming compared to conventional irrigation. Methods The canals on 48 single canal lower bicuspids were cleaned and shaped using rotary instrumentation. All roots were autoclaved for 20 min. Thirty-six of the roots were placed in glass flasks with blood heart infusion media and Enterococcus faecalis (ATCC 4082) for 4 weeks. The remaining 12 roots were placed in a sterile environment and served as negative controls. The contaminated roots were irrigated by conventional means using a buffered 0.5 % sodium hypochlorite solution with or without photon-induced photoacoustic streaming (PIPS) activation (n = 12 each group). The remaining 12 roots did not receive any treatment and served as positive controls. The apical 3 mm of each tooth was sectioned and pulverized. The pulverized samples were collected and placed in Eppendorf micro-centrifuge tubes with sterile phosphate-buffered saline. Thirty MicroLiters of the collected samples was then placed in the blood heart infusion media and incubated for 24 h at 37 °C. Colony forming units (CFU) were compared with Wilcoxon signed ranked test. Mann-Whitney U test was used to assess PIPS effectiveness in comparison with conventional irrigation. Results Both regimens reduced significantly the number of CFU; however, reduction was significantly higher for the PIPS group (p = 0.002). Conclusion Buffered 0.5 % sodium hypochlorite delivered by conventional method was effective in removing E. faecalis from contaminated root canals; however, activation of a buffered 0.5 % sodium hypochlorite solution by PIPS significantly increased its antimicrobial capacity

Therapeutic effect of TSG-6 engineered iPSC-derived MSCs on experimental periodontitis in rats: a pilot study.

BACKGROUND: We derived mesenchymal stem cells (MSCs) from rat induced pluripotent stem cells (iPSCs) and transduced them with tumor necrosis factor alpha-stimulated gene-6 (TSG-6), to test whether TSG-6 overexpression would boost the therapeutic effects of iPSC-derived MSCs in experimental periodontitis. METHODS: A total of 30 female Sprague-Dawley (SD) rats were randomly divided into four groups: healthy control group (Group-N, n = 5), untreated periodontitis group (Group-P, n = 5), iPS-MSCs-treated and iPSC-MSCs/TSG-6-treated periodontitis groups (Group-P1 and P2, n = 10 per group). Experimental periodontitis was established by ligature and infection with Porphyromonas gingivalis around the maxillae first molar bilaterally. MSC-like cells were generated from rat iPSCs, and transducted with TSG-6. iPSC-MSCs or iPSC-MSCs/TSG-6 were administrated to rats in Group-P1 or P2 intravenously and topically, once a week for three weeks. Blood samples were obtained one week post-injection for the analysis of serum pro-inflammatory cytokines. All animals were killed 3 months post-treatment; maxillae were then dissected for histological analysis, tartrate-resistant acid phosphatase (TRAP) staining, and morphological analysis of alveolar bone loss. RESULTS: Administration of iPSC-MSC/TSG-6 significantly decreased serum levels of IL-1β and TNF-α in the Group-P2 rats (65.78 pg/ml and 0.56 pg/ml) compared with those in Group-P (168.31 pg/ml and 1.15 pg/ml respectively) (p<0.05). Both alveolar bone loss and the number of TRAP-positive osteoclasts showed a significant decrease in rats that received iPSC-MSC/TSG-6 treatment compared to untreated rats in Group-P (p<0.05). CONCLUSIONS: We demonstrated that overexpression of TSG-6 in rat iPSC-derived MSCs were capable of decreasing inflammation in experimental periodontitis and inhibiting alveolar bone resorption. This may potentially serve as an alternative stem-cell-based approach in the treatment and regeneration of periodontal tissues

Alveolar bone loss: Alveolar bone loss analysis.

<p>(A) Healthy control group. (B) Untreated periodontitis group. (C) iPSC-MSCs treated periodontitis group. (D) iPSC-MSCs/TSG-6 treated periodontitis group. Scale bar, 1 mm. (E) Alveolar bone loss (ABL) was analyzed by measuring the distance between the cementum-enamel junction (CEJ) and the palatal alveolar bone crest (ABC) at 9 sites from first to second molar. The ABL in iPSC-MSCs/TSG-6 treated group showed a significant difference compared to the other groups.</p

Cell culture: Morphology of cells under light microscopy.

<p>(A) Rat induced pluripotent stem cells (iPSCs). (B) Rat bone marrow mesenchymal stem cells (BM-MSCs), passage 3. (C) iPSC derived-MSCs, passage 3. Scale bar, 200 µm.</p

Overexpression TSG-6 in iPSC-derive MSCs in vitro.

<p>Overexpression TSG-6 in iPSC-MSCs in vitro. After 24 hrs transfection, TSG-6 was overexpressed in iPSC-MSCs in vitro, detected by PCR.</p

TRAP staining: Osteoclasts formation in different groups.

<p>(A) Healthy control group. (B) Untreated periodontitis group. (C) iPSC-MSCs treated periodontitis group. (D) iPSC-MSCs/TSG-6 treated periodontitis group. TRAP-positive osteoclasts in rat maxillae showed a significant decrease in iPSC-MSCs/TSG-6 treated group. (AB = alveolar bone, PDL = periodontal ligament, R = tooth root, black arrow = TRAP-positive osteoclasts) (TRAP staining, scale bar, 50 µm). (E) Quantitative analysis of TRAP-positive osteoclasts (nuclei≥3) showed a significantly decreased number of osteoclasts in iPSC-MSCs and iPSC-MSCs/TSG-6 treated group, compared to untreated periodontitis group; the decrease number of osteoclasts showed a significant differences between iPSC-MSCs/TSG-6 and iPSC-MSCs treated group.</p

Flow cytometry assay.

<p>The characteristic cell surface makers of rat MSCs were detected by FACS. The iPSC derived MSCs at passage 5 revealed positivity for CD29 and CD90, negativity for CD34 and CD45.</p

Pro-inflammation cytokine.

<p>Pro-inflammatory cytokine IL-1β and TNF-α in serum was detected by ELISA. The serum concentration of IL-1β and TNF-α showed a significant decrease after iPS-MSCs/TSG-6 treated when compared to untreated periodontitis group; and no significant differences compared to healthy control group.</p