Bactericidal Effects of Diode Laser Irradiation on Enterococcus faecalis Using Periapical Lesion Defect Model.

Objective. Photodynamic therapy has been expanded for use in endodontic treatment. The aim of this study was to investigate the antimicrobial effects of diode laser irradiation on endodontic pathogens in periapical lesions using an in vitro apical lesion model. Study Design. Enterococcus faecalis in 0.5% semisolid agar with a photosensitizer was injected into apical lesion area of in vitro apical lesion model. The direct effects of irradiation with a diode laser as well as heat produced by irradiation on the viability of microorganisms in the lesions were analyzed. Results. The viability of E. faecalis was significantly reduced by the combination of a photosensitizer and laser irradiation. The temperature caused by irradiation rose, however, there were no cytotoxic effects of heat on the viability of E. faecalis. Conclusion. Our results suggest that utilization of a diode laser in combination with a photosensitizer may be useful for clinical treatment of periapical lesions.

Enumeration of viable Enterococcus faecalis, a predominant apical periodontitis pathogen, using propidium monoazide and quantitative real-time polymerase chain reaction.

To discriminate between viable and non-viable Enterococcus faecalis, the predominant pathogen in apical periodontitis, a real-time PCR method combined with propidium monoazide (PMA) was developed and evaluated. PMA had no antimicrobial effect on E. faecalis cells and permitted enumeration of both viable and non-viable cells. Therefore, E. faecalis cells from the root canals of nine patients with apical periodontitis were analyzed to evaluate the diagnostic usefulness of this approach. Viable and non-viable E. faecalis cells were successfully discriminated in these clinical specimens. A real-time PCR assay combined with PMA will contribute to the precise diagnosis of apical periodontitis.

Effects of hyaluronic acid sponge as a scaffold on odontoblastic cell line and amputated dental pulp.

It is important to develop a suitable three-dimensional scaffold for the regeneration therapy of dental pulp. In the present study, the effects of hyaluronic acid (HA) sponge on responses of the odontoblastic cell line (KN-3 cells) in vitro, as well as responses of amputated dental pulp of rat molar in vivo, were examined. In vitro, KN-3 cells adhered to the stable structure of HA sponge and that of collagen sponge. In vivo, dental pulp proliferation and vessel invasion were observed in both sponges implanted at dentin defect area above amputated dental pulp, and the cell-rich reorganizing tissue was observed in the dentin defect when HA sponge was implanted as compared with collagen sponge. Expression levels of IL-6 and TNF-alpha in KN-3 cells seeded in HA sponge were nearly the same with those in the cells seeded in collagen sponge, while the numbers (0.67 x 10(3) at 1 week and 0.7 x 10(3) at 3 weeks) of granulated leukocytes that invaded into HA sponge from amputated dental pulp was significantly lower than those (1.22 x 10(3) at 1 week and 1.1 x 10(3) at 3 weeks) of collagen sponge (p < 0.01 at 1 week and p < 0.05 at 3 weeks). These results suggest that HA sponge has an appropriate structure, biocompatibility, and biodegradation for use as a scaffold for dental pulp regeneration.

Ozonated water improves lipopolysaccharide-induced responses of an odontoblast-like cell line.

INTRODUCTION: It is important to develop an antimicrobial agent without any damage on dental pulp. In the present study, we examined whether pretreatment of bacterial lipopolysaccharides (LPS) with ozonated water (O(3)aq) improves LPS-induced responses of rat odontoblastic cell line, KN-3. METHODS: After the pretreatment of LPS with O(3)aq, effects of LPS and O(3)aq-treated LPS on cell viability; calcification ability; expression of cyclooxygenase 2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha); and activation of p38 of KN-3 cells were examined. RESULTS: The formation of mineralized nodules by KN-3 cells was suppressed by LPS, whereas that suppression was inhibited by the pretreatment of LPS with ozonated water. We also found that LPS-induced expression of COX-2, IL-6, and TNF-alpha and p38 activation were markedly suppressed when LPS was pretreated with ozonated water. Furthermore, expression of COX-2, IL-6, and TNF-alpha by LPS were mainly induced through p38 activation. CONCLUSION: These results suggest that odontoblastic cells exhibit inflammatory responses against LPS and that ozonated water has the ability to improve LPS-induced inflammatory responses and suppression of odontoblastic properties of KN-3 cells through direct inhibition of LPS.

Effects of lipopolysaccharide on newly established rat dental pulp-derived cell line with odontoblastic properties.

To clarify mechanisms of pulp wound healing and regeneration, it is important to establish continuous odontoblast-lineage cell lines. In this study, we established the proliferating pulp progenitor cell lines from dental papilla cells of rat incisor. These cell lines showed high levels of alkaline phosphatase (ALP) activity, expression of Runx2 and dentin sialophosphoprotein (DSPP), and extracellular formation of mineralized nodules. By using the cell line with high expression level of DSPP and the prominent mineral deposition, we examined whether bacterial lipopolysaccharide (LPS) had effects on its odontoblastic properties and found that ALP activity, expression of DSPP and Runx2, and the formation of mineralized nodules were suppressed in LPS dose-dependent manner. These results indicate that our established pulp progenitor cell line exhibits odontoblastic properties, which were suppressed by LPS, suggesting that gram-negative bacterial infection might downregulate the odontoblast function.

Effects of sequential exposure to lipopolysaccharide and heat stress on dental pulp cells.

In the present study, we examined the effects of sequential exposure to bacterial lipopolysaccharide (LPS) and heat stress on dental pulp cells. LPS induced the proliferation of pulp cells through the activation of p38 MAPK. HSP27 was expressed in cells with or without LPS during the entire period of heat stress, while transiently phosphorylated by short-term heat stress. In LPS-treated cells, short-term heat stress also induced the phosphorylation of HSF1. The immediate phosphorylation of HSF1 and HSP27 in LPS-treated cells by short-term heat stress occurred dependent on the activation of p38 MAPK. However, with long-term heat stress, the activation of HSF1 and induction of HSP27 occurred independent of p38 MAPK. Further, full activation of Akt in LPS-treated cells was immediately induced by short-term heat stress and lasted during the entire period of heat stress. IkappaB alpha was induced and phosphorylated throughout sequential exposure to LPS and heat stress. These results suggest that LPS has the unique effects on the cytoprotection and the cell death of pulp cells during heat stress through the modification and the activation of heat stress responsive molecules, HSF1 and HSP27, and cell survival molecules, Akt and NF-kappaB/IkappaB alpha.

Thermotolerance of pulp cells and phagocytosis of apoptotic pulp cells by surviving pulp cells following heat stress.

Apoptosis is known to be associated with wound healing and regeneration of dental pulp. We examined the effects of heat stress on clonal dental pulp cell line (RPC-C2A cells) to clarify the pulp wound healing process. RPC-C2A cells were exposed to heat stress at 43 degrees C for 45 min. After several time intervals, the inhibition of cell proliferation and apoptosis induction were analyzed by cell viability assay, DNA gel electrophoresis, nuclear staining, and terminal deoxynucleotidyl transferase mediated labeling assay. RPC-C2A cells showed the thermotolerance following heat stress. We found that apoptosis was induced in some RPC-C2A cells, whereas others remained alive, and observed the engulfment of apoptotic cells by scavenger-like RPC-C2A cells following heat stress. We also analyzed the phagocytotic activity of RPC-C2A cells and found that they had an ability to engulf apoptotic RPC-C2A cells, which was stimulated by heat stress. These results suggest that heat stress induces apoptosis of RPC-C2A cells, which are phagocytosed by the surviving RPC-C2A cells.

Antimicrobial effect of ozonated water on bacteria invading dentinal tubules.

Ozone is known to act as a strong antimicrobial agent against bacteria, fungi, and viruses. In the present study, we examined the effect of ozonated water against Enterococcus faecalis and Streptcoccus mutans infections in vitro in bovine dentin. After irrigation with ozonated water, the viability of E. faecalis and S. mutans invading dentinal tubules significantly decreased. Notably, when the specimen was irrigated with sonication, ozonated water had nearly the same antimicrobial activity as 2.5% sodium hypochlorite (NaOCl). We also compared the cytotoxicity against L-929 mouse fibroblasts between ozonated water and NaOCl. The metabolic activity of fibroblasts was high when the cells were treated with ozonated water, whereas that of fibroblasts significantly decreased when the cells were treated with 2.5% NaOCl. These results suggest that ozonated water application may be useful for endodontic therapy.