Reduced Pressure Drop in Viscoelastic Polydimethylsiloxane Wall Channels.

Polydimethylsiloxane (PDMS) is an important viscoelastic material that finds applications in a large number of engineering systems, particularly lab-on-chip microfluidic devices built with a flexible substrate. Channels made of PDMS, used for transporting analytes, are integral to these applications. The PDMS viscoelastic nature can induce additional hydrodynamic contributions at the soft wall/fluid interface compared to rigid walls. In this research, we investigated the pressure drop within PDMS channels bounded by rigid tubes (cellulose tubes). The bulging effect of the PDMS was limited by the rigid tubes under flowing fluids. The PDMS viscoelasticity was modulated by changing the ratio of the base to the cross-linker from 10:1 to 35:1. We observed that the pressure drop of the flowing fluids within the channel decreased with the increased loss tangent of the PDMS in the examined laminar regime [Reynolds number (Re) ∼ 23-58.6 for water and Re ∼ 0.69-8.69 for glycerol solution]. The elastic PDMS 10:1 wall channels followed the classical Hagen Poiseuille's equation, but the PDMS walls with lower cross-linker concentrations and thicker walls decreased pressure drops. The friction factor (f) for the PDMS channels with the two working fluids could be approximated as f = 47/Re. We provide a correlation between the pressure drop and PDMS viscoelasticity based on experimental findings. In the correlation, the loss tangent predominates; the larger the loss tangent, the smaller is the pressure drop. The research findings appear to be unexpected if only considering the energy dissipation of viscoelastic PDMS walls. We attributed the reduction in the pressure drop to a lubricating effect of the viscoelastic PDMS walls in the presence of the working fluids. Our results reveal the importance of the subtle diffusion of the residual oligomers and water from the bulk to the soft wall/fluid interface for the observed pressure drop in soft wall channels.

Synthetic Human ß Defensin-3-C15 Peptide in Endodontics: Potential Therapeutic Agent in Streptococcus gordonii Lipoprotein-Stimulated Human Dental Pulp-Derived Cells.

Human ß defensin-3-C15, an epithelium-derived cationic peptide that has antibacterial/antifungal and immuno-regulatory properties, is getting attention as potential therapeutic agent in endodontics. This study aimed to investigate if synthetic human ß defensin-3-C15 (HBD3-C15) peptides could inhibit inflammatory responses in human dental pulp cells (hDPCs), which had been induced by gram-positive endodontic pathogen. hDPC explant cultures were stimulated with Streptococcus gordonii lipoprotein extracts for 24 h to induce expression of pro-inflammatory mediators. The cells were then treated with either HBD3-C15 (50 µg/mL) or calcium hydroxide (CH, 100 µg/mL) as control for seven days, to assess their anti-inflammatory effects. Quantitative RT-PCR analyses and multiplex assays showed that S. gordonii lipoprotein induced the inflammatory reaction in hDPCs. There was a significant reduction of IL-8 and MCP-1 within 24 h of treatment with either CH or HBD3-C15 (p < 0.05), which was sustained over 1 week of treatment. Alleviation of inflammation in both medications was related to COX-2 expression and PGE2 secretion (p < 0.05), rather than TLR2 changes (p > 0.05). These findings demonstrate comparable effects of CH and HDB3-C15 as therapeutic agents for inflamed hDPCs.

Enterococcus Phage vB_EfaS_HEf13 as an Anti-Biofilm Agent Against Enterococcus faecalis.

Enterococcus faecalis is a Gram-positive bacterium that is frequently found in the periapical lesion of patients with apical periodontitis. Its biofilm formation in root canal is closely related to the development of refractory apical periodontitis by providing increased resistance to endodontic treatments. Phage therapy has recently been considered as an efficient therapeutic strategy in controlling various periodontal pathogens. We previously demonstrated the bactericidal capacities of Enterococcus phage vB_EfaS_HEf13 (phage HEf13) against clinically-isolated E. faecalis strains. Here, we investigated whether phage HEf13 affects biofilm formation and pre-formed biofilm of clinically-isolated E. faecalis, and its combinatory effect with endodontic treatments, including chlorhexidine (CHX) and penicillin. The phage HEf13 inhibited biofilm formation and disrupted pre-formed biofilms of E. faecalis in a dose- and time-dependent manner. Interestingly, phage HEf13 destroyed E. faecalis biofilm exopolysaccharide (EPS), which is known to be a major component of bacterial biofilm. Furthermore, combined treatment of phage HEf13 with CHX or penicillin more potently inhibited biofilm formation and disrupted pre-formed biofilm than either treatment alone. Confocal laser scanning microscopic examination demonstrated that these additive effects of the combination treatments on disruption of pre-formed biofilm are mediated by relatively enhanced reduction in thickness distribution and biomass of biofilm. Collectively, our results suggest that the effect of phage HEf13 on E. faecalis biofilm is mediated by its EPS-degrading property, and its combination with endodontic treatments more potently suppresses E. faecalis biofilm, implying that phage HEf13 has potential to be used as a combination therapy against E. faecalis infections.

Short-chain fatty acids inhibit the biofilm formation of Streptococcus gordonii through negative regulation of competence-stimulating peptide signaling pathway.

Streptococcus gordonii, a Gram-positive commensal bacterium, is an opportunistic pathogen closely related to initiation and progression of various oral diseases, such as periodontitis and dental caries. Its biofilm formation is linked with the development of such diseases by enhanced resistance against antimicrobial treatment or host immunity. In the present study, we investigated the effect of short-chain fatty acids (SCFAs) on the biofilm formation of S. gordonii. SCFAs, including sodium acetate (NaA), sodium propionate (NaP), and sodium butyrate (NaB), showed an effective inhibitory activity on the biofilm formation of S. gordonii without reduction in bacterial growth. SCFAs suppressed S. gordonii biofilm formation at early time points whereas SCFAs did not affect its preformed biofilm. A quorum-sensing system mediated by competence-stimulating peptide (CSP) is known to regulate biofilm formation of streptococci. Interestingly, SCFAs substantially decreased mRNA expression of comD and comE, which are CSP-sensor and its response regulator responsible for CSP pathway, respectively. Although S. gordonii biofilm formation was enhanced by exogenous synthetic CSP treatment, such effect was not observed in the presence of SCFAs. Collectively, these results suggest that SCFAs have an anti-biofilm activity on S. gordonii through inhibiting comD and comE expression which results in negative regulation of CSP quorum-sensing system. SCFAs could be an effective anti-biofilm agent against S. gordonii for the prevention of oral diseases.

Candida albicans Virulence Factors and Pathogenicity for Endodontic Infections.

Candida albicans (C. albicans) is the fungus most frequently isolated from endodontic root canal infections. Although recognized by dental pulp and periradicular tissue cells that elicit immune responses, it eludes host defenses and elicits cell death. Then, C. albicans binds tooth dentin, forms biofilms, and invades dentinal tubules to resist intracanal disinfectants and endodontic treatments. Insensitive to most common medicaments, it survives sequestered within biofilms and intratubular dentin. Thus, C. albicans has been associated with cases of persistent or refractory root canal infections. Its treatment strategies may require alternative intracanal irrigants, intracanal medicaments such as chlorhexidine gel or human beta defensin-3 (HBD3), Ca-Si-based obturating materials, and microsurgical procedures.

A Study on the Correlation between C-Reactive Protein Concentration and Teeth with a ≥5 mm Periodontal Pocket in Chronic Periodontitis Patients.

OBJECTIVE: To evaluate the relationship between CRP levels and teeth with ≥5 mm PD in chronic periodontitis patients. MATERIALS AND METHODS: We evaluated 49 patients with chronic periodontitis who visited the Department of Periodontology at Wonkwang University Dental Hospital. At the first visit, high-sensitive CRP testing of venous blood samples was performed, and correlations were statistically evaluated. RESULTS: The mean hs-CRP level of patients diagnosed with severe periodontitis was 2.0 mg/L (0.13-13.95 mg/L). Statistically, patients with a high rate of teeth diagnosed with severe periodontitis are more likely to have higher hs-CRP level. CONCLUSION: Within the limits of this study, the number and proportion of teeth showing ≥5 mm PD was positively correlated with CRP concentration.

Effect of Etching Procedures on the Adhesion of Biofilm-Coated Dentin.

Oral biofilms coat all surfaces in the oral cavity including the exposed dentin surface. This study aimed to investigate biofilm removal by acid etching procedures and the effects of the residual biofilm on dentin surfaces on composite-dentin adhesion. Dentin discs were assigned to five groups: no biofilm formation (C); biofilm formation and no surface treatment (BF); biofilm formation and acid etching (BF-E); biofilm formation and acid etching followed by chlorhexidine soaking (BF-EC); and biofilm formation and rubbing with pumice, followed by acid etching (BF-RE). Biofilms were formed on saliva-precoated dentin discs by soaking the discs in Streptococcus mutans (S. mutans) suspension. Biofilm removal from the dentin surface was evaluated quantitatively and qualitatively by confocal laser scanning microscopy and scanning electron microscopy, respectively. To compare the bond strength of the biofilm-coated dentin discs with the surface treatments, specimens were assigned to four groups: no biofilm formation and acid etching (C-E); BF-E; BF-EC; and BF-RE. Assessments of the micro-shear bond strength and subsequent failure modes were performed. BF-E and BF-EC did not remove the biofilm, whereas BF-RE partially removed the biofilm attached to the dentin (p < 0.05). The bond strength of BF-RE was significantly higher than those of BF-E and BF-EC, but lower than that of C-E (p < 0.05). In conclusion, mechanical biofilm removal is recommended before etching procedures to enhance adhesion to the biofilm-coated dentin.

Lactobacillus plantarum lipoteichoic acid disrupts mature Enterococcus faecalis biofilm.

Apical periodontitis is caused by biofilm-mediated root canal infection. Early phase oral bacterial biofilms are inhibited by Lactobacillus plantarum lipoteichoic acid (Lp.LTA). However, mature biofilms that develop over 3 weeks are more resistant to traditional endodontic medicaments. Therefore, this study examined the effectiveness of Lp.LTA on disrupting mature Enterococcus faecalis biofilms, and on enhancing the effects of endodontic medicaments. LTA was purified from L. plantarum through butanol extraction followed by hydrophobic and ion-exchange chromatography. E. faecalis biofilms were formed over 3 weeks on glass bottom dishes and in dentin blocks obtained from human single-rooted premolars. These mature biofilms were treated with or without Lp.LTA for 1 h, followed by additional treatment with either chlorhexidine digluconate (CHX), calcium hydroxide (CH), or triple antibiotics for 24 h. Biofilms on glass were live/dead stained and quantified by ZEN through confocal laser microscopy. Bio-films in dentin were fixed, sputter coated and analyzed by ImageJ with scanning electron microscopy. Preformed E. faecalis mature biofilms on the culture dishes were dose-dependently disrupted by Lp.LTA. Lp.LTA potentiated the effects of CHX or CH on the disruption of mature biofilm. Interestingly, CHX-induced disruption of preformed E. faecalis mature biofilms was synergistically enhanced only when pre-treated with Lp.LTA. Furthermore, in the dentin block model, Lp.LTA alone reduced E. faecalis mature biofilm and pre-treatment with Lp.LTA promoted the anti-biofilm activity of CHX. Lp.LTA could be an anti-biofilm or supplementary agent that can be effective for E. faecalis-biofilm-induced diseases.

Enhanced biofilm formation of Streptococcus gordonii with lipoprotein deficiency.

Streptococcus gordonii is a commensal Gram-positive bacterium that acts as an opportunistic pathogen that can cause apical periodontitis, endocarditis, and pneumonia. Biofilm formation of bacteria is important for the initiation and progression of such diseases. Although lipoproteins play key roles in physiological functions, the role of lipoproteins of S. gordonii in its biofilm formation has not been clearly understood. In this study, we investigated the role of lipoproteins of S. gordonii in the bacterial biofilm formation using its lipoprotein-deficient strain (Δlgt). The S. gordonii Δlgt exhibited increased biofilm formation on the human dentin slices or on the polystyrene surfaces compared to the wild-type strain, while its growth rate did not differ from that of the wild-type. In addition, the S. gordonii Δlgt strain exhibited the enhanced LuxS mRNA expression and AI-2 production, which is known to be a positive regulator of biofilm formation, compared to the wild-type. Concordantly, the augmented biofilm formation of S. gordonii Δlgt was attenuated by an AI-2 inhibitor, D-ribose. In addition, lipoproteins from purified S. gordonii inhibited the biofilm formation of S. gordonii wild-type and Δlgt. Taken together, these results suggest that lipoprotein-deficient S. gordonii form biofilms more effectively than the wild-type strain, which might be related to the AI-2 quorum-sensing system.

Endodontic biofilms: contemporary and future treatment options.

Apical periodontitis is a biofilm-mediated infection. The biofilm protects bacteria from host defenses and increase their resistance to intracanal disinfecting protocols. Understanding the virulence of these endodontic microbiota within biofilm is essential for the development of novel therapeutic procedures for intracanal disinfection. Both the disruption of biofilms and the killing of their bacteria are necessary to effectively treat apical periodontitis. Accordingly, a review of endodontic biofilm types, antimicrobial resistance mechanisms, and current and future therapeutic procedures for endodontic biofilm is provided.

Lactobacillus plantarum Lipoteichoic Acid Inhibits Oral Multispecies Biofilm.

INTRODUCTION: Apical periodontitis is an inflammatory disease in the periradicular region of teeth that results from infection by multispecies bacterial biofilm residing in the root canal system. In this study, we investigated whether Lactobacillus plantarum lipoteichoic acid (Lp.LTA) could inhibit multispecies oral pathogenic bacterial biofilm formation. METHODS: Highly pure and structurally intact Lp.LTA was purified from L. plantarum. Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and Enterococcus faecalis were co-cultured to form oral multispecies biofilm in the presence or absence of Lp.LTA on culture plates or human dentin slices. Preformed biofilm was treated with or without Lp.LTA, followed by additional treatment with intracanal medicaments such as calcium hydroxide or chlorhexidine digluconate. Confocal microscopy and crystal violet assay were performed to determine biofilm formation. Biofilm on human dentin slices was visualized with a scanning electron microscope. RESULTS: Biofilm formation of multispecies bacteria on the culture dishes was dose-dependently reduced by Lp.LTA compared with the nontreatment control group. Lp.LTA also inhibited multispecies biofilm formation on the dentin slices in a dose-dependent manner. Interestingly, Lp.LTA was shown to reduce preformed multispecies biofilm compared with the nontreatment group. Moreover, Lp.LTA potentiated the effectiveness of the intracanal medicaments in the removal of preformed multispecies biofilm. CONCLUSIONS: These results suggest that Lp.LTA is a potential anti-biofilm agent for treatment or prevention of oral infectious disease, including apical periodontitis, which is mainly caused by multispecies bacterial biofilm.

Lipoteichoic acids of lactobacilli inhibit Enterococcus faecalis biofilm formation and disrupt the preformed biofilm.

Enterococcus faecalis, a Gram-positive bacterium commonly isolated in patients with refractory apical periodontitis, invades dentin tubules easily and forms biofilms. Bacteria in biofilms, which contribute to recurrent and/or chronic inflammatory diseases, are more resistant to antimicrobial agents than planktonic cells and easily avoid phagocytosis. Although Lactobacillus plantarum lipoteichoic acid (Lp.LTA) is associated with biofilm formation, the effect of Lp.LTA on biofilm formation by E. faecalis is not clearly understood. In this study, we investigated whether Lp.LTA inhibits E. faecalis biofilm formation. The degree of biofilm formation was determined by using crystal violet assay and LIVE/DEAD bacteria staining. The quantification of bacterial growth was determined by measuring the optical density at 600 nm with a spectrophotometer. Formation of biofilms on human dentin slices was observed under a scanning electron microscope. E. faecalis biofilm formation was reduced by Lp.LTA treatment in a dose-dependent manner. Lp.LTA inhibited biofilm development of E. faecalis at the early stage without affecting bacterial growth. LTA from other Lactobacillus species such as Lactobacillus acidophilus, Lactobacillus casei, or Lactobacillus rhamnosus GG also inhibited E. faecalis biofilm formation. In particular, among LTAs from various lactobacilli, Lp.LTA showed the highest inhibitory effect on biofilms formed by E. faecalis. Interestingly, LTAs from lactobacilli could remove the biofilm preformed by E. faecalis. These inhibitory effects were also observed on the surface of human dentin slices. In conclusion, Lactobacillus species LTA inhibits biofilm formation caused by E. faecalis and it could be used as an anti-biofilm agent for prevention or treatment against E. faecalis-associated diseases.

The synthetic human beta-defensin-3 C15 peptide exhibits antimicrobial activity against Streptococcus mutans, both alone and in combination with dental disinfectants.

Streptococcus mutans is a major etiologic agent of human dental caries that forms biofilms on hard tissues in the human oral cavity, such as tooth and dentinal surfaces. Human ß-defensin-3 (HBD3) is a 45-amino-acid natural antimicrobial peptide that has broad spectrum antimicrobial activity against bacteria and fungi. A synthetic peptide consisting of the C-terminal 15 amino acids of HBD3 (HBD3-C15) was recently shown to be sufficient for its antimicrobial activity. Thus, clinical applications of this peptide have garnered attention. In this study, we investigated whether HBD3-C15 inhibits the growth of the representative cariogenic pathogen Streptococcus mutans and its biofilm formation. HBD3-C15 inhibited bacterial growth, exhibited bactericidal activity, and attenuated bacterial biofilm formation in a dose-dependent manner. HBD3-C15 potentiated the bactericidal and anti-biofilm activity of calcium hydroxide (CH) and chlorhexidine digluconate (CHX), which are representative disinfectants used in dental clinics, against S. mutans. Moreover, HBD3-C15 showed antimicrobial activity by inhibiting biofilm formation by S. mutans and other dentinophilic bacteria such as Enterococcus faecalis and Streptococcus gordonii, which are associated with dental caries and endodontic infection, on human dentin slices. These effects were observed for HBD3-C15 alone and for HBD3-C15 in combination with CH or CHX. Therefore, we suggest that HBD3-C15 is a potential alternative or additive disinfectant that can be used for the treatment of oral infectious diseases, including dental caries and endodontic infections.

Streptococcus gordonii lipoproteins induce IL-8 in human periodontal ligament cells.

Streptococcus gordonii, a Gram-positive oral bacterium, is a life-threatening pathogen that causes infective endocarditis. It is frequently isolated from the periapical lesions of patients with apical periodontitis and has thus been implicated in inflammatory responses. However, little is known about the virulence factors of S. gordonii responsible for the induction of inflammatory responses in the periapical areas. Here, we investigated the role of S. gordonii cell wall-associated virulence factors on interleukin (IL)-8 induction in human periodontal ligament (PDL) cells using ethanol-inactivated wild-type S. gordonii, a lipoteichoic acid (LTA)-deficient mutant (ΔltaS), and a lipoprotein-deficient mutant (Δlgt). Wild-type S. gordonii induced IL-8 expression at both the protein and mRNA levels in human PDL cells in a dose- and time-dependent manner. A transient transfection and reporter gene assay demonstrated that wild-type S. gordonii activated Toll-like receptor 2 (TLR2). Additionally, IL-8 production induced by wild-type S. gordonii was substantially inhibited by anti-TLR2-neutralizing antibodies. Both wild-type S. gordonii and the ΔltaS mutant induced IL-8 production; however, this response was not observed when cells were stimulated with the Δlgt mutant. Interestingly, lipoproteins purified from S. gordonii induced IL-8 production, whereas purified LTA did not. In addition, purified lipoproteins stimulated TLR2 more potently than LTA. Furthermore, S. gordonii-induced IL-8 expression was specifically inhibited by blocking p38 kinase, while lipoprotein-induced IL-8 expression was inhibited by blocking p38 kinase, ERK, or JNK. Of particular note, exogenous addition of purified S. gordonii lipoproteins enhanced Δlgt-induced IL-8 production in human PDL cells to an extent similar to that induced by the wild-type strain. Collectively, these results suggest that lipoproteins are an important component of S. gordonii for the induction of IL-8 production in human PDL cells through TLR2 activation. Therefore, lipoproteins potentially contribute to inflammatory apical periodontitis.

Serine-rich Repeat Adhesin Gordonii Surface Protein B is Important for Streptococcus gordonii Biofilm Formation.

INTRODUCTION: Streptococcus gordonii is a predominant member of the oral microflora and has been isolated from root canals of teeth with refractory apical periodontitis. Biofilm formation is important for various dental diseases, and S. gordonii is involved in dental biofilm formation as an early colonizer. Although serine-rich repeat (SRR) adhesins of S. gordonii such as gordonii surface protein B (GspB) are associated with bacterial colonization, the role of GspB in biofilm formation is not clearly understood. In the present study, we investigated the effect of S. gordonii GspB on biofilm formation using wild-type and GspB-deficient mutant S. gordonii strains. METHODS: Confocal microscopy and crystal violet assay were used to determine biofilm formation. Bacterial growth was examined by measuring optical density with spectrometry. Bacterial adherence and biofilm on the culture plate and human dentin slices were visualized with a scanning electron microscope. RESULTS: The GspB-deficient S. gordonii mutant strain was less potent than the wild-type strain in biofilm formation. Of note, there was no difference in the bacterial growth rate between the mutant and wild-type strains. Differences in biofilm-forming ability between the wild-type and mutant strains were more distinct in the sucrose-supplemented media. Furthermore, the GspB-deficient mutant exhibited attenuated formation of aggregates on the surface of the culture plate and human dentin slices. CONCLUSIONS: These results suggest that GspB is important for S. gordonii biofilm formation, which may contribute to the development of dental biofilm-associated diseases.

Increased humoral antibody response of foot-and-mouth disease virus vaccine in growing pigs pre-treated with poly-γ-glutamic acid.

This study was conducted to determine if humoral antibody response of foot-and-mouth disease (FMD) vaccine improved in 8-week-old growing pigs born to well-vaccinated sows pre-treated with 60 mg of poly-γ-glutamic acid (γ-PGA) three days before vaccination. Antibody against FMD virus serotype O was measured 0, 2, 4 and 6 weeks post-vaccination, using a PrioCHECK FMDV type O ELISA kit. The results showed that positive antibody reactions against FMDV serotype O antigen among a component of the vaccine significantly increased in response to pre-injection with γ-PGA.