Zhongchun Tong

Basic Fibroblast Growth Factor Enhances Stemness of Human Stem Cells from the Apical Papilla

INTRODUCTION Stem cells from the apical papilla (SCAP) are a type of mesenchymal stem cells found in the developing tissue, apical papilla, of immature permanent teeth. Studies have shown that SCAP are likely to be a source of primary odontoblasts that are responsible for the formation of root dentin. Basic fibroblast growth factor (bFGF) is a signaling molecule and pleiotropic growth factor involved in tooth root development, and it promotes proliferation of a variety of cell types. The effects of bFGF on SCAP, however, have not been examined. METHODS We investigated the regulatory effects of bFGF on the proliferation and differentiation potential of human SCAP in vitro. Changes in the cell cycle and proliferation, colony-forming unit-fibroblastic formation, alkaline phosphatase (ALP) activity, osteogenic/dentinogenic differentiation, and stem cell gene makers of SCAP, cultured in the presence or absence of bFGF, were evaluated. RESULTS Treatment with 5 ng/mL bFGF significantly increased SCAP proliferation and their colony-forming unit-fibroblastic formation efficiency. The growth factor also increased the expression of STRO-1 and the stem cell gene makers Nanog, Oct4, Sox2, and Rex1 in SCAP. In contrast, bFGF reduced the ALP activity, mineral nodule formation, and the expression of ALP, osteocalcin, bone sialoprotein, and dentin sialophosphoprotein. When SCAP cultures were expanded in the presence of bFGF for 1 week, subsequent stimulation of the osteogenic/dentinogenic condition resulted in enhanced differentiation. CONCLUSIONS Under certain conditions, bFGF enhances SCAP stemness by up-regulating stem cell gene expression, increasing proliferation ability, and potentiating differentiation potency.

Effect of a novel antimicrobial peptide chrysophsin-1 on oral pathogens and Streptococcus mutans biofilms

Dental caries and pulpal diseases are common oral bacterial infectious diseases. Controlling and reducing the causative pathogens, such as Streptococcus mutans and Enterococcus faecalis, is a key step toward prevention and treatment of the two diseases. Chrysophsin-1 is a cationic antimicrobial peptide having broad-spectrum bactericidal activity against both Gram-positive and Gram-negative bacteria. In this study, we investigated the antibacterial activity of chrysophsin-1 against several oral pathogens and S. mutans biofilms and performed a preliminary study of the antimicrobial mechanism. Cytotoxic activity of chrysophsin-1 against human gingival fibroblasts (HGFs) was investigated. Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and time-kill assay were used to evaluate the killing effect of chrysophsin-1. Scanning electron microscopy (SEM) was used to analyze morphological and membrane change in oral pathogens. Live/Dead staining, in conjunction with confocal scanning laser microscopy (CSLM), was used to observe and analyze S. mutans biofilms. MIC and MBC results demonstrated that chrysophsin-1 had different antimicrobial activities against the tested oral microbes. Lysis and pore formation of the cytomembrane were observed following treatment of the bacteria with chrysophsin-1 for 4h or 24h by SEM. Furthermore, CLSM images showed that chrysophsin-1 remarkably reduced the viability of cells within biofilms and had a significantly lethal effect against S. mutans biofilms. Toxicity studies showed that chrysophsin-1 at concentration between 8 μg/ml and 32 μg/ml had little effect on viability of HGFs in 5 min. Our findings suggest that chrysophsin-1 may have potential clinical applications in the prevention and treatment of dental caries and pulpal diseases.

Effects of sub-minimum inhibitory concentrations of antimicrobial agents on Streptococcus mutans biofilm formation.

Many studies have demonstrated that sub-minimum inhibitory concentrations (sub-MICs) of antimicrobial agents can inhibit bacterial biofilm formation. However, the mechanisms by which antimicrobial agents at sub-MICs inhibit biofilm formation remain unclear. At present, most studies are focused on Gram-negative bacteria; however, the effects of sub-MICs of antimicrobial agents on Gram-positive bacteria may be more complex. Streptococcus mutans is a major cariogenic bacterium. In this study, the S. mutans growth curve as well as the expression of genes related to S. mutans biofilm formation were evaluated following treatment with 0.5× MIC of chlorhexidine (CHX), tea polyphenols and sodium fluoride (NaF), which are common anticaries agents. The BioFlux system was employed to generate a biofilm under a controlled flow. Morphological changes of the S. mutans biofilm were observed and analysed using field emission scanning electron microscopy and confocal laser scanning microscopy. The results indicated that these three common anticaries agents could significantly upregulate expression of the genes related to S. mutans biofilm formation, and S. mutans exhibited a dense biofilm with an extensive extracellular matrix following treatment with sub-MICs of NaF and CHX. These findings suggest that sub-MICs of anticaries agents favour S. mutans biofilm formation, which might encourage dental caries progression.

Nisin inhibits dental caries-associated microorganism in vitro

Nisin, produced by Lactococcus lactis, is an antibiotic peptide to effectively antagonize a broad spectrum of Gram-positive bacteria, and is widely used as a safe food antimicrobial agent. In the present study, we investigated whether nisin could be used as an effective antibiotic peptide against the nine common cariogenic microorganisms, and its antimicrobial activity could be affected by the ingredients of saliva in oral cavity. In the minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC) and spot-on-lawn assay, nisin displayed different MIC, MBC and antimicrobial activity against the nine tested strains. There was statistical difference between the inhibitory zone diameters of nisin against the different tested bacteria (p<0.05), but no statistical difference between the inhibitory zone diameters of nisin dissolved in PBS and saliva (p>0.05). Furthermore, morphology and membranes of Streptococcus sanguinis, Streptococcus mutans, Lactobacillus fermenti and Lactobacillus acidophilus with nisin treatment were observed and showed different degrees of variation by a Field Emission Scanning Electron Microscope (FE-SEM). Our findings suggested that nisin has considerable potential for prevention and treatment of dental caries.

Parylene coating hinders Candida albicans adhesion to silicone elastomers and denture bases resin.

OBJECTIVE To investigate whether parylene coatings over denture bases and silicone elastomers can effectively reduce Candida albicans adhesion and thus to decrease the incidence of denture stomatitis. DESIGN Specimens of silicone elastomers A-2186 or lucitone 199 resin were prepared, and the measurements of contact angle, assay of XTT reduction and cell count of C. albicans adhesion were taken before and after parylene treatment. Furthermore, morphology of C. albicans adhesion for 48 h was observed by scanning electron microscope (SEM), and C. albicans adhesion for 4h was illustrated by confocal scanning laser microscopy (CLSM) in combination with fluorescent dyes FUN-1 and Concanavalin A. RESULT There was a statistical difference between mean contact angles of silicone elastomer A-2186 before and after parylene coating (P<0.05). The amount of C. albicans adhesion to the surface of silicone elastomer A-2186 and lucitone 199 resin after parylene treatment was significantly less than before parylene treatment by cells count and XTT reduction assay (P<0.05). In SEM and CLSM analysis, C. albicans biofilm was more apt to generate on the surface of silicone elastomer A-2186 than other three groups, and more C. albicans aggregation formed on the surface of silicone elastomer A-2186 and lucitone 199 resin before parylene treatment than after parylene treatment. CONCLUSION Parylene coating reduced C. albicans adhesion and aggregation on the surface of silicone elastomer A-2186 and lucitone 199 resin, and improved the wettability of silicone elastomer A-2186.

Antimicrobial and antibiofilm activity of pleurocidin against cariogenic microorganisms.

Dental caries is a common oral bacterial infectious disease of global concern. Prevention and treatment of caries requires control of the dental plaque formed by pathogens such as Streptococcus mutans and Streptococcus sobrinus. Pleurocidin, produced by Pleuronectes americanus, is an antimicrobial peptide that exerts broad-spectrum activity against pathogenic bacteria and fungi. Moreover, pleurocidin shows less hemolysis and is less toxic than other natural peptides. In the present study, we investigated whether pleurocidin is an effective antibiotic peptide against common cariogenic microorganisms and performed a preliminary study of the antimicrobial mechanism. We assayed minimal inhibitory concentration (MIC), minimal bactericide concentration (MBC) and bactericidal kinetics and performed a spot-on-lawn assay. The BioFlux system was used to generate bacterial biofilms under controllable flow. Fluorescence microscopy and confocal laser scanning microscopy (CLSM) were used to analyze and observe biofilms. Scanning electron microscopy was used to observe the bacterial membrane. MIC and MBC results showed that pleurocidin had different antimicrobial activities against the tested oral strains. Although components of saliva could affect antimicrobial activity, pleurocidin dissolved in saliva still showed antimicrobial effects against oral microorganisms. Furthermore, pleurocidin showed a favorable killing effect against BioFlux flow biofilms in vitro. Our findings suggest that pleurocidin has the potential to kill dental biofilms and prevent dental caries.

An in vitro synergetic evaluation of the use of nisin and sodium fluoride or chlorhexidine against Streptococcus mutans.

The objective of this study is to investigate the synergetic action between nisin and sodium fluoride or chlorhexidine against Streptococcus mutans, a primary cariogenic pathogen. In the antibacterial assay, a synergetic effect on S. mutans was found between nisin and sodium fluoride, but there was no interaction between nisin and chlorhexidine by the checkerboard, the fractional inhibitory concentration (FIC) and the fractional bactericidal concentration (FBC) tests. S. mutans survival rates showed a significant decline after treatment with a combination of nisin and sodium fluoride in a time-kill study. Scanning electron microscopy showed that the damage to S. mutans with the combined nisin and sodium fluoride treatment was the most severe among all of the different single and combined antimicrobial treatments. Furthermore, in the antibiofilm test, nisin in combination with sodium fluoride produced a stronger bactericidal effect on a S. mutans biofilm for 4 h and 16 h compared with sodium fluoride alone by confocal laser scanning microscopy. Nisin in combination with sodium fluoride exerted a high bactericidal effect on S. mutans and thereby has the potential to be used as an effective drug combination to prevent dental caries.

In Vitro Evaluation of the Antibacterial Activities of MTAD in Combination with Nisin against Enterococcus faecalis

INTRODUCTION MTAD is a common intracanal irrigant. Although MTAD inhibits Enterococcus faecalis in root canals, its bactericidal effect against E. faecalis remains to be improved. Nisin, an antibacterial peptide, possesses a strong bactericidal effect. The study evaluated the synergetic action between MTAD and nisin against E. faecalis. METHODS The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were used to measure the antibacterial activities of MTAD, MTAN (substitution of doxycycline with nisin), and MTADN (nisin in combination with doxycycline), respectively. The synergetic effect between nisin and doxycycline was evaluated by fractional inhibitory concentration (FIC) and time-killing curves. Furthermore, morphologic changes in E. faecalis were observed by scanning electron microscopy after E. faecalis was treated with MTAD, MTAN, or MTADN for 24 hours. RESULTS The MBC of MTADN against E. faecalis was lower than that of MTAD and MTAN. The combination of nisin and doxycycline had a significantly synergetic antibacterial effect on E. faecalis. Among the 3 antimicrobial treatments, MTADN caused the most severe damage to E. faecalis. CONCLUSIONS The combination of nisin and doxycycline has a synergetic antibacterial effect on E. faecalis, and MTAD in conjunction with nisin inhibits E. faecalis better than MTAD alone.

An in vitro investigation of Lactococcus lactis antagonizing cariogenic bacterium Streptococcus mutans.

OBJECTIVES The present study tested the antagonism between Lactococcus lactis and Streptococcus mutans and evaluated the feasibility of the application of L. lactis for the inhibition of S. mutans in the oral cavity. METHODS Competition assays on plates were employed to determine whether L. lactis antagonises S. mutans under different nutritional conditions, and real-time reverse-transcriptase PCR was used to evaluate the effects of metabolites of S. mutans on the bacteriocin nisin genes in L. lactis. Furthermore, the colonisation and effects of L. lactis on the surface of a tooth were examined by scanning electron microscopy. RESULTS L. lactis competitively inhibited S. mutans growth under nutritional deficiency, and the metabolites of S. mutans, including several exogenous molecular signals, enhanced the expression of genes related to nisin synthesis, nisA, nisB and nisI. Additionally, L. lactis effectively colonised the surface of tooth enamel, which showed substantially less decay with L. lactis adhesion compared to S. mutans adhesion. CONCLUSIONS These findings suggest avenues of research into a new strategy to reduce major cariogenic S. mutans adhesion on the surfaces of teeth.

In Vitro Evaluation of MTAD and Nisin in Combination against Common Pathogens Associated with Root Canal Infection

INTRODUCTION Many pathogenic microorganisms were found in an infected root canal. The object of this study was to evaluate the effect of MTAD in combination with nisin on the pathogens associated with root canal infection. METHODS The survival rates of 9 pathogenic bacteria were determined after 1-, 5-, and 10-minute treatment with MTAD, MTAN (substitution of doxycycline with nisin), and MTADN (nisin in combination with doxycycline). The survival rates of Enterococcus faecalis in the starvation phase and pretreatment alkalization as well as in the normal physiological state under MTAD, MTAN, and MTADN challenge for 1, 5, and 10 minutes were evaluated and compared. Furthermore, scanning electron microscopy was used to observe the morphologic modification of Actinomyces naeslundii, Lactobacillus paracasei, and Porphyromonas gingivalis after MTAD and MTADN treatment. RESULTS L. fermenti, L. paracasei, A. viscosus, A. naeslundii, Streptococcus gordonii, and Peptostreptococcus were more sensitive to MTADN and MTAN than to MTAD. MTAD, MTAN, and MTADN showed a rapid antibacterial effect on P. gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. Enterococcus faecalis in the stress state was as sensitive to MTAD, MTAN, and MTADN as the control E. faecalis. Furthermore, in the observation of scanning electron microscopy, the membranes in A. naeslundii and L. paracasei presented significant rupture, and P. gingivalis did not exhibit significant damage after MTADN treatment. CONCLUSIONS MTAD in combination with nisin improved antibacterial efficacy against pathogens, especially for some gram-positive bacteria associated with persistent intracanal infection. Therefore, the combination had the potential to be used as an effective intracanal irrigation.