Longxing Ni

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.

Root canal morphology of permanent three-rooted mandibular first molars--part I: pulp floor and root canal system.

INTRODUCTION Racial variations in root form and canal anatomy present endodontic challenges for clinicians. This study examined root canal morphology of three-rooted mandibular first molars by micro-computed tomography scans. METHODS A total of 122 extracted mandibular first molars were collected from a native Chinese population. After calculating the frequency of occurrence, 20 three-rooted (experimental group) and 25 two-rooted first molars (control group) were scanned and reconstructed three-dimensionally. RESULTS The frequency of three-rooted mandibular first molars was 31.97% (39/122). The mean interorifice distances from the distolingual (DL) canal to the distobuccal (DB) and mesiolingual canal were 2.93 mm and 2.86 mm, respectively. The mesial root predominately contained a type 2-2 root canal, with an incidence of 65% in the experimental group and 64% in the control group. Type 1-1 canals were seen more frequently in the DL and DB roots of the three-rooted first molars as well as in the distal roots of the two-rooted first molars. The incidences were 100% (20/20), 95% (19/20), and 72% (18/25), respectively. Accessory and lateral canals rarely occurred in the extra DL roots. The incidence was only 10% (2/20). A furcation canal extending from the floor to the furcation region was not observed. CONCLUSION Three-rooted mandibular first molars commonly have 4 separate canals with high incidences of accessory canals in the mesial and DB root. The geometric data of pulp floors are useful for locating the extra DL canal.

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.

The role of runt-related transcription factor 2 (Runx2) in the late stage of odontoblast differentiation and dentin formation

Runx2, of the Runx family, is an essential transcription factor that controls bone and tooth development by regulating osteoblast and odontoblast differentiation. However, the function of Runx2 in late stage odontoblast differentiation is not clear. We studied the function of Runx2 in dentinogenesis by generating transgenic mice expressing Runx2 specifically in odontoblasts. We observed dentin formation in postnatal day 3 (P3), P7 and P28 mice and measured the expression levels of Runx2 and matrix proteins in dentin. The odontoblasts in transgenic mice (Tg) lost their tall columnar shape and polarization and dentinal tubules were absent. The dental pulp chamber was dramatically enlarged and the dentin in Tg mice was thinner. Osteoblast-like cells were seen instead of normal odontoblasts and were embedded in a bone-like matrix, indicating that dentin formation was replaced with bone. Predentin was disorganized possessing lacunae that contained odontoblasts. The mandibular molars of Tg mice showed noticeable defects by Micro-CT. Using quantitative real-time PCR, the expression of dentin matrix proteins, particularly dentin sialophosphoprotein (DSPP), was found to be upregulated in 3-day-old Tg mice and downregulated at 1 month of age. These findings indicate that Runx2 inhibited odontoblast terminal differentiation and induced transdifferentiation of odontoblasts to osteoblasts at the late cell differentiation stage. Therefore, Runx2 should be inhibited in odontoblasts to encourage normal cell maturation, differentiation and dentinogenesis.

Root canal morphology of permanent three-rooted mandibular first molars: Part II--measurement of root canal curvatures.

INTRODUCTION The distolingual (DL) roots of three-rooted mandibular molars often challenge clinicians during root canal therapy. This study investigated canal curvatures in permanent three-rooted mandibular first molars by using micro-computed tomography (micro-CT) scans. METHODS Twenty three-rooted (group 1) and twenty-five two-rooted mandibular first molars (group 2) were scanned by micro-CT. The specimens were reconstructed 3-dimensionally by the software Mimics 10.01 and shown in a parallel projection mode. The images of the root canals in clinical view (CV) and proximal view (PV) were analyzed by the software Image-Pro Plus. Schneider method and a modified Pruett method were used to measure the angles and radius of canal curvatures. RESULTS In the three-rooted molar group in a CV, the average angles of primary curvatures were 24.34 degrees for the mesiobuccal, 22.39 degrees for the mesiolingual, 13.71 degrees for the distobuccal (DB), and 13.81 degrees for the DL canal. In a PV, the average angles were 16.60 degrees for the DB and 36.06 degrees for the DL canal, respectively. Secondary curvatures were frequently seen in a CV (60%) for the DB canals, with an average angle of 26.94 degrees. In a PV, the average central angle of curvature was 59.04 degrees for the DL canal, and the average radius and curve length were 6.17 and 5.73 mm, respectively. In general, no statistically significant difference was found for canal curvatures in the mesial roots between the three-rooted and two-rooted molar groups (P > .05). CONCLUSIONS A better understanding of the canal curvatures is essential for successful endodontic treatment of three-rooted mandibular first molars.

Down-regulated non-coding RNA (lncRNA-ANCR) promotes osteogenic differentiation of periodontal ligament stem cells

OBJECTIVE Our studies aimed to figure out how anti-differentiation noncoding RNA (ANCR) regulates the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). DESIGN In this study, we used lentivirus infection to down-regulate the expression of ANCR in PDLSCs. Then we compared the proliferation of control cells and PDLSC/ANCR-RNAi cells by Cell Counting Kit-8. And the osteogenic differentiation of control cells and PDLSC/ANCR-RNAi cells were evaluated by Alkaline phosphatase (ALP) activity quantification and Alizarin red staining. WNT inhibitor was used to analyze the relationship between ANCR and canonical WNT signalling pathway. The expression of osteogenic differentiation marker mRNAs, DKK1, GSK3-β and β-catenin were evaluated by qRT-PCR. RESULTS The results showed that down-regulated ANCR promoted proliferation of PDLSCs. Down-regulated ANCR also promoted osteogenic differentiation of PDLSCs by up-regulating osteogenic differentiation marker genes. After the inhibition of canonical WNT signalling pathway, the osteogenic differentiation of PDLSC/ANCR-RNAi cells was inhibited too. qRT-PCR results also demonstrated that canonical WNT signalling pathway was activated for ANCR-RNAi on PDLSCs during the procedure of proliferation and osteogenic induction. CONCLUSIONS These results indicated that ANCR was a key regulator of the proliferation and osteogenic differentiation of PDLSCs, and its regulating effects was associated with the canonical WNT signalling pathway, thus offering a new target for oral stem cell differentiation studies that could also facilitate oral tissue engineering.

Antibacterial peptide nisin: A potential role in the inhibition of oral pathogenic bacteria

Although the antimicrobial peptide nisin has been extensively studied in the food industry for decades, its application in the oral cavity remains to develop and evaluate its feasibility in treating oral common diseases. Nisin is an odorless, colorless, tasteless substance with low toxicity and with antibacterial activities against Gram-positive bacteria. These biologic properties may establish its use in promising products for oral diseases. This article summarizes the antibacterial efficiency of nisin against pathogenic bacteria related to dental caries and root canal infection and discusses the combination of nisin and common oral drugs.

Antimicrobial and anti-biofilm effect of Bac8c on major bacteria associated with dental caries and Streptococcus mutans biofilms

Dental caries is a common oral bacterial infectious disease. Its prevention and treatment requires control of the causative pathogens within dental plaque, especially Streptococcus mutans (S. mutans). Antimicrobial peptides (AMPs), one of the promising substitutes for conventional antibiotics, have been widely tested and used for controlling bacterial infections. The present study focuses on evaluating the potential of the novel AMPs cyclic bactenecin and its derivatives against bacteria associated with dental caries. The results indicate that Bac8c displayed highest activity against the bacteria tested, whereas both cyclic and linear bactenecin had weak antimicrobial activity. The cytotoxicity assay showed that Bac8c did not cause detectable toxicity at concentrations of 32-128μg/ml for 5min or 32-64μg/ml for 60min. S. mutans and Lactobacillus fermenti treated with Bac8c showed variable effects on bacterial structure via scanning electron microscopy and transmission electron microscopy. There appeared to be a large amount of extracellular debris and obvious holes on the cell surface, as well as loss of cell wall and nucleoid condensation. The BioFlux system was employed to generate S. mutans biofilms under a controlled flow, which more closely resemble the formation process of natural biofilms. Bac8c remarkably reduced the viability of cells in biofilms formed in the BioFlux system. This phenomenon was further analyzed and verified by real-time PCR results of a significant suppression of the genes involved in S. mutans biofilm formation. Taken together, this study suggests that Bac8c has a potential clinical application in preventing and treating dental caries.