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author:("Fan, wuwei")
1.  Psr is involved in regulation of glucan production, and double deficiency of BrpA and Psr is lethal in Streptococcus mutans 
Microbiology  2013;159(Pt 3):493-506.
Streptococcus mutans, the primary causative agent of dental caries, contains two paralogues of the LytR-CpsA-Psr family proteins encoded by brpA and psr, respectively. Previous studies have shown that BrpA plays an important role in cell envelope biogenesis/homeostasis and affects stress responses and biofilm formation by Strep. mutans, traits critical to cariogenicity of this bacterium. In this study, a Psr-deficient mutant, TW251, was constructed. Characterization of TW251 showed that deficiency of Psr did not have any major impact on growth rate. However, when subjected to acid killing at pH 2.8, the survival rate of TW251 was decreased dramatically compared with the parent strain UA159. In addition, TW251 also displayed major defects in biofilm formation, especially during growth with sucrose. When compared to UA159, the biofilms of TW251 were mainly planar and devoid of extracellular glucans. Real-time-PCR and Western blot analyses revealed that deficiency of Psr significantly decreased the expression of glucosyltransferase C, a protein known to play a major role in biofilm formation by Strep. mutans. Transmission electron microscopy analysis showed that deficiency of BrpA caused alterations in cell envelope and cell division, and the most significant defects were observed in TW314, a Psr-deficient and BrpA-down mutant. No such effects were observed with Psr mutant TW251 under similar conditions. These results suggest that while there are similarities in functions between BrpA and Psr, distinctive differences also exist between these two paralogues. Like Bacillus subtilis but different from Staphylococcus aureus, a functional BrpA or Psr is required for viability in Strep. mutans.
doi:10.1099/mic.0.063032-0
PMCID: PMC3709821  PMID: 23288544
2.  Formulation and Characterization of Antibacterial Fluoride-releasing Sealants 
Pediatric dentistry  2013;35(1):13-18.
Purpose
The purpose of this study was to formulate and characterize experimental antibacterial fluoride-releasing sealants and compare them with commercial sealants for fluoride release, recharge, adhesion, and microleakage.
Methods
Two experimental sealants (Exp-1, Exp-2) containing a synthesized antibacterial fluoride-releasing monomer and fluoride-releasing filler were formulated. Exp-2 also contained NovaMin nanoparticles. Commercial sealants Clinpro (CL) FluoroShield (FS), and SeLECT Defense (E34) were also included. Fluoride release from disk samples in deionized water was measured daily using an ion-selective electrode for 14 days, and after recharging with Neutra-Foam (2.0% sodium fluoride), fluoride was measured for 5 days. Microtensile bonding strengths (MTBS) to enamel were tested after 24-hour storage in water at 37°C or thermocycling 5-55°C for 1,000 cycles. A microleakage test was conducted on extracted teeth using a dye-penetration method. The data were analyzed using analysis of variance with the Tukey’s honestly significant difference test and Kruskal-Wallis test.
Results
Exp-1 and Exp-2 had significantly higher fluoride release and recharge capabilities than CL and FL (P<.05). All tested sealants had similar MTBS before and after thermocycling. Exp-2 and Exp-1 had significantly lower microleakage scores (P<.05) than other groups.
Conclusion
The experimental sealants had higher fluoride release and recharge capabilities and similar or better retention than commercial sealants.
PMCID: PMC3644993  PMID: 23635887
ANTIBACTERIAL; FLUORIDE RELEASE; SEALANTS; MICROLEAKAGE; MICROTENSILE BOND STRENGTH
3.  Novel amelogenin-releasing hydrogel for remineralization of enamel artificial caries 
Recently, the use of recombinant full-length amelogenin protein in combination with fluoride has shown promising results in the formation of densely packed enamel-like structures. In this study, amelogenin (rP172)-releasing hydrogels containing calcium, phosphate, and fluoride were investigated for remineralization efficacy using in vitro early enamel caries models. The hydrogels were applied to artificial caries lesions on extracted human third molars, and the remineralization efficacy was tested in different models: static gel remineralization in the presence of artificial saliva, pH cyclic treatment at pH 5.4 acetic buffer and pH 7.3 gel remineralization, and treatment with multispecies oral biofilms grown in a continuous flowing constant-depth film fermenter. The surface microhardness of remineralized enamel increased significantly when amelogenin was released from hydrogel. No cytotoxicity was observed when periodontal ligament cells were cultured with the mineralized hydrogels.
doi:10.1177/0883911512458050
PMCID: PMC3548329  PMID: 23338820
Remineralization; biocompatibility; enamel-like crystals; amelogenin; oral bacterial biofilm
4.  Synthesis and characterization of antibacterial dental monomers and composites 
The objective of this study is to synthesize antibacterial methacrylate and methacrylamide monomers and formulate antibacterial fluoride-releasing dental composites. Three antibacterial methacrylate or methacrylamide monomers containing long-chain quaternary ammonium fluoride, 1,2-methacrylamido-N,N,N-trimethyldodecan-1-aminium fluoride (monomer I), N-benzyl-11-(methacryloyloxy)-N,N-dimethylundecan-1-aminium fluoride (monomer II), and methacryloxyldecylpyridinium fluoride (monomer III) have been synthesized and analyzed by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The cytotoxicity test and bactericidal test against Streptococcus mutans indicate that antibacterial monomer II is superior to monomers I and III. A series of dental composites containing 0–6% of antibacterial monomer II have been formulated and tested for degree of conversion (DC), flexure strength, water sorption, solubility, and inhibition of S. mutans biofilms. An antibacterial fluoride-releasing dental composite has also been formulated and tested for flexure strength and fluoride release. The dental composite containing 3% of monomer II has a significant effect against S. mutans biofilm formation without major adverse effects on its physical and mechanical properties. The new antibacterial monomers can be used together with the fluoride-releasing monomers containing a ternary zirconiun- fluoride chelate to formulate a new antibacterial fluoride- releasing dental composite. Such a new dental composite is expected to have higher anticaries efficacy and longer service life.
doi:10.1002/jbm.b.32683
PMCID: PMC3407682  PMID: 22447582
synthesis; antibacterial monomers; dental composites; biofilm; mechanical properties
5.  Novel Dental Composites Reinforced with Zirconia-Silica Ceramic Nanofibers 
Dental Materials  2011;28(4):360-368.
Objective
To fabricate and characterize dental composites reinforced with various amounts of zirconia-silica (ZS) or zirconia-yttria-silica (ZYS) ceramic nanofibers.
Methods
Control composites (70 wt% glass particle filler, no nanofibers) and experimental composites (2.5, 5.0, and 7.5 wt% ZS or ZYS nanofibers replacing glass particle filler) were prepared by blending 29 wt% dental resin monomers, 70 wt% filler, and 1.0 wt% initiator, and polymerized by either heat or dental curing light. Flexural strength (FS), flexural modulus (FM), energy at break (EAB), and fracture toughness (FT) were tested after the specimens were stored in 37 °C deionized water for 24 h, 3 months, or 6 months. Degree of conversion (DC) of monomers in composites was measured using Fourier transformed near-infrared (FT-NIR) spectroscopy. Fractured surfaces were observed by field-emission scanning electron microscope (FE-SEM). The data were analyzed using ANOVA with Tukey’s Honestly Significant Differences test used for post hoc analysis.
Results
Reinforcement of dental composites with ZS or ZYS nanofibers (2.5% or 5.0%) can significantly increase the FS, FM and EAB of dental composites over the control. Further increase the content of ZS nanofiber (7.5%), however, decreases these properties (although they are still higher than those of the control). Addition of nanofibers did not decrease the long-term mechanical properties of these composites. All ZS reinforced composites (containing 2.5%, 5.0% and 7.5% ZS nanofibers) exhibit significantly higher fracture toughness than the control. The DC of the composites decreases with ZS nanofiber content.
Significance
Incorporation of ceramic nanofibers in dental composites can significantly improve their mechanical properties and fracture toughness and thus may extend their service life.
doi:10.1016/j.dental.2011.11.006
PMCID: PMC3299838  PMID: 22153326
reinforcement; zirconia; ceramic nanofiber; dental composites; mechanical properties; flexural strength; fracture toughness
6.  Transcriptional Repressor Rex Is Involved in Regulation of Oxidative Stress Response and Biofilm Formation by Streptococcus mutans 
FEMS microbiology letters  2011;320(2):110-117.
The transcriptional repressor Rex has been implicated in regulation of energy metabolism and fermentative growth in response to redox potential. Streptococcus mutans, the primary causative agent of human dental caries, possesses a gene that encodes a protein with high similarity to members of the Rex family of proteins. In this study, we showed that Rex-deficiency compromised the ability of S. mutans to cope with oxidative stress and to form biofilms. The Rex-deficient mutant also accumulated less biofilm after 3-days than the wild-type strain, especially when grown in sucrose-containing medium, but produced more extracellular glucans than the parental strain. Rex-deficiency caused substantial alterations in gene transcription, including those involved in heterofermentative metabolism, NAD+ regeneration and oxidative stress. Among the up-regulated genes was gtfC, which encodes glucosyltransferase C, an enzyme primarily responsible for synthesis of water-insoluble glucans. These results reveal that Rex plays an important role in oxidative stress responses and biofilm formation by S. mutans.
doi:10.1111/j.1574-6968.2011.02293.x
PMCID: PMC3115380  PMID: 21521360
Redox sensing; oxidative stress; biofilm formation; Streptococcus mutans
7.  Amelogenin-assisted ex vivo remineralization of human enamel: effects of supersaturation degree and fluoride concentration 
Acta biomaterialia  2011;7(5):2293-2302.
The formation of organized nanocrystals that resemble enamel is crucial for successful enamel remineralization. Calcium, phosphate and fluoride ions and amelogenin are important ingredients for the formation of organized hydroxyapatite (HAP) crystals in vitro. However, the effects of these remineralization agents on the enamel crystal morphology have not been thoroughly studied. The objective of this study was to investigate the effects of fluoride ions, supersaturation degree and amelogenin on the crystal morphology and organization of ex vivo remineralized human enamel. Extracted third molars were sliced thin and acid-etched to provide the enamel surface for immersion in different remineralization solutions. The crystal morphology and mineral phase of the remineralized enamel surface were analyzed by FE-SEM, ATR-FTIR and XRD. The concentration of fluoride and supersaturation degree of hydroxyapatite had significant effects on the crystal morphology and crystal organization, which varied from plate-like loose crystals to rod-like densely packed nanocrystal arrays. Densely packed arrays of fluoridated hydroxyapatite nanorods were observed under the following conditions: σ(HAP) = 10.2±2.0 with fluoride 1.5±0.5 mg/L and amelogenin 40±10 µg/mL, pH 6.8±0.4. A phase diagram summarized the conditions that form dense or loose hydroxyapatite nanocrystal structures. This study provides the basis for the development of novel dental materials for caries management.
doi:10.1016/j.actbio.2011.01.028
PMCID: PMC3074030  PMID: 21256987
Supersaturation degree; Enamel-like crystals; Fluoride; Remineralization; Amelogenin
8.  Fabrication and Characterization of Dense Zirconia and Zirconia-Silica Ceramic Nanofibers 
The objective of this study was to prepare dense zirconia-yttria (ZY), zirconia-silica (ZS) and zirconia-yttria-silica (ZYS) nanofibers as reinforcing elements for dental composites. Zirconium (IV) propoxide, yttrium nitrate hexahydrate, and tetraethyl orthosilicate (TEOS) were used as precursors for the preparation of zirconia, yttria, and silica sols. A small amount (1–1.5 wt%) of polyethylene oxide (PEO) was used as a carry polymer. The sols were preheated at 70 °C before electrospinning and their viscosity was measured with a viscometer at different heating time. The gel point was determined by viscosity–time (η–t) curve. The ZY, ZS and ZYS gel nanofibers were prepared using a special reactive electrospinning device under the conditions near the gel point. The as-prepared gel nanofibers had diameters between 200 and 400 nm. Dense (nonporous) ceramic nanofibers of zirconia-yttria (96/4), zirconia-silica (80/20) and zirconia-yttria-silica (76.8/3.2/20) with diameter of 100–300 nm were obtained by subsequent calcinations at different temperatures. The gel and ceramic nanofibers obtained were characterized by scanning electron microscope (SEM), high-resolution field-emission scanning electron microscope (FE-SEM), thermogravimetric analyzer (TGA), differential scanning calorimeter (DSC), Fourier transform infrared spectrometer (FT-IR), and X-ray diffraction (XRD). SEM micrograph revealed that ceramic ZY nanofibers had grained structure, while ceramic ZS and ZYS nanofibers had smooth surfaces, both showing no visible porosity under FE-SEM. Complete removal of the polymer PEO was confirmed by TGA/DSC and FT-IR. The formation of tetragonal phase of zirconia and amorphous silica was proved by XRD. In conclusion, dense zirconia-based ceramic nanofibers can be fabricated using the new reactive sol–gel electrospinning technology with minimum organic polymer additives.
PMCID: PMC3181101  PMID: 21133090
Reactive Electrospinning; Zirconia; Silica; Ceramic Nanofiber; Dental Composite
9.  Controlled remineralization of enamel in the presence of amelogenin and fluoride 
Biomaterials  2008;30(4):478-483.
Reconstructing enamel-like structures on teeth has been an important topic of study in the material sciences and dentistry. The important role of amelogenin in modulating the mineralization of organized calcium phosphate crystals has been previously reported. We used amelogenin and utilized a modified biomimetic deposition method to remineralize the surface of etched enamel to form mineral layers containing organized needle-like fluoridated hydroxyapatite crystals. The effect of a recombinant amelogenins (rP172) on the microstructure of the mineral in the coating was analyzed by SEM, XRD and FT-IR. At rP172 concentrations below 33 μg/mL, no significant effect was observed. In the presence of 1 mg/L F and at a concentration of 33 μg/mL rP172, formation of fused crystals growing from the enamel surface was initiated. Amelogenin promoted the oriented bundle formation of needle-like fluoridated hydroxyapatite in a dose dependent manner. Biomimetic synthesis of the amelogenin fluoridated hydroxyapatite nano-composite is one of the primary steps towards the development and design of novel biomaterial for future application in reparative and restorative dentistry.
doi:10.1016/j.biomaterials.2008.10.019
PMCID: PMC2642519  PMID: 18996587
amelogenin; fluoride; enamel remineralization; fluoridated hydroxyapatite
10.  Immunogold Labeling of Amelogenin in Developing Porcine Enamel Revealed by Field Emission Scanning Electron Microscopy 
Cells, tissues, organs  2008;189(1-4):207-211.
The present study describes a method using immunohistochemical labeling in combination with high-resolution imaging (field emission scanning electron microscopy) to investigate the spatial localization of amelogenins on apatite crystallites in developing porcine enamel. Cross-sections of developing enamel tissue from freeze-fractured pig third molar were treated with antiserum against recombinant mouse amelogenin and immunoreactivity confirmed by Western blot analysis. The samples were then treated with the goat anti-rabbit IgG conjugated with 10-nm gold particles. The control samples were treated with the secondary antibody only. The in-lens secondary electrons detector and quadrant back-scattering detector were employed to reveal the high-resolution morphology of enamel structures and gold particle distribution. The immunolabeling showed a preference of the gold particle localization along the side faces of the ribbon-like apatite crystals. The preferential localization of amelogenin in vivo on enamel crystals strongly supports its direct function in controlling crystal morphology.
doi:10.1159/000151385
PMCID: PMC2633245  PMID: 18701812
Amelogenin; Developing porcine enamel; Immunogold labeling; Field emission scanning electron microscopy; Biomineralization
11.  Immunogold Labeling of Amelogenin in Developing Porcine Enamel Revealed by Field Emission Scanning Electron Microscopy 
Cells, Tissues, Organs  2008;189(1-4):207-211.
The present study describes a method using immunohistochemical labeling in combination with high-resolution imaging (field emission scanning electron microscopy) to investigate the spatial localization of amelogenins on apatite crystallites in developing porcine enamel. Cross-sections of developing enamel tissue from freeze-fractured pig third molar were treated with antiserum against recombinant mouse amelogenin and immunoreactivity confirmed by Western blot analysis. The samples were then treated with the goat anti-rabbit IgG conjugated with 10-nm gold particles. The control samples were treated with the secondary antibody only. The in-lens secondary electrons detector and quadrant back-scattering detector were employed to reveal the high-resolution morphology of enamel structures and gold particle distribution. The immunolabeling showed a preference of the gold particle localization along the side faces of the ribbon-like apatite crystals. The preferential localization of amelogenin in vivo on enamel crystals strongly supports its direct function in controlling crystal morphology.
doi:10.1159/000151385
PMCID: PMC2633245  PMID: 18701812
Amelogenin; Developing porcine enamel; Immunogold labeling; Field emission scanning electron microscopy; Biomineralization
12.  Enamel inspired nano-composite fabrication through amelogenin supramolecular assembly 
Biomaterials  2007;28(19):3034-3042.
Fabricating the structures similar to dental enamel through the in vitro preparation method is of great interest in the field of dentistry and material science. Developing enamel is composed of calcium phosphate mineral, water, and enamel matrix proteins, mainly amelogenins. To prepare a material mimicking such composition a novel approach of simultaneously assembling amelogenin and calcium phosphate precipitates by electrolytic deposition was established. It was found that recombinant full-length amelogenin (rP172) self-assembled into nanochain structures during electrolytic deposition (following increase in solution pH), and had significant effect on the induction of the parallel bundles of calcium phosphate nanocrystals, grown on semiconductive silicon wafer surface. When a truncated amelogenin (rP148) was used; no nano-chain assembly was observed, neither parallel bundles were formed. The coating obtained in the presence of rP172 had improved elastic modulus and hardness when compared to the coating incorporated with rP148. Our data suggest that the formation of organized bundles in amelogenin-apatite composites is mainly driven by amelogenin nanochain assembly and highlights the potential of such composite for future application as dental restorative materials.
doi:10.1016/j.biomaterials.2007.02.016
PMCID: PMC1995434  PMID: 17382381
Electrolytic deposition; Amelogenin; Self-assembly; Enamel; Nanoindentation

Results 1-12 (12)