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author:("Zhou, xuelong")
1.  Oral Microbiota Distinguishes Acute Lymphoblastic Leukemia Pediatric Hosts from Healthy Populations 
PLoS ONE  2014;9(7):e102116.
In leukemia, oral manifestations indicate aberrations in oral microbiota. Microbiota structure is determined by both host and environmental factors. In human hosts, how health status shapes the composition of oral microbiota is largely unknown. Taking advantage of advances in high-throughput sequencing, we compared the composition of supragingival plaque microbiota of acute lymphoblastic leukemia (ALL) pediatric patients with healthy controls. The oral microbiota of leukemia patients had lower richness and less diversity compared to healthy controls. Microbial samples clustered into two major groups, one of ALL patients and another of healthy children, with different structure and composition. Abundance changes of certain taxa including the Phylum Firmicutes, the Class Bacilli, the Order Lactobacillales, the Family Aerococcaceae and Carnobacteriaceae, as well as the Genus Abiotrophia and Granulicatella were associated with leukemia status. ALL patients demonstrated a structural imbalance of the oral microbiota, characterized by reduced diversity and abundance alterations, possibly involved in systemic infections, indicating the importance of immune status in shaping the structure of oral microbiota.
PMCID: PMC4099009  PMID: 25025462
2.  Regenerative Endodontics: Barriers and Strategies for Clinical Translation 
Dental clinics of North America  2012;56(3):639-649.
Despite a great deal of enthusiasm and effort, regenerative endodontics has encountered substantial challenges towards clinical translation. Recent adoption by the American Dental Association (ADA) of evoked pulp bleeding in immature permanent teeth is an important step for regenerative endodontics. However, there is no regenerative therapy for the majority of endodontic diseases. Simple recapitulation of cell therapy and tissue engineering strategies that are under development for other organ systems has not led to clinical translation in regeneration endodontics. Dental pulp stem cells may appear to be a priori choice for dental pulp regeneration. However, dental pulp stem cells may not be available in a patient who is in need of pulp regeneration. Even if dental pulp stem cells are available autologously or perhaps allogeneically, one must address a multitude of scientific, regulatory and commercialization barriers, and unless these issues are resolved, transplantation of dental pulp stem cells will remain a scientific exercise, rather than a clinical reality. Recent work using novel biomaterial scaffolds and growth factors that orchestrate the homing of host endogenous cells represents a departure from traditional cell transplantation approaches and may accelerate clinical translation. Given the functions and scale of dental pulp and dentin, regenerative endodontics is poised to become one of the early biological solutions in regenerative dental medicine.
PMCID: PMC4093795  PMID: 22835543
regenerative; endodontics; pulp; dentin; regeneration; stem cells; tissue engineering
3.  Growth Phase and pH Influence Peptide Signaling for Competence Development in Streptococcus mutans 
Journal of Bacteriology  2014;196(2):227-236.
The development of competence by the dental caries pathogen Streptococcus mutans is mediated primarily through the alternative sigma factor ComX (SigX), which is under the control of multiple regulatory systems and activates the expression of genes involved in DNA uptake and recombination. Here we report that the induction of competence and competence gene expression by XIP (sigX-inducing peptide) and CSP (competence-stimulating peptide) is dependent on the growth phase and that environmental pH has a potent effect on the responses to XIP. A dramatic decline in comX and comS expression was observed in mid- and late-exponential-phase cells. XIP-mediated competence development and responses to XIP were optimal around a neutral pH, although mid-exponential-phase cells remained refractory to XIP treatment, and acidified late-exponential-phase cultures were resistant to killing by high concentrations of XIP. Changes in the expression of the genes for the oligopeptide permease (opp), which appears to be responsible for the internalization of XIP, could not entirely account for the behaviors observed. Interestingly, comS and comX expression was highly induced in response to endogenously overproduced XIP or ComS in mid-exponential-phase cells. In contrast to the effects of pH on XIP, competence induction and responses to CSP in complex medium were not affected by pH, although a decreased response to CSP in cells that had exited early-exponential phase was observed. Collectively, these results indicate that competence development may be highly sensitive to microenvironments within oral biofilms and that XIP and CSP signaling in biofilms could be spatially and temporally heterogeneous.
PMCID: PMC3911236  PMID: 24163340
4.  Antibacterial Effect of Dental Adhesive Containing Dimethylaminododecyl Methacrylate on the Development of Streptococcus mutans Biofilm 
Antibacterial bonding agents and composites containing dimethylaminododecyl methacrylate (DMADDM) have been recently developed. The objectives of this study were to investigate the antibacterial effect of novel adhesives containing different mass fractions of DMADDM on Streptococcus mutans (S. mutans) biofilm at different developmental stages. Different mass fractions of DMADDM were incorporated into adhesives and S. mutans biofilm at different developmetal stages were analyzed by MTT assays, lactic acid measurement, confocal laser scanning microscopy and scanning electron microscopy observations. Exopolysaccharides (EPS) staining was used to analyze the inhibitory effect of DMADDM on the biofilm extracellular matrix. Dentin microtensile strengths were also measured. Cured adhesives containing DMADDM could greatly reduce metabolic activity and lactic acid production during the development of S. mutans biofilms (p < 0.05). In earlier stages of biofilm development, there were no significant differences of inhibitory effects between the 2.5% DMADDM and 5% DMADDM group. However, after 72 h, the anti-biofilm effects of adhesives containing 5% DMADDM were significantly stronger than any other group. Incorporation of DMADDM into adhesive did not adversely affect dentin bond strength. In conclusion, adhesives containing DMADDM inhibited the growth, lactic acid production and EPS metabolism of S. mutans biofilm at different stages, with no adverse effect on its dentin adhesive bond strength. The bonding agents have the potential to control dental biofilms and combat tooth decay, and DMADDM is promising for use in a wide range of dental adhesive systems and restoratives.
PMCID: PMC4139875  PMID: 25046750
antibacterial adhesive; DMADDM; S. mutans biofilm; microtensile
5.  Adiponectin Ameliorates Experimental Periodontitis in Diet-Induced Obesity Mice 
PLoS ONE  2014;9(5):e97824.
Adiponectin is an adipokine that sensitizes the body to insulin. Low levels of adiponectin have been reported in obesity, diabetes and periodontitis. In this study we established experimental periodontitis in male adiponectin knockout and diet-induced obesity mice, a model of obesity and type 2 diabetes, and aimed at evaluating the therapeutic potential of adiponectin. We found that systemic adiponectin infusion reduced alveolar bone loss, osteoclast activity and infiltration of inflammatory cells in both periodontitis mouse models. Furthermore, adiponectin treatment decreased the levels of pro-inflammatory cytokines in white adipose tissue of diet-induced obesity mice with experimental periodontitis. Our in vitro studies also revealed that forkhead box O1, a key transcriptional regulator of energy metabolism, played an important role in the direct signaling of adiponectin in osteoclasts. Thus, adiponectin increased forkhead box O1 mRNA expression and its nuclear protein level in osteoclast-precursor cells undergoing differentiation. Inhibition of c-Jun N-terminal kinase signaling decreased nuclear protein levels of forkhead box O1. Furthermore, over-expression of forkhead box O1 inhibited osteoclastogenesis and led to decreased nuclear levels of nuclear factor of activated T cells c1. Taken together, this study suggests that systemic adiponectin application may constitute a potential intervention therapy to ameliorate type 2 diabetes-associated periodontitis. It also proposes that adiponectin inhibition of osteoclastogenesis involves forkhead box O1.
PMCID: PMC4023953  PMID: 24836538
6.  Inhibition of Rgs10 Expression Prevents Immune Cell Infiltration in Bacteria-induced Inflammatory Lesions and Osteoclast-mediated Bone Destruction 
Bone research  2013;1(3):267-281.
Regulator of G-protein Signaling 10 (Rgs10) plays an important function in osteoclast differentiation. However, the role of Rgs10 in immune cells and inflammatory responses, which activate osteoclasts in inflammatory lesions, such as bacteria-induced periodontal disease lesions, remains largely unknown. In this study, we used an adeno-associated virus (AAV-) mediated RNAi (AAV-shRNA-Rgs10) knockdown approach to study Rgs10’s function in immune cells and osteoclasts in bacteria-induced inflammatory lesions in a mouse model of periodontal disease. We found that AAV-shRNA-Rgs10 mediated Rgs10 knockdown impaired osteoclastogenesis and osteoclast-mediated bone resorption, in vitro and in vivo. Interestingly, local injection of AAV-shRNA-Rgs10 into the periodontal tissues in the bacteria-induced inflammatory lesion greatly decreased the number of dendritic cells, T-cells and osteoclasts, and protected the periodontal tissues from local inflammatory damage and bone destruction. Importantly, AAV-mediated Rgs10 knockdown also reduced local expression of osteoclast markers and pro-inflammatory cytokines. Our results demonstrate that AAV-shRNA-Rgs10 knockdown in periodontal disease tissues can prevent bone resorption and inflammation simultaneously. Our data indicate that Rgs10 may regulate dendritic cell proliferation and maturation, as well as the subsequent stimulation of T-cell proliferation and maturation, and osteoclast differentiation and activation. Our study suggests that AAV-shRNA-Rgs10 can be useful as a therapeutic treatment of periodontal disease.
PMCID: PMC3994128  PMID: 24761229
Rgs10; immune cell; AAV-mediated RNAi knockdown; gene therapy; periodontal disease; gingival inflammation; bone resorption
7.  Development of HuMiChip for Functional Profiling of Human Microbiomes 
PLoS ONE  2014;9(3):e90546.
Understanding the diversity, composition, structure, function, and dynamics of human microbiomes in individual human hosts is crucial to reveal human-microbial interactions, especially for patients with microbially mediated disorders, but challenging due to the high diversity of the human microbiome. Here we have developed a functional gene-based microarray for profiling human microbiomes (HuMiChip) with 36,802 probes targeting 50,007 protein coding sequences for 139 key functional gene families. Computational evaluation suggested all probes included are highly specific to their target sequences. HuMiChip was used to analyze human oral and gut microbiomes, showing significantly different functional gene profiles between oral and gut microbiome. Obvious shifts of microbial functional structure and composition were observed for both patients with dental caries and periodontitis from moderate to advanced stages, suggesting a progressive change of microbial communities in response to the diseases. Consistent gene family profiles were observed by both HuMiChip and next generation sequencing technologies. Additionally, HuMiChip was able to detect gene families at as low as 0.001% relative abundance. The results indicate that the developed HuMiChip is a useful and effective tool for functional profiling of human microbiomes.
PMCID: PMC3942451  PMID: 24595026
8.  A Comparative Study on Root Canal Repair Materials: A Cytocompatibility Assessment in L929 and MG63 Cells 
The Scientific World Journal  2014;2014:463826.
Cytocompatibility of repair materials plays a significant role in the success of root canal repair. We conducted a comparative study on the cytocompatibility among iRoot BP Plus, iRoot FS, ProRoot MTA, and Super-EBA in L929 cells and MG63 cells. The results revealed that iRoot FS was able to completely solidify within 1 hour. iRoot BP Plus required 7-day incubation, which was much longer than expected (2 hours), to completely set. ProRoot MTA and Super-EBA exhibited a similar setting duration of 12 hours. All the materials except Super-EBA possessed negligible in vitro cytotoxicity. iRoot FS had the best cell adhesion capacity in both L929 and MG63 cells. With rapid setting, negligible cytotoxicity, and enhanced cell adhesion capacity, iRoot FS demonstrated great potential in clinical applications. Future work should focus on longer-term in vitro cytocompatibility and an in vivo assessment.
PMCID: PMC3913516  PMID: 24526893
9.  Inhibition of TGF–β signaling in subchondral bone mesenchymal stem cells attenuates osteoarthritis 
Nature medicine  2013;19(6):704-712.
Osteoarthritis is a highly prevalent and debilitating joint disorder. There is no effective medical therapy for osteoarthritis due to limited understanding of osteoarthritis pathogenesis. We show that TGF–β1 is activated in the subchondral bone in response to altered mechanical loading in an anterior cruciate ligament transection (ACLT) osteoarthritis mouse model. TGF–β1 concentrations also increased in human osteoarthritis subchondral bone. High concentrations of TGF–β1 induced formation of nestin+ mesenchymal stem cell (MSC) clusters leading to aberrant bone formation accompanied by increased angiogenesis. Transgenic expression of active TGF–β1 in osteoblastic cells induced osteoarthritis. Inhibition of TGF–β activity in subchondral bone attenuated degeneration of osteoarthritis articular cartilage. Notably, knockout of the TGF–β type II receptor (TβRII) in nestin+ MSCs reduced development of osteoarthritis in ACLT mice. Thus, high concentrations of active TGF–β1 in the subchondral bone initiated the pathological changes of osteoarthritis, inhibition of which could be a potential therapeutic approach.
PMCID: PMC3676689  PMID: 23685840
10.  Hyperosmotic response of streptococcus mutans: from microscopic physiology to transcriptomic profile 
BMC Microbiology  2013;13:275.
Oral streptococci metabolize carbohydrate to produce organic acids, which not only decrease the environmental pH, but also increase osmolality of dental plaque fluid due to tooth demineralization and consequent calcium and phosphate accumulation. Despite these unfavorable environmental changes, the bacteria continue to thrive. The aim of this study was to obtain a global view on strategies taken by Streptococcus mutans to deal with physiologically relevant elevated osmolality, and perseveres within a cariogenic dental plaque.
We investigated phenotypic change of S. mutans biofilm upon hyperosmotic challenge. We found that the hyperosmotic condition was able to initiate S. mutans biofilm dispersal by reducing both microbial content and extracellular polysaccharides matrix. We then used whole-genome microarray with quantitative RT-PCR validation to systemically investigate the underlying molecular machineries of this bacterium in response to the hyperosmotic stimuli. Among those identified 40 deferentially regulated genes, down-regulation of gtfB and comC were believed to be responsible for the observed biofilm dispersal. Further analysis of microarray data showed significant up-regulation of genes and pathways involved in carbohydrate metabolism. Specific genes involved in heat shock response and acid tolerance were also upregulated, indicating potential cross-talk between hyperosmotic and other environmental stress.
Hyperosmotic condition induces significant stress response on S. mutans at both phenotypic and transcriptomic levels. In the meantime, it may take full advantage of these environmental stimuli to better fit the fluctuating environments within oral cavity, and thus emerges as numeric-predominant bacterium under cariogenic conditions.
PMCID: PMC4219374  PMID: 24289739
Streptococcus mutans; Hyperosmotic condition; Transcriptional profile; Biofilm dispersal; Environmental fitness; Dental plaque
11.  Phenotypic characterization of the foldase homologue PrsA in Streptococcus mutans 
Molecular oral microbiology  2012;28(2):10.1111/omi.12014.
Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Many of the proteins necessary for its colonization of the oral cavity and pathogenesis are exported to the cell surface or the extracellular matrix, a process that requires the assistance of the export machineries. Bioinformatic analysis revealed that the S. mutans genome contains a prsA gene, whose counterparts in other gram positive bacteria, including Bacillus and Lactococcus encode functions involved in protein post-export. In this study, we constructed a PrsA-deficient derivative of S. mutans and demonstrated that the prsA mutant displayed an altered cell wall/ membrane protein profile as well as cell surface related phenotypes, including auto-aggregation, increased surface hydrophobicity, and abnormal biofilm formation. Further analysis revealed that the disruption of the prsA gene resulted in reduced insoluble glucan production by cell surface localized glucosyltransferases, and mutacin as well as cell surface-display of a heterologous expressed GFP fusion to the cell surface protein SpaP. Our study suggested that PrsA in S. mutans encodes functions similar to the ones identified in Bacillus, and thus is likely involved in protein post-export.
PMCID: PMC3819222  PMID: 23241367
foldase protein PrsA; protein secretion; Streptococcus mutans
12.  Dental plaque microcosm biofilm behavior on calcium phosphate nanocomposite with quaternary ammonium 
Dental Materials  2012;28(8):853-862.
Half of dental restorations fail in 10 years, with secondary caries as the main reason. Calcium phosphate composites could remineralize tooth lesions. The objectives of this study were to: (1) Impart antibacterial activity to a composite with nanoparticles of amorphous calcium phosphate (NACP); and (2) investigate the effect of quaternary ammonium dimethacrylate (QADM) on mechanical and dental plaque microcosm biofilm properties for the first time.
The NACP and glass particles were filled into a dental resin that contained bis(2-methacryloyloxy-ethyl) dimethyl-ammonium bromide, the QADM. NACP nanocomposites containing 0%, 7%, 14%, and 17.5% of QADM by mass, respectively, were photo-cured. A commercial composite with no antibacterial activity was used as control. Mechanical properties were measured in three-point flexure. A human saliva microcosm model was used to grow biofilms on composites. Live/dead assay, metabolic activity, colony-forming unit (CFU) counts, and lactic acid production of biofilms on the composites were measured.
Increasing QADM mass fraction monotonically reduced the biofilm viability, CFU and lactic acid. Biofilms on NACP nanocomposite with 17.5% QADM had metabolic activity that was 30% that on a commercial composite control (p<0.05). Total microorganisms, total streptococci, and mutans streptococci CFU counts (mean±sd; n=6) on composite control was 6-fold those on NACP+17.5% QADM nanocomposite. Composite control had long strings of cells with normal short-rod shapes, while some cells on NACP-QADM nanocomposites disintegrated into pieces. Adding QADM to NACP did not decrease the strength and elastic modulus, which matched (p>0.1) those of a commercial composite without Ca-PO4 or antibacterial activity.
A dental plaque microcosm model was used to evaluate the novel NACP-QADM nanocomposite. The nanocomposite greatly reduced the biofilm viability, metabolic activity and lactic acid, while its mechanical properties matched those of a commercial composite. NACP-QADM nanocomposite with calcium phosphate fillers, good mechanical properties and a strong antibacterial activity may have potential for anti-biofilm and anti-caries restorations.
PMCID: PMC3393817  PMID: 22578992
Antibacterial nanocomposite; amorphous calcium phosphate nanoparticles; quaternary ammonium; dental plaque microcosm biofilm; stress-bearing; dental caries
13.  Histone Demethylases KDM4B and KDM6B Promotes Osteogenic Differentiation Of Human MSCs 
Cell Stem Cell  2012;11(1):50-61.
Human bone marrow mesenchymal stem/stromal cells (MSCs) are multipotent progenitor cells with multilineage differentiation potentials including osteogenesis and adipogenesis. While significant progress has been made in understanding transcriptional controls of MSC fate, little is known about how MSC differentiation is epigenetically regulated. Here we show that the histone demethylases KDM4B and KDM6B play critical roles in osteogenic commitment of MSCs by removing H3K9me3 and H3K27me3. Depletion of KDM4B or KDM6B significantly reduced osteogenic differentiation and increased adipogenic differentiation. Mechanistically, while KDM6B controlled HOX expression by removing H3K27me3, KDM4B promoted DLX expression by removing H3K9me3. Importantly, H3K27me3- and H3K9me3-positive MSCs of bone marrow were significantly elevated in ovariectomized and aging mice in which adipogenesis was highly active. Since histone demethylases are chemically modifiable, KDM4B and KDM6B may present as novel therapeutic targets for controlling MSC fate choices, and lead to clues for new treatment in metabolic bone diseases such as osteoporosis.
PMCID: PMC3392612  PMID: 22770241
14.  C-Jun N-Terminal Kinase (JNK) Mediates Wnt5a-Induced Cell Motility Dependent or Independent of RhoA Pathway in Human Dental Papilla Cells 
PLoS ONE  2013;8(7):e69440.
Wnt5a plays an essential role in tissue development by regulating cell migration, though the molecular mechanisms are still not fully understood. Our study investigated the pathways involved in Wnt5a-dependent cell motility during the formation of dentin and pulp. Over-expression of Wnt5a promoted cell adhesion and formation of focal adhesion complexes (FACs) in human dental papilla cells (hDPCs), while inhibiting cell migration. Instead of activating the canonical Wnt signal pathway in hDPCs, Wnt5a stimulation induced activation of the JNK signal in a RhoA-dependent or independent manner. Inhibiting JNK abrogated Wnt5a-induced FACs formation but not cytoskeletal rearrangement. Both dominant negative RhoA (RhoA T19N) and constitutively active RhoA mutants (RhoA Q63L) blocked the Wnt5a-dependent changes in hDPCs adhesion, migration and cytoskeletal rearrangement here too, with the exception of the formation of FACs. Taken together, our study suggested that RhoA and JNK signaling have roles in mediating Wnt5a-dependent adhesion and migration in hDPCs, and the Wnt5a/JNK pathway acts both dependently and independently of the RhoA pathway.
PMCID: PMC3700942  PMID: 23844260
15.  Effect of amorphous calcium phosphate and silver nanocomposites on dental plaque microcosm biofilms 
A dental composite containing amorphous calcium phosphate nanoparticles (NACP) was developed that released calcium (Ca) and phosphate (PO4) ions and possessed acid-neutralization capability. There has been little study on incorporation of antibacterial agents into calcium phosphate composites. The objective of this study was to investigate the effect of silver nanoparticle (NAg) mass fraction in NACP nanocomposite on mechanical properties and dental plaque microcosm biofilm for the first time. NACP nanoparticles of 116 nm were synthesized via a spray-drying technique. NAg nanoparticles were synthesized using Ag 2-ethylhexanoate and 2-(tert-butylamino)ethyl methacrylate, yielding NAg of particle size of 2.7 nm that were well-dispersed in the resin. Five NACP nanocomposites were fabricated with NAg mass fractions of 0, 0.028, 0.042, 0.088, and 0.175%, respectively. Mechanical properties of NACP nanocomposites containing 0–0.042% of NAg matched those of a commercial composite without antibacterial activity. Live/dead assay of dental plaque microcosm biofilms showed complete coverage with live bacteria on commercial composite. However, there were increasingly more dead bacteria with higher NAg content in the NACP nanocomposite. Colony-forming unit (CFU) counts for total microorganisms, total Streptococci, and mutans Streptococci for NACP nanocomposite with 0.042% NAg were about 1/4 those of commercial composite. Lactic acid production on NACP nanocomposite with 0.042% NAg was 1/3 that on commercial composite. In conclusion, novel NACP–NAg nanocomposites were developed which possessed good mechanical properties and potent antibacterial properties, with substantially reduced biofilm viability and lactic acid production. Hence, the NACP–NAg nanocomposites are promising for dental restorations with remineralizing and antibacterial capabilities.
PMCID: PMC3373271  PMID: 22566464
antibacterial nanocomposite; amorphous calcium phosphate nanoparticles; silver nanoparticles; human dental plaque microcosm biofilm; stress-bearing; tooth caries inhibition
16.  Antibacterial and physical properties of calcium-phosphate and calcium-fluoride nanocomposites with chlorhexidine 
Dental Materials  2012;28(5):573-583.
Previous studies have developed calcium phosphate and fluoride releasing composites. Other studies have incorporated chlorhexidine (CHX) particles into dental composites. However, CHX has not been incorporated in calcium phosphate and fluoride composites. The objectives of this study were to develop nanocomposites containing amorphous calcium phosphate (ACP) or calcium fluoride (CaF2) nanoparticles and CHX particles, and investigate S. mutans biofilm formation and lactic acid production for the first time.
Chlorhexidine was frozen via liquid nitrogen and ground to obtain a particle size of 0.62 µm. Four nanocomposites were fabricated with fillers of: Nano ACP; nano ACP+10% CHX; nano CaF2; nano CaF2+10% CHX. Three commercial materials were tested as controls: A resin-modified glass ionomer, and two composites. S. mutans live/dead assay, colony-forming unit (CFU) counts, biofilm metabolic activity, and lactic acid were measured.
Adding CHX fillers to ACP and CaF2 nanocomposites greatly increased their antimicrobial capability. ACP and CaF2 nanocomposites with CHX that were inoculated with S. mutans had a growth medium pH > 6.5 after 3 d, while the control commercial composites had a cariogenic pH of 4.2. Nanocomposites with CHX reduced the biofilm metabolic activity by 10–20 folds and reduced the acid production, compared to the controls. CFU on nanocomposites with CHX were three orders of magnitude less than that on commercial composite. Mechanical properties of nanocomposites with CHX matched a commercial composite without fluoride.
The novel calcium phosphate and fluoride nanocomposites could be rendered antibacterial with CHX to greatly reduce biofilm formation, acid production, CFU and metabolic activity. The antimicrobial and remineralizing nanocomposites with good mechanical properties may be promising for a wide range of tooth restorations with anti-caries capabilities.
PMCID: PMC3322264  PMID: 22317794
dental nanocomposite; calcium phosphate; calcium fluoride; chlorhexidine; stress-bearing; S. mutans biofilm; caries inhibition
17.  Antibacterial amorphous calcium phosphate nanocomposites with a quaternary ammonium dimethacrylate and silver nanoparticles 
Dental Materials  2012;28(5):561-572.
Calcium and phosphate ion-releasing resin composites are promising for remineralization. However, there has been no report on incorporating antibacterial agents to these composites. The objective of this study was to develop antibacterial and mechanically-strong nanocomposites incorporating a quaternary ammonium dimethacrylate (QADM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP).
The QADM, bis(2-methacryloyloxyethyl) dimethylammonium bromide (ionic dimethacrylate-1), was synthesized from 2-(N,N-dimethylamino)ethyl methacrylate and 2-bromoethyl methacrylate. Ng was synthesized by dissolving Ag 2-ethylhexanoate salt in 2-(tertbutylamino)ethyl methacrylate. Mechanical properties were measured in three-point flexure with bars of 2×2×25 mm (n = 6). Composite disks (diameter = 9 mm, thickness = 2 mm) were inoculated with Streptococcus mutans. The metabolic activity and lactic acid production of biofilms were measured (n = 6). Two commercial composites were used as controls.
Flexural strength and elastic modulus of NACP+QADM, NACP+NAg, and NACP+QADM+NAg matched those of commercial composites with no antibacterial property (p > 0.1). The NACP+QADM+NAg composite decreased the titer counts of adherent S. mutans biofilms by an order of magnitude, compared to the commercial composites (p < 0.05). The metabolic activity and lactic acid production of biofilms on NACP+QADM+NAg composite were much less than those on commercial composites (p < 0.05). Combining QADM and NAg rendered the nanocomposite more strongly antibacterial than either agent alone (p < 0.05).
QADM and NAg were incorporated into calcium phosphate composite for the first time. NACP+QADM+NAg was strongly-antibacterial and greatly reduced the titer counts, metabolic activity, and acid production of S. mutans biofilms, while possessing mechanical properties similar to commercial composites. These nanocomposites are promising to have the double benefits of remineralization and antibacterial capabilities to inhibit dental caries.
PMCID: PMC3322309  PMID: 22305716
Antibacterial nanocomposite; amorphous calcium phosphate; quaternary ammonium salt; silver nanoparticles; Streptococcus mutans biofilm; stress-bearing; tooth caries inhibition
18.  Effects of antibacterial primers with quaternary ammonium and nano-silver on S. mutans impregnated in human dentin blocks 
Recent studies developed antibacterial bonding agents and composites containing a quaternary ammonium dimethacrylate (QADM) and nanoparticles of silver (NAg). The objectives of this study were to investigate: (1) the effect of antibacterial primers containing QADM and NAg on the inhibition of Streptococcus mutans (S. mutans) impregnated into dentin blocks for the first time, and (2) the effect of QADM or NAg alone or in combination, and the effect of NAg mass fraction, on S. mutans viability in dentin.
Scotchbond Multi-Purpose (SBMP) bonding agent was used. QADM and NAg were incorporated into SBMP primer. Six primers were tested: SBMP primer control, control + 10% QADM (mass %), control + 0.05% NAg, control + 10% QADM + 0.05% NAg, control + 0.1% NAg, and control + 10% QADM + 0.1% NAg. S. mutans were impregnated into dentin blocks, then a primer was applied. The viable colony-forming units (CFU) were then measured by harvesting the bacteria in dentin using a sonication method.
Control + 10% QADM + 0.1% NAg had bacteria inhibition zone 8-fold that of control (p < 0.05). The sonication method successfully harvested bacteria from dentin blocks. Control + 10% QADM + 0.1% NAg inhibited S. mutans in dentin blocks, reducing the viable CFU in dentin by three orders of magnitude, compared to control dentin without primer. Using QADM+NAg was more effective than QADM alone. Higher NAg content increased the potency. Dentin shear bond strength was similar for all groups (p > 0.1).
Antibacterial primer with QADM and NAg were shown to inhibit the S. mutans impregnated into dentin blocks for the first time. Bonding agent containing QADM and NAg is promising to eradicate bacteria in tooth cavity and inhibit caries. The QADM and NAg may have applicability to other adhesives, cements, sealants and composites.
PMCID: PMC3631006  PMID: 23422420
Antibacterial dental primer; bacteria impregnation in dentin; quaternary ammonium dimethacrylate; silver nanoparticles; caries inhibition
19.  Dental primer and adhesive containing a new antibacterial quaternary ammonium monomer dimethylaminododecyl methacrylate 
Journal of dentistry  2013;41(4):345-355.
The main reason for restoration failure is secondary caries caused by biofilm acids. Replacing the failed restorations accounts for 50–70% of all operative work. The objectives of this study were to incorporate a new quaternary ammonium monomer (dimethylaminododecyl methacrylate, DMADDM) and nanoparticles of silver (NAg) into a primer and an adhesive, and to investigate their effects on antibacterial and dentin bonding properties.
Scotchbond Multi-Purpose (SBMP) served as control. DMADDM was synthesized and incorporated with NAg into primer/adhesive. A dental plaque microcosm biofilm model with human saliva was used to investigate metabolic activity, colony-forming units (CFU), and lactic acid. Dentin shear bond strengths were measured.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the new DMADDM were orders of magnitude lower than those of a previous quaternary ammonium dimethacrylate (QADM). Uncured primer with DMADDM had much larger inhibition zones than QADM (p<0.05). Cured primer/adhesive with DMADDM-NAg greatly reduced biofilm metabolic activity (p<0.05). Combining DMADDM with NAg in primer/adhesive resulted in less CFU than DMADDM alone (p<0.05). Lactic acid production by biofilms was reduced by 20-fold via DMADDM-NAg, compared to control. Incorporation of DMADDM and NAg into primer/adhesive did not adversely affect dentin bond strength.
A new antibacterial monomer DMADDM was synthesized and incorporated into primer/adhesive for the first time. The bonding agents are promising to combat residual bacteria in tooth cavity and invading bacteria at tooth-restoration margins to inhibit caries. DMADDM and NAg are promising for use into a wide range of dental adhesive systems and restoratives.
PMCID: PMC3631010  PMID: 23353068
Antibacterial dental adhesive; dentin bond strength; silver nanoparticles; quaternary ammonium methacrylate; human saliva microcosm biofilm; caries inhibition
20.  Tetracalcium phosphate composite containing quaternary ammonium dimethacrylate with antibacterial properties 
Tooth caries is a carbohydrate-modified bacterial infectious disease, and recurrent caries is a frequent reason for restoration failure. The objective of this study was to develop a novel antibacterial composite using tetracalcium phosphate (TTCP) fillers and bis(2-methacryloyloxy-ethyl) dimethyl-ammonium bromide, which is a quaternary ammonium dimethacrylate (QADM). QADM was synthesized using 2-(N,N-dimethylamino)ethyl methacrylate and 2-bromoethyl methacrylate and incorporated into a resin. The resin was filled with 40% TTCP and 30% glass particles. The following QADM mass fractions in the composite were tested: 0%, 6%, 12%, and 18%. Streptococcus mutans biofilms were formed on the composites and the colony-forming units (CFUs), metabolic activity, and lactic acid production were measured. The TTCP-QADM composite had flexural strength and elastic modulus similar to those of two commercial composites (p > 0.1). Increasing the QADM content in TTCP composite greatly decreased the bacteria growth and biofilm matrix production. There were significantly more dead bacteria with increasing QADM content. TTCP composite containing 18% QADM had biofilm CFU, metabolic activity, and acid production about half of those without QADM. Inversely linear relationships were established between QADM mass fraction and S. mutans biofilm CFU, metabolic activity, and acid production, with correlation coefficients R2 ≥ 0.98. In conclusion, TTCP-QADM composites were developed and the effect of QADM mass fraction on the antibacterial properties of the composite was determined for the first time. The novel TTCP-QADM composites possessing a strong antibacterial capability, together with calcium phosphate ion release and good mechanical properties, are promising for dental restorations to reduce biofilm growth and recurrent caries.
PMCID: PMC3373275  PMID: 22190356
resin composite; tetracalcium phosphate; antibacterial; quaternary ammonium salt; Streptococcus mutans biofilm; tooth caries inhibition
21.  BMP Receptor 1A Determines the Cell Fate of the Postnatal Growth Plate 
Bone morphogenic proteins (BMPs) are critical for both chondrogenesis and osteogenesis. Previous studies reported that embryos deficient in Bmp receptor (Bmpr)1a or Bmpr1b in cartilage display subtle skeletal defects; however, double mutant embryos develop severe skeletal defects, suggesting a functional redundancy that is essential for early chondrogenesis. In this study, we examined the postnatal role of Bmpr1a in cartilage. In the Bmpr1a conditional knockout (cKO, a cross between Bmpr1a flox and aggrecan-CreERT2 induced by a one-time-tamoxifen injection at birth and harvested at ages of 2, 4, 8 and 20 weeks), there was essentially no long bone growth with little expression of cartilage markers such as SOX9, IHH and glycoproteins. Unexpectedly, the null growth plate was replaced by bone-like tissues, supporting the notions that the progenitor cells in the growth plate, which normally form cartilage, can form other tissues such as bone and fibrous; and that BMPR1A determines the cell fate. A working hypothesis is proposed to explain the vital role of BMPR1A in postnatal chondrogenesis.
PMCID: PMC3807016  PMID: 24163588
BMPR1A; Growth plate; Cell fate; Chondrogenesis; Endochondral Bone.
22.  microRNA miR-34a Regulates Cytodifferentiation and Targets Multi-signaling Pathways in Human Dental Papilla Cells 
PLoS ONE  2012;7(11):e50090.
Odontogenesis relies on the reciprocal signaling interactions between dental epithelium and neural crest-derived mesenchyme, which is regulated by several signaling pathways. Subtle changes in the activity of these major signaling pathways can have dramatic effects on tooth development. An important regulator of such subtle changes is the fine tuning function of microRNAs (miRNAs). However, the underlying mechanism by which miRNAs regulate tooth development remains elusive. This study determined the expression of miRNAs during cytodifferentiation in the human tooth germ and studied miR-34a as a regulator of dental papilla cell differentiation. Using microarrays, miRNA expression profiles were established at selected times during development (early bell stage or late bell stage) of the human fetal tooth germ. We identified 29 differentially expressed miRNAs from early bell stage/late bell stage comparisons. Out of 6 miRNAs selected for validation by qPCR, all transcripts were confirmed to be differentially expressed. miR-34a was selected for further investigation because it has been previously reported to regulate organogenesis. miR-34a mimics and inhibitors were transfected into human fetal dental papilla cells, mRNA levels of predicted target genes were detected by quantitative real-time PCR, and levels of putative target proteins were examined by western blotting. ALP and DSPP expression were also tested by qPCR, western blotting, and immunofluorescence. Findings from these studies suggested that miR-34a may play important roles in dental papilla cell differentiation during human tooth development by targeting NOTCH and TGF-beta signaling.
PMCID: PMC3511455  PMID: 23226240
23.  Molecular Characterization of the Microbiota Residing at the Apical Portion of Infected Root Canals of Human Teeth 
Journal of endodontics  2011;37(10):1359-1364.
This study investigated the bacterial communities residing in the apical portion of human teeth with apical periodontitis in primary and secondary infections using a culture-independent molecular biology approach.
Root canal samples from the apical root segments of extracted teeth were collected from 18 teeth with necrotic pulp and 8 teeth with previous endodontic treatment. Samples were processed for amplification via polymerase chain reaction (PCR) and separated with denaturing gradient gel electrophoresis (DGGE). Selected bands were excised from the gel and sequenced for identification.
Comparable to previous studies of entire root canals, the apical bacterial communities in primary infections were significantly more diverse than in secondary infections (p=0.0003). Inter- and intra-patient comparisons exhibited similar variations in profiles. Different roots of the same teeth with secondary infections displayed low similarity in bacterial composition, while an equivalent sample collected from primary infection contained almost identical populations. Sequencing revealed a high prevalence of fusobacteria, Actinomyces sp. and oral Anaeroglobus geminatus in both types of infection. Many secondary infections contained Burkholderiales or Pseudomonas sp. both of which represent opportunistic environmental pathogens.
Certain microorganisms exhibit similar prevalence in primary and secondary infection indicating that they are likely not eradicated during endodontic treatment. The presence of Burkholderiales and Pseudomonas sp. underscores the problem of environmental contamination. Treatment appears to affect the various root canals of multi-rooted teeth differently, resulting in local changes of the microbiota.
PMCID: PMC3415298  PMID: 21924182
Apical periodontitis; endodontic infections; community profiling; polymerase chain reaction; denaturing gradient gel electrophoresis
24.  The influence of iron availability on human salivary microbial community composition 
Microbial Ecology  2012;64(1):152-161.
It is a well-recognized fact that the composition of human salivary microbial community is greatly affected by its nutritional environment. However, most studies are currently focused on major carbon or nitrogen sources with limited attention to trace elements like essential mineral ions. In this study, we examined the effect of iron availability on the bacterial profiles of an in vitro human salivary microbial community as iron is an essential trace element for the survival and proliferation of virtually all microorganisms. Analysis via a combination of PCR with denaturing gradient gel electrophoresis (DGGE) demonstrated a drastic change in species composition of an in vitro human salivary microbiota when iron was scavenged from the culture medium by addition of the iron chelator 2,2’- bipyridyl (Bipy). This shift in community profile was prevented by the presence of excessive ferrous iron (Fe2+). Most interestingly, under iron deficiency, the in vitro grown salivary microbial community became dominated by several hemolytic bacterial species, including Streptococcus spp., Gemella spp. and Granulicatella spp.all of which have been implicated in infective endocarditis. These data provide evidence that iron availability can modulate host-associated oral microbial communities, resulting in a microbiota with potential clinical impact.
PMCID: PMC3376180  PMID: 22318873
iron availability; microbial flora; oral cavity
25.  Oral Microbiology: Past, Present and Future 
Since the initial observations of oral bacteria within dental plaque by van Leeuwenhoek using his primitive microscopes in 1680, an event that is generally recognized as the advent of oral microbiological investigation, oral microbiology has gone through phases of “reductionism” and “holism”. From the small beginnings of the Miller and Black period, in which microbiologists followed Koch’s postulates, took the reductionist approach to try to study the complex oral microbial community by analyzing individual species; to the modern era when oral researchers embrace “holism” or “system thinking”, adopt new concepts such as interspecies interaction, microbial community, biofilms, poly-microbial diseases, oral microbiological knowledge has burgeoned and our ability to identify the resident organisms in dental plaque and decipher the interactions between key components has rapidly increased, such knowledge has greatly changed our view of the oral microbial flora, provided invaluable insight into the etiology of dental and periodontal diseases, opened the door to new approaches and techniques for developing new therapeutic and preventive tools for combating oral poly-microbial diseases.
PMCID: PMC2949409  PMID: 20687296

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